Mass Spectrometry-Based Proteomic Analysis of Selected Bacteria from the Human Gut Microbiome

Mass Spec ome y-Based P o eomic Analysis o Selec ed Bac e ia om he Human Gu Mic obiome Disse a ion zu E langung des Dok o g ades de Ma hema isch-Na u wissenscha lichen Fakul ä de Ch is ian-Alb ech s-Uni e si ä zu Kiel o geleg on Je ome Gen h Kiel, 2024 E s e Gu ach e (Fi s e iewe ): P o . D . And eas Tholey Zwei e Gu ach e (Second e iewe ): P o . D . Ru h Schmi z-S ei Tag de Dispu a ion (Da e o de ense): 16.10.2024 Zum D uck genehmig (App o ed o publica ion): 16.10.2024 „Who you a e is de ined by wha you' e willing o s uggle o .” - Ma k Manson P A G E | i ABSTRACT Human gu bac e ia li e in a highly dynamic en i onmen wi h equen changes such as a ia ions in pH o nu ien a ailabili y. To ensu e hei su i al and unc ionali y unde hese luc ua ing condi ions, hey cons an ly adap hei p o eomes by adjus ing he abundance o essen ial p o eins. Howe e , he speci ic p o eomic changes in indi idual human gu mic obiome (HGM) membe s unde di e en in i o condi ions emain la gely unexplo ed. This s udy employs mass spec ome y-based p o eomic analysis o add ess his gap on he selec ed membe s o he human gu mic obiome. Ini ially, he ep oducibili y and accu acy o bo om-up label- ee quan i ica ion (LFQ) and andem mass ag (TMT)-based quan i ica ion we e assessed in quan i ying p o eomic changes o Bac e oides he aio aomic on induced by suc ose and glucose. Bo h me hods achie ed compa able esul s, indica ing ha his bac e ium p ima ily al e s he abundance o p o eins in ol ed in he machine y equi ed o u ilize he p o ided ca bon sou ces. The LFQ app oach was hen applied o examine p o eomic changes in B. he aio aomic on, Blau ia p oduc a, and Bi idobac e ium longum in esponse o di e en en i onmen al pH le els. Dis inc and concu en al e a ions in pa hway- ela ed and s ess-associa ed p o eins we e iden i ied, including his idine biosyn hesis in B. p oduc a, ni ogen me abolism in B. he aio aomic on, and inosi ol ca bohyd a e me abolism in B. he aio aomic on and B. p oduc a. The compa a i e analysis o bo om-up and op-down p o eomics in B. p oduc a demons a ed hei abili y o quan i y he same p o eins, whe he di e en ially abundan o no . The hi d p ojec ocused on iden i ying no el p o eins, speci ically sho open eading ame- encoded pep ides (SEP), in B. p oduc a. A p o eogenomic app oach was used o analyze hei p esence unde a ious cul i a ion condi ions, including di e en media (BHI and YCFA), pH le els, and supplemen ed ac o s (yeas ex ac , SCFAs, and LPS). S ingen alida ion c i e ia ensu ed accu a e iden i ica ion o SEP, and biochemical p edic ions explo ed hei po en ial unc ional oles. The combined bo om-up and op-down p o eomics analyses iden i ied a o al o 45 SEP, including p e iously epo ed SEP (BP1 o BP14). This s udy demons a ed ha he p oduc ion o ce ain SEP in B. p oduc a is in luenced by speci ic en i onmen al ac o s a he han solely by in e species in e ac ions, as p e iously sugges ed. The las p ojec es ablished and op imized an isola ion p o ocol o he LC-MS-based p o eomic analysis o ex acellula esicles. Bo om-up p o eomic analysis o ou e memb ane esicles (OMVs) isola ed om E. coli unde di e en cul u e condi ions and g ow h phases quan i ied OMV ma ke p o eins and classi ied he subcellula opological dis ibu ion o he P A G E | ii E. coli OMV p o eomes. Nanopa icle acking analysis alida ed OMV nanopa icles, and an in i o wound healing assay wi h human colonic Caco-2 cells sugges ed a dose-dependen inhibi o y e ec o OMVs on wound closu e. Va ious sample p epa a ion p o ocols we e e alua ed, and he in eg a ion o non-ionic de e gen s o OMV lysis and p o eoly ic diges ion imp o ed p o ein and pep ide iden i ica ions, making he imp o ed in-solu ion diges ion p o ocol he p e e ed me hod o u u e analyses. In mos p ojec s, a p o eo o m-di ec ed op-down analysis, including a disco e y-based open modi ica ion sea ch, was applied. This analysis iden i ied se e al po en ial pos - ansla ional modi ica ions (e.g., β-me hyl hio-aspa ic acid on B. he aio aomic on ibosomal p o ein S12 and se yl-phospho yla ions on B. p oduc a HP p o eins) and neo- e mini (po en ial a i icial clea age e en s, ini ia o me hionine excisions, and al e na i e ini ia ion si es). P A G E | ix TABLE OF CONTENTS Abs ac ..................................................................................................................................... i Zusammen assung .................................................................................................................. iii Danksagung ............................................................................................................................. Lis o Publica ions and Con e ence Con ibu ions ................................................................. ii Table o Con en s .................................................................................................................... ix I GENERAL INTRODUCTION .................................................................................................. 1 II GENERAL METHODS ....................................................................................................... 17 III PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON ........................................................... 37 IV INFLUENCE OF PH ON BACTERIAL PROTEOMES ............................................................... 63 V PROTEOGENOMIC ANALYSIS OF B. PRODUCTA .............................................................. 103 VI OUTER MEMBRANE VESICLES ANALYSIS ....................................................................... 121 Bibliog aphy ............................................................................................................................... I Lis o Abb e ia ions ........................................................................................................... XXIII Lis o Figu es ..................................................................................................................... XXV Lis o Tables ................................................................................................................... XXVIII Appendix ............................................................................................................................ XXIX P A G E | 1 I GENERAL INTRODUCTION 1 The Dynamic Na u e o he P o eome .............................................................................. 2 1.1 P o eome Va ia ion .................................................................................................... 3 2 P inciples o Mass Spec ome y ..................................................................................... 5 2.1 Online LC-MS ............................................................................................................ 5 2.2 Mass- o-cha ge Analysis ........................................................................................... 5 2.3 Tandem Mass Spec ome y and F agmen a ion Analysis ........................................ 7 3 Analysis o Mass Spec ome y Da a ............................................................................... 8 3.1 Da a Acquisi ion Techniques ..................................................................................... 9 3.2 Pep ido o m and P o ein Iden i ica ion .................................................................... 10 3.3 Quan i a i e Techniques in P o eomics ................................................................... 11 4 In lamma o y Bowel Disease .......................................................................................... 12 4.1 The Human Gu Mic obiome as a The apeu ic Ta ge ............................................ 12 4.2 P o eomics in IBD Resea ch ................................................................................... 14 5 Objec i e o he Thesis ................................................................................................... 15 I | GENERAL INTRODUCTION P A G E | 2 1 The Dynamic Na u e o he P o eome The genome con ains all gene ic in o ma ion and ypically emains la gely s able wi hin a speci ic cell. In con as , he p o eome, ep esen ing he comple e se o p o eins (Wasinge e al., 1995), is cons an ly changing. This cons an al e a ion o he p o eomic landscape is cha ac e ized by con inuous p o ein syn hesis, deg ada ion, and modi ica ion. The abili y o cells o adjus hei p o ein composi ion con inuously in esponse o changing needs and in luences, bo h in e nal and ex e nal, is a key aspec o cellula lexibili y and c ucial o a cell o e ec i ely ca y ou i s unc ions. The undamen al p ocess o ansc ibing DNA (deoxy ibonucleic acid) in o mRNA (messenge ibonucleic acid) molecules and hen ansla ing hem in o amino acid sequences esul s in a wide a ie y o p o eins. Each p o ein is assembled om he epe oi e o 22 p o einogenic amino acids, wi h each amino acid impa ing unique physico-chemical cha ac e is ics o he esul ing p o ein. The e m 'p o ein' is essen ially a gene ic e m e e ing o a canonical amino acid sequence (Cassidy e al., 2023). Wi h an inc eased unde s anding o he p o eome, i becomes e iden ha he adi ional no ion o 'one-gene, one-p o ein, one- unc ion' is inadequa e o ully comp ehend he complexi y o he p o eome (Ca bona a e al., 2021; Cassidy e al., 2023). Recognizing his complexi y, e ms such as 'p o ein species' (Schlü e e al., 2009) and 'p o eo o ms' (Smi h and Kellehe , 2013) ha e eme ged, wi h 'p o eo o ms' gaining widesp ead accep ance (Ca bona a e al., 2021; Ma x, 2024). The p o eo o m concep includes a b oad spec um o molecula a ia ions esul ing om co- and pos - ansla ional modi ica ions (PTMs) and sequence a ian s, which go beyond ansc ip ion and ansla ion e o s (FIGURE I-1). Unde s anding he unc ion o di e se p o eo o ms is essen ial o comp ehending cellula dynamics and p ocesses (Ma x, 2024). FIGURE I-1 | Sou ces o P o eome Complexi y. Iso o m a ia ion o he same gene combined wi h si e- speci ic changes gene a e a a ie y o p o eo o ms. Abb e ia ion: SAV (single amino acid a ia ion). Adap ed wi h pe mission om (Aebe sold e al., 2018). DNA Iso o ms Si e-speci ic ea u es P o eo o ms SAV Glycosyla ion Phospho yla ion GENERAL INTRODUCTION | I P A G E | 3 1.1 P o eome Va ia ion O ganisms ha e de eloped many e ec i e s a egies o di e si y he p o eome wi hou inc easing he size o he genome, using mechanisms ha gene a e mul iple p o eins om a single gene. Gene- and T ansc ip -le el Va ia ion – T ansc ip ional ead- h ough, a undamen al mechanism, allows RNA polyme ases o bypass e mina ion signals and ansc ibe mul iple ope ons wi hin a single mRNA molecule (Wade and G ainge , 2014). In esponse o en i onmen al cues, bac e ia dynamically exhibi ansc ip ional ead- h ough o adap o changing condi ions (Junie and Ri oi e, 2016). In euka yo es, ansc ip ional complexi y a ises no only om ansc ip elonga ion bu also om mRNA splicing, du ing which in e nal exons a e emo ed. Va ia ions in ansc ip s, such as ibosomal ameshi ing, can lead o ansla ion ini ia ion and e mina ion a di e en si es wi hin a single mRNA molecule (A kins e al., 2016), esul ing in polypep ides wi h sequences di e ing om he main open eading ame (ORF) (Ko niy e al., 2019). In pa icula , al e na i e ansla ion ini ia ion and syn hesis o N- e minally unca ed polypep ides a e associa ed wi h he de elopmen o ce ain diseases (Bogae e al., 2020). Addi ionally, RNA edi ing, in ol ing p ocesses such as nucleo ide deamina ion o adenosine in o inosine o nucleo ide inse ions and dele ions, can al e mRNA sequences and lead o he o ma ion o dis inc mRNA iso o ms (Knoop, 2011). T ansla ion-le el Va ia ion – Co- and pos - ansla ional modi ica ions can apidly modi y p o ein p ope ies and unc ions. Cu en ly, o e 200 ypes o PTMs o biological and chemical modi ica ions, p ima ily a ge ing speci ic amino acid esidues (FIGURE I-2), ha e been documen ed (C easy and Co ell, 2004; Mon ecchi-Palazzi e al., 2008). Fo example, phospho yla ion and lipida ion, can edi ec p o eins o speci ic cellula loca ions o modula e hei in e ac ions wi h o he molecules, he eby signi ican ly in luencing a ious physiological p ocesses such as ansc ip ion, ansla ion, and me abolic unc ions (Jiang e al., 2018; Macek e al., 2019). Addi ionally, o he PTMs such as glycosyla ion, can in luence p o ein olding and s abili y (Jayap akash and Su olia, 2017). Con olled p o eolysis, by endopep idases, can p ecisely clea e p o eins, con e ing inac i e zymogens in o hei biologically ac i e o ms (Neu a h and Walsh, 1976). Zymogens ypically con ain inhibi o y p opep ides a hei N- e mini, ensu ing bo h he p o ec ion o p o ein unc ion and hei di ec ed anspo o speci ic cellula compa men s. Upon eaching hei des ina ion, he p opep ide is emo ed, ac i a ing he ca aly ic ac i i ies o he p o ein. Fo example, ca hepsins, a amily o lysosomal p o eases, achie e hei op imal unc ionali y wi hin he highly acidic en i onmen o lysosomes upon ac i a ion (Jo dans e al., 2009). This selec i e, compa men -speci ic ac i a ion isola es po en ially de imen al eac ions, he eby sa egua ding he cell. In con as , exopep idases ha I | GENERAL INTRODUCTION P A G E | 4 p ima ily emo e e minal amino acids play a signi ican ole in p o ein deg ada ion p ocesses. Bo h p o eoly ic p ocesses possess he po en ial o gene a e unca ed p o eo o m a ian s, capable o modula ing p o ein ac i i ies and po en ially in luencing he de elopmen o speci ic diseases (Bogae e al., 2020). The analysis o PTMs equi es p ecise mass spec ome y (MS) analysis because isoba ic PTMs sha e simila molecula masses and, consequen ly, compa able mass- o-cha ge a ios. This simila i y inc eases he isk o misiden i ica ions due o measu ing e o s (Kim e al., 2016). Fo example, ime hyla ion (C3H6, 42.047 Da) and ace yla ion (C2H2O, 42.011 Da) on lysine esidues, commonly ound on his one p o eins, ha e ema kably simila masses, di e ing by only 0.036 Da. In he case o a ypical 1 kDa yp ic pep ide (F icke , 2015), his co esponds o a di e ence o 36 ppm (pa s pe million). While mode n high- esolu ion MS mass analyze s ha e mi iga ed his p oblem o pep ides, challenges emain o in ac p o eins. Fo a 15 kDa co e his one, his mass di e ence is 2.4 ppm, highligh ing he c i ical ole o accu a e mass measu emen s in de e mining he co ec p o eo o m. FIGURE I-2 | P o ein Modi ica ions in Bac e ia. O e iew o he mos commonly occu ing p o ein pos - ansla ional modi ica ions, co esponding amino acid esidue, and co esponding modi ying enzymes. Reac i e g oups on amino acid side chains a e highligh ed. PTS, phospho ans e ase sys em; TCS, wo-componen sys em. Adap ed wi h pe mission om (Macek e al., 2019). GENERAL INTRODUCTION | I P A G E | 5 2 P inciples o Mass Spec ome y Mass spec ome y is a powe ul analy ical echnique used o iden i ying molecules based on hei mass- o-cha ge a io (m/z). 2.1 Online LC-MS The p e e ed me hod in p o eomics in ol es he online coupling o a sepa a ion echnique, such as liquid ch oma og aphy (LC), wi h MS. Pep ide and p o ein mix u es a e ypically sepa a ed based on hei hyd ophobic in e ac ions wi h a non-pola s a iona y phase, employing e e sed-phase high-pe o mance liquid ch oma og aphy (RP HPLC). Commonly used mic opa icula e columns consis o silica beads wi h co alen ly bonded hyd ophobic chains, such as oc adecyl alkane chains (C18) o pep ides and bu yl alkane chains (C4) o p o ein mix u es. This esul s in species wi h di e ing hyd ophobici y elu ing a di e en imes due o hei a ini ies o he s a iona y phase. In con as , monoli hic columns ea u e a con inuous po ous s uc u e o he sepa a ion o pep ides o p o eins. Re en ion, a me ic ha quan i ies how long an analy e emains on he column, and esolu ion, he abili y o dis inguish adjacen peaks, a e de e mined by he balance be ween he analy e's hyd ophobic in e ac ions wi h he s a iona y phase and i s solubili y in he mobile phase. The g adual inc ease in he concen a ion o o ganic sol en , ypically ace oni ile, in he mobile phase ini ia es elu ion. The u iliza ion o an acidic mobile phase, o en con aining an ion-pai ing modi ie such as o mic acid o TFA, can enhance ch oma og aphic sepa a ion, aid in con olling e en ion imes, imp o e peak shapes, and imp o e he o e all de ec ion e iciency and pe o mance o he LC- MS sys em (Ga cía, 2005; Lenčo e al., 2022). Elec osp ay ioniza ion (ESI) is a widely used echnique o ans e ing analy es om he liquid phase o he gas phase in online LC-MS se ups. I enables he gen le apo iza ion o molecules wi hou causing signi ican agmen a ion (Keba le and Ve ke k, 2009). Du ing he ESI p ocess, he analy e solu ion is di ec ed h ough a conduc i e capilla y ( e e ed o as he emi e ), o which a ol age is applied. In posi i e ion mode, he p e e ed pola i y o he analysis o p o eins and pep ides, ions a e con e ed in o p o ona ed molecula ions [M+nH]n+ wi h a ious cha ge and p o ona ion s a es. P o ona ion p ima ily occu s a he ee amino e minus and he basic side-chain unc ionali ies o a ginine, lysine, and his idine esidues. 2.2 Mass- o-cha ge Analysis Va ious mass analyze s and de ec o s, each wi h unique cha ac e is ics such as mass accu acy, speed, sensi i i y, and esolu ion ( he abili y o dis inguish closely spaced m/z a ios), ha e been de eloped o measu ing ion m/z a ios. High- esolu ion and accu a e mass analyze s allow o he iden i ica ion o cha ac e is ic iso ope pa e ns, pa icula ly in pep ides I | GENERAL INTRODUCTION P A G E | 6 con aining na u ally occu ing s able, hea y iso opes such as 13C and 15N. Mass analyze s ope a e on di e se p inciples: some (e.g., linea o quad upole ion aps) u ilize di e en elec ic cu en s o manipula e ions o selec i e isola ion, apping, and agmen a ion, while o he s (e.g., ime-o - ligh o O bi ap analyze s) accele a e ions owa ds a de ec o in an elec ic ield o measu e hei ime-o - ligh o adial oscilla ion equencies o de e mine m/z (Sa a yn e al., 2016). Today, hyb id ins umen s combine di e en analyze s allowing lexibili y in expe imen al design. A no able example o such hyb idiza ion is he Fusion Lumos T ib id mass spec ome e , in eg a ing quad upole, O bi ap, and dual-p essu e linea ion ap analyze s (FIGURE I-3). Be o e en e ing he mass spec ome e , ions can be in oduced in o a high- ield asymme ic wa e o m ion mobili y spec ome y (FAIMS) module, o sepa a e hem based on ion mobili y a ia ions in he p esence o high and low elec ic ields (Gue emon , 2004). Ac ing as a mass il e , FAIMS can elimina e singly cha ged con aminan ions and enhance he dep h o p o eome co e age by applying mul iple compensa ion ol ages (CVs) (Swea ingen and Mo i z, 2012; Kaulich e al., 2022a). A e en e ing he Fusion Lumos T ib id mass spec ome e , ions pass h ough ion op ics, which ocus and di ec hem owa d he quad upole. The quad upole hen ac s as a mass il e , allowing o selec p ecu so ions o in e es based on hei m/z alues (Sa a yn e al., 2016). Ions wi hin a speci ic isola ion window a e collec ed, s o ed in he ion- ou ing mul ipole, and hen ans e ed h ough he C- ap in o he O bi ap. F agmen spec a can be acqui ed in ei he he O bi ap o he ion ap mass analyze . FIGURE I-3 | The O bi ap Fusion Lumos T ib id Mass Spec ome e . A e ioniza ion, ion op ics ocus and di ec gas phase analy e ions owa d he quad upole. In he quad upole, ions a e selec i ely il e ed based on hei mass- o-cha ge a io (m/z) wi hin a speci ic isola ion window. The selec ed p ecu so ions a e hen di ec ed o he ion- ou ing mul ipole o he linea ion ap mass analyze o agmen a ion. F agmen ion spec a a e hen acqui ed in ei he he O bi ap o he linea ion ap. Adap ed om h p://plane o bi ap.com. Ion Sou ce T ans e Tube Ion Op ics Quad upole O bi ap C-T ap Ion Rou ing Mul ipole Pe o ms HCD Dual-P essu e Linea Ion T ap Pe o ms CID and ETD GENERAL INTRODUCTION | I P A G E | 7 2.3 Tandem Mass Spec ome y and F agmen a ion Analysis Analyzing samples ia andem mass spec ome y (MS/MS) wi h da a-dependen acquisi ion (DDA) in ol es ob aining p ecu so MS1 spec a, agmen ing isola ed ions, and de e mining he m/z alues o esul ing agmen ions in he MS2 spec a. This p ocess p o ides essen ial insigh s in o he amino acid sequence o he pep ide (Biemann, 1992), acili a ing he di e en ia ion o isoba ic pep ides wi h dis inc amino acid sequences o PTMs (Kim e al., 2016). T ib id ins umen s o e a ious ion ac i a ion me hods, wi h collision-induced dissocia ion (CID) (Hun e al., 1986) o highe -ene gy collisional dissocia ion (HCD) (Olsen e al., 2007) being he mos common. CID and HCD in ol e collisions wi h ine gases (e.g., ni ogen, helium, o a gon), esul ing in cha ge-di ec ed agmen a ion, d i en by he kine ic ene gy o ionizing p o ons, which mos ly esul s in clea age o he pep ide backbone. In he Fusion Lumos, CID is pe o med in he high-p essu e collision cell o he linea ion ap, while HCD is conduc ed sepa a ely in he ion ou ing mul ipole. This sepa a ion allows o enhanced esolu ion and imp o ed mass accu acy measu emen s o agmen ions by enabling highe kine ic ene gies and sho e impac imes du ing agmen a ion (Michalski e al., 2012). Ano he impo an me hod is he EThcD app oach, combining elec on ans e dissocia ion (ETD) (Syka e al., 2004) in he linea ion ap wi h subsequen ans e o p ecu so s and p oduc ions o he collision cell o HCD agmen a ion. F agmen a ion o p ecu so ions by ETD esul s in adical-d i en agmen a ion o pep ide bonds while limi ing he neu al loss o labile g oups and p ese ing PTMs (Syka e al., 2004). F agmen ions esul ing om pep ide dissocia ion a e in luenced by se e al ac o s, including he mass and cha ge s a e o he p ecu so ion, amino acid composi ion, and adjacen esidues a he backbone clea age si e (Reid e al., 2001; Tabb e al., 2003; Ha e land e al., 2017). Typically, he cha ge is dis ibu ed be ween bo h agmen s, wi h agmen ions e aining hei cha ge ei he a he C- e minus (x-, y-, and z-ions) o he N- e minus (a-, b-, and c-ions) acco ding o he Roeps o -Fohlmann-Biemann nomencla u e (Roeps o and Fohlman, 1984; Biemann, 1992) (FIGURE I-4). FIGURE I-4 | F agmen Ion Nomencla u e. (A) Roeps o –Fohlmann–Biemann nomencla u e o agmen ions (Roeps o and Fohlman, 1984; Biemann, 1992). (B) De e mina ion o pep ide sequence using y-ion se ies. y11 z11 x11 b2c2 a2 y2z2 x2 b11 c11 a11 a2-ion b2-ion c2-ion x2-ion z2-ion y2-ion A B y1 y2 y3 y10 y9 y8 y7 y6 y5 y4 y12 y11 R A V E A D H P T D A N 200 1,2001,0008006004000 m/z Rela i e in ensi y (%) 100 50 0 TNADTPHDAEVAR I | GENERAL INTRODUCTION P A G E | 8 3 Analysis o Mass Spec ome y Da a Unlike genomics and ansc ip omics, which can ampli y DNA o RNA using echniques like polyme ase chain eac ion, espec i ely, p o eomics lacks a compa able me hod o ampli ying p o eins. This limi a ion necessi a es ca e ul a en ion o he p o eomics wo k low o maximize in o ma ion om he po en ially limi ed s a ing ma e ial. In gene al, a p o eomics wo k low can be di ided in o h ee majo s eps (FIGURE I-5). The ini ial s ep (i) in ol es sample p epa a ion, which includes isola ing he p o ein mix u e om he s udied biological sample. The complexi y o samples can be educed and p o eome co e age enhanced by employing p o ein o pep ide p e- ac iona ion echniques (Zhang e al., 2010). Following sample cleanup, he second s ep (ii) in ol es online sepa a ion and MS measu emen o analy es, ypically using e e sed-phase LC-MS/MS. In p inciple, pep ide o p o ein masses can be de e mined om MS1 spec a by u ilizing he m/z alue and spacing be ween iso ope peaks. The co esponding amino acid sequences a e hen de e mined based on he MS2 mass di e ence be ween he agmen s. Wi h mode n mass spec ome e s gene a ing housands o spec a in a sho ime, manual anno a ion becomes imp ac ical, necessi a ing an au oma ed app oach o pep ide and p o ein iden i ica ion. Thus, he inal s ep (iii) in ol es employing a ious so wa e applica ions and compu a ional ools o iden i y pep ides and p o eins, along wi h pe o ming pos -p ocessing asks like quan i ica ion, s a is ical es ing, and me abolic mapping o e alua e signi ican di e ences be ween samples. In his s udy, P o eome Disco e e se ed as he p ima y so wa e ool o p o eomic da a analysis. No ably, P o eome Disco e e demons a ed imp o ed pe o mance compa ed o he widely used so wa e MaxQuan , pa icula ly in e ms o quan i ica ion yield, dynamic ange, and ep oducibili y o label- ee quan i ica ion (Palomba e al., 2021). FIGURE I-5 | Gene alized Bo om-up P o eomics Wo k low. P o eins a e ex ac ed om cells ea ed wi h a ious s imuli. Sample complexi y can be educed h ough p o ein p e ac iona ion, pep ide ac iona ion, o isoba ic labeling a e enzyma ic diges ion. Following sample clean-up using solid- phase ex ac ion, samples a e sepa a ed by e e sed-phase liquid ch oma og aphy (LC) and analyzed by mass spec ome y (MS). Analy es a e iden i ied om MS/MS spec a using P o eome Disco e e (PD) da abase ma ching. Pos -p ocessing includes quan i ica ion, s a is ical es ing, and me abolic mapping o assess signi ican di e ences be ween samples. (Adap ed wi h pe mission om (Me gne and Kus e , 2022)). m / z m / z m / z PD SAMPLE PREPARATION Cell cul u e Cell Lysis LC-MS/MS DATA ANALYSIS P o ein diges ion Sample cleanup Iden i ica ion Da abase sea ch Pos p ocessing Op ional P o ein ac iona ion Pep ide ac iona ion Isoba ic labeling MS/MS MS Theo e ical spec um Acqui ed spec um Quan i ica ion Di e ence Signi icance GENERAL INTRODUCTION | I P A G E | 15 Al hough s udies on single bac e ial isola es in esponse o di e se in i o condi ions a e limi ed, hey ha e yielded di e se p o eomic indings. Fo ins ance, p o eomic analyses we e u ilized o alida e B. he aio aomic on-speci ic p o ein equi emen s unde a ious g ow h condi ions (Liu e al., 2021a), obse e he selec i e packing o acidic glycosidases and p o eases in o Bac e oides ou e memb ane esicles (Elhenawy e al., 2014), and cha ac e ize p o eins equi ed o mucin deg ada ion by human gu bac e ia (C ouch e al., 2020). Howe e , how p o eomic p o iles change in single membe s o he mic obio a a IBD ini ia ion, p og ession, and du ing he apeu ic in e en ions emains la gely unknown. To imp o e unc ional knowledge o he gu mic obiome in he con ex o IBD, i is essen ial o gene a e de ailed p o eomic p o iles. 5 Objec i e o he Thesis This s udy aims o analyze he p o eomic adap a ions o selec ed bac e ia o he human gu mic obiome in esponse o a ying en i onmen al condi ions, ocusing on he speci ic objec i es: E alua ion o Quan i a i e P o eomic App oaches (Chap e III) – Assess he ep oducibili y and accu acy o bo om-up label- ee quan i ica ion (LFQ) and andem mass ag (TMT)-based quan i ica ion me hods o de ec ing p o eomic changes induced by suc ose and glucose in B. he aio aomic on. Explo a ion o pH-Dependen P o eomic Al e a ions (Chap e IV) – Analyze p o eomic changes in B. he aio aomic on, B. p oduc a, and B. longum in esponse o di e en en i onmen al pH le els. Iden i ica ion o Sho Open Reading F ame-Encoded Pep ides (Chap e V) – Employ a p o eogenomic app oach o iden i y and analyze he ansla ion o p e iously undisco e ed SEP in B. p oduc a unde a ious cul i a ion condi ions. Op imiza ion o OMV Isola ion P o ocol (Chap e VI) – Es ablish and op imize a p o ocol o he LC-MS-based p o eomic analysis o ex acellula esicles, speci ically ou e memb ane esicles (OMVs) isola ed om E. coli. In eg a ion o P o eo o m-Di ec ed Analysis (Chap e s III o IV) – Apply a p o eo o m- di ec ed op-down analysis, including a disco e y-based open modi ica ion sea ch, o iden i y po en ial pos - ansla ional modi ica ions and neo- e mini in HGM membe s. P A G E | 16 P A G E | 17 II GENERAL METHODS 1 Gene al Ma e ials ............................................................................................................. 18 1.1 Chemicals and Reagen s ........................................................................................ 18 1.2 LC-column Packing ................................................................................................. 18 2 P o ein and Pep ide Sou ces .......................................................................................... 19 2.1 Cul i a ion o Human Gu Bac e ia .......................................................................... 19 2.2 Cul i a ion o E. coli and OMV Isola ion .................................................................. 20 2.3 Caco-2 Wound Healing Assay ................................................................................ 22 3 Sample P epa a ion o LC-MS Analysis ....................................................................... 23 3.1 P o eome Clean-up and Diges ion .......................................................................... 23 3.2 Molecula Weigh -based P e ac iona ion ............................................................... 25 3.3 SDC Remo al by Phase- ans e ............................................................................ 26 3.4 Solid-phase Ex ac ion ............................................................................................ 27 3.5 Tandem Mass Tag Labelling ................................................................................... 27 3.6 P o ein Gel and S aining ......................................................................................... 29 4 Mass Spec ome y Da a Acquisi ion ............................................................................ 30 4.1 Bo om-up LC-MS Measu emen s ........................................................................... 30 4.2 Top-down LC-MS Measu emen s ........................................................................... 31 4.3 MALDI-TOF Measu emen s .................................................................................... 32 5 Da a P ocessing and Analysis ....................................................................................... 32 5.1 Da abase Sea ch ..................................................................................................... 32 5.2 Genome and P o eome Sequence-based P edic ions ............................................ 34 5.3 Func ional in silico Analysis ..................................................................................... 34 5.4 Func ional and S a is ical Da a Analyses ................................................................ 35 II | GENERAL METHODS P A G E | 18 1 Gene al Ma e ials 1.1 Chemicals and Reagen s Deionized wa e (18.2 MΩ/cm) was ob ained using an A ium611 VF sys em (Sa o ius). Comple e p o ease inhibi o cock ail was pu chased om Roche Diagnos ics. Pie ce Coomassie and BCA p o ein assay ki s (bo h The mo) we e used o p o ein quan i ica ion. Single-po , solid-phase-enhanced (SP3) bead-based pu i ica ion was pe o med using Se a- Mag SpeedBead ca boxyla e-modi ied magne ic pa icles (GE Li e Sciences). Sequencing g ade modi ied ypsin was pu chased om P omega. Lyophilized p o ein s anda ds o cy och ome C (Equus caballus), myoglobin (Equus caballus), be a-casein (Bos au us), ca bonic anhyd ase (Bos au us), bo ine se um albumin (Bos au us), and alcohol dehyd ogenase (Saccha omyces ce e isiae) we e pu chased om Sigma-Ald ich. HeLa and cy och ome C diges we e pu chased om The mo Fishe . Syn he ic pep ides we e pu chased om JPT Pep ide Technologies GmbH. Addi ional chemicals equi ed o a ious s ages o media p epa a ion, sample handling, and LC-MS/MS analysis we e pu chased om a ious supplie s, including Sigma-Ald ich, Me ck, and Se a. 1.2 LC-column Packing F i s o column packing we e p epa ed by c oss-linking Kasil (po assium silica e solu ion) wi h o mamide (Co es e al., 1987). Kasil 1, a 29.1% (w/w) po assium silica e solu ion in wa e , was solubilized a 80°C and 2000 pm o 1 hou . Equal pa s o Kasil 1 and a 25% w/ o mamide solu ion in wa e we e combined o c ea e he inal i solu ion. A e mixing and cen i uging a 21,000 g o 5 minu es a 20°C, ub used silica capilla ies (360 µm OD x 75 o 150 µm ID) we e gen ly p essed on o a glass mic o ibe il e (GF/C, Wha man), which had been p e iously soaked wi h 2 µL o he i solu ion (Maiolica e al., 2005). Capilla ies we e incuba ed a 85°C o 20 hou s o polyme iza ion. Using a high-p essu e bomb loade , capilla ies we e packed wi h PLRP-S beads (5 µm, 1000 Å), which we e collec ed om a PLRP-S column (4.6 x 50 mm, Agilen ). The collec ed beads we e washed wi h 50% and hen 100% ace oni ile (ACN), d ied a 70°C and suspended in me hanol (60 mg/ml). A e allowing he ma e ial o se le by g a i y o 20-30 minu es and mixing o 1 minu e and sonica ion, columns we e packed unde low-speed s i ing (400-500 pm) wi h con inuous mechanical apping (Ko alchuk e al., 2019). The bomb con aining he capilla y was p essu ized o 100 ba wi h ni ogen immedia ely a e moun ing he capilla y o p e en passi e illing o he capilla y wi h he sol en . A e he packing p ocess was comple ed, he p essu e was slowly eleased o 10 minu es o p e en bubble o ma ion wi hin he column. The eshly packed columns we e connec ed o an Ul ima e 3000 sys em and lushed wi h 95% ACN a a low a e o 600 GENERAL METHODS | II P A G E | 19 nl/min o 30 minu es o comp ess he so ben bed. The columns we e hen cu o he desi ed leng h, and connec ions we e made using ZIRCOFIT UHPLC i ings wi h a 1/16" 13-mm bo e (MS Wil). This esul ed in wo di e en ypes o columns: p e-columns (150 µm x 4 cm) and analy ical columns (75 µm x 17 cm). 2 P o ein and Pep ide Sou ces De ails on he numbe o bac e ial cell cul u e eplica e and speci ic cul u e condi ions o pa icula expe imen s a e desc ibed in he expe imen al p ocedu e sec ions o he co esponding chap e s. 2.1 Cul i a ion o Human Gu Bac e ia The cul i a ion o Bac e oides he aio aomic on VPI-5482, Blau ia p oduc a ATCC 27340, and Bi idobac e ium longum NCC 2705 was pe o med by Ka h in Schä e (Depa men o In ec ious Diseases and Mic obiology, Uni e si y o Lübeck, UKSH Lübeck; chai : P o . D . Jan Rupp). Yeas ex ac , casein, and a y acid (YCFA) medium (Duncan e al., 2009) (TABLE II-1) o b ain-hea in usion (BHI) medium adjus ed o di e en pH alues (pH 6.0, pH 7.0, and pH 8.0) and supplemen ed wi h di e en ca bon sou ces (27.8 mM glucose o suc ose) o a ious g ow h supplemen s (lipopolysaccha ide (LPS), SCFA o yeas ex ac ) we e u ilized o bac e ial cul i a ion. Bac e ial cells we e cul u ed a 37°C unde s ic anae obic condi ions in an anoxic chambe (H35, Don Whi ley Scien i ic Limi ed) con aining 85% ( / ) N2, 10% ( / ) CO2 and 5% ( / ) H2. A single colony was ans e ed o 5 ml o YCFA o BHI medium, incuba ed o e nigh and 0.1% ( / ) was used o inoculum. TABLE II-1 | Modi ied YCFA Medium COMPONENTS [g/l] COMPONENTS [mg/l] COMPONENTS [g/l] Casi one 10 Bio in 0,02 Ace ic acid 1900 Yeas ex ac 2,5 Folic acid 0,10 P opionic acid 700 Ca bon Sou ce 5 Py idoxine hyd ochlo ide 0,05 iso-Bu y ic acid 90 MgSO4 x 7 H2O 0,45 Thiamine-HCl x 2 H2O 0,05 n-Vale ic acid 100 CaCl2 x 2 H2O 0,90 Ribo la in 0,05 iso-Vale ic acid 100 K2HPO4 0,45 Nico inic acid 0,05 KH2PO4 0,45 D-Calcium pan o hena e 0,001 NaCl 0,90 Vi amin B12 0,05 Resazu in 0,01 p-Aminobenzoic acid 0,05 Dis illed wa e 4 Lipoic acid 10 NaHCO3 1 L-Cys eine HCl 0,1 Hemin 0,02 II | GENERAL METHODS P A G E | 20 2.2 Cul i a ion o E. coli and OMV Isola ion G ow h Media – Lysogeny b o h (LB) medium (1% yp one, 0.5% yeas ex ac , 1% NaCl, pH 7.0, Sigma-Ald ich) was p epa ed acco ding o he manu ac u e 's ins uc ions. Aga pla es we e p epa ed by adding 1% aga and au ocla ing a 121°C o 15 minu es. M9 cul u e media we e p epa ed as indica ed in TABLE II-2 and TABLE II-3 and s e ilized by au ocla ing a 121°C o 12 minu es. Glucose and ace a e solu ions we e au ocla ed sepa a ely, and hea -sensi i e componen s such as bio in and hiamine we e il e -s e ilized (0.2 μm il e ) and added o he media a e wa d. TABLE II-2 | 100X T ace Elemen s Solu ion COMPONENT [g/l] CONCENTRATION EDTA 5 g/l 13.4 mM FeCl3-6H20 0.83 g/l 3.1 mM ZnCl2 84 mg/l 0.62 mM CuCl2-2H20 13 mg/l 76 µM CoCl2-2H20 10 mg/l 42 µM H3BO3 10 mg/l 162 µM MnCl2-4H20 1.6 mg/l 8.1 µM TABLE II-3 | M9 Mine al Medium VOLUME COMPONENT COMPONENT CONCENTRATION 100 ml M9 sal solu ion (10X) Na2HPO4-2H20 KH2PO4 NaCl NH4Cl 33.7 mM 22.0 mM 8.55 mM 9.35 mM 20 ml Ca bon sou ce Glucose o ace a e 15 mM o 45 mM 1 ml MgS04 (1M) MgS04 1 mM 0.3 ml CaCl2 (1M) CaCl2 0.3 mM 1 ml* Bio in (1 mg/ml) Bio in 1 µg 1 ml* Thiamin (1 mg/ml) Thiamin 1 µg 10 ml* T ace elemen s (100X) T ace elemen s 1X * 0.22-µm il e s e iliza ion Cul i a ion – A single colony o Esche ichia coli K-12 s ain MG1655 was cul u ed in 100 ml o LB medium o 18 h a 37°C and 150 pm in a non-ba led 250 ml shake lask. A e wa d, he cul u e medium was emo ed by cen i uga ion a 7,000 g o 3 min a 4°C, and cells we e washed wice wi h il e -s e ilized M9 minimal medium. Then, cells we e inocula ed wi h 1/100 dilu ions o 18 h p e-cul u e a an ini ial OD600 o 0.1 in M9 media con aining ei he 15 mM glucose o 45 mM ace a e. The cul u es we e incuba ed ae obically a 37°C, 150 pm in ba led 1 l shake lasks wi h 300 ml o media. Du ing he cul i a ion cul u e samples we e aken o ack cell g ow h ia OD600 measu emen s. GENERAL METHODS | II P A G E | 21 Isola ion o Ou e Memb ane Vesicles (OMV) – OMV isola ion om E. coli supe na an was pe o med using he ExoBac e ia OMV Isola ion Ki (Sys em Biosciences). Cul i a ions we e ans e ed in o s e ile 50 ml cen i uge ubes and cen i uged a 8,000 g o 20 min a 4°C. The supe na an was ans e ed o a new s e ile 50 ml cen i uge ube and spun again a 8,000 g o 20 min a 4°C. The supe na an was il e -s e ilized (0.2 μm il e ) and he esul ing cell- ee supe na an was used o isola e OMVs. The binding column was p epa ed by adding 1 ml o OMV binding esin and equilib a ing i wi h 10 ml o OMV binding bu e . A e equilib a ion, he binding bu e was allowed o comple ely low h ough he column. Subsequen ly, he bo om o he column was sealed, and 20 ml o supe na an was added. The op o he column was sealed, and he uni was placed on a o a ing ack o 30 min a 4°C o allow o mixing and binding o he OMVs o he esin. A e 30 min, he op and bo om o he column we e opened o allow he supe na an o low h ough he esin. Depending on he expe imen , loading o supe na an was epea ed up o 2 addi ional imes so ha a o al o 40 ml o 60 ml o cul u e supe na an , was incuba ed wi h he OMV binding esin. A e OMV binding, he supe na an was allowed o low h ough he column, and he esin was washed wi h 15 ml OMV binding bu e pe 20 ml o loaded supe na an . A e wa d, he bo om o he column was sealed, and 1.5 ml OMV elu ion bu e was added. Columns we e allowed o incuba e a 20°C o 2 min wi h gen le agi a ion e e y 30 s, a e which he bo om o he column was unsealed and he OMV isola e was collec ed. Samples we e ozen a -80°C un il u he analysis. Nanopa icle T acking Analysis – Fo nanopa icle acking analysis (NTA) a NanoSigh NS300 sys em (NanoSigh L d), equipped wi h a 488 nm lase and a high sensi i i y digi al came a sys em sCMOS (scien i ic complemen a y me al oxide semiconduc o came a) was employed. Videos we e acqui ed and analyzed using he NTA so wa e ( e sion 3.3), wi h minimum ack leng h and blu se ing, all se o au oma ic. The came a shu e was se manually in dependency o he pa icle in ensi y and came a gain was se o 366. Came a le els we e se o 14 o 15 and he de ec ion h eshold was se o 5, o e eal small pa icles. The ambien empe a u e was main ained a 25°C. Samples we e adminis e ed and eco ded unde con olled low, using he NanoSigh sy inge pump and sc ip con ol sys em. Fo each sample, six ideos o 60 seconds du a ion we e eco ded, wi h a 10-second delay be ween eco dings, gene a ing six eplica e his og ams ha we e a e aged. Las ly, he hyd odynamic diame e s and pa icle size dis ibu ions we e analyzed by he so wa e using he S okes- Eins ein equa ion. A summa y o he comple e lis o pa ame e s used in he nanopa icle acking analysis is p o ided in TABLE II-4. The ins umen was calib a ed p io o each expe imen al un using s anda dized nanopa icle dilu ions pu chased om he manu ac u e . II | GENERAL METHODS P A G E | 22 TABLE II-4 | Pa ame e s o Nanopa icle T acking Analysis PARAMETER SETTING Ins umen NanoSigh NS300 NTA Ve sion NTA 3.3 De Build 3.3.301 Diluen Wa e (1:1) Came a Type sCMOS Came a Le el 14-15 Lase Type Blue 488 Slide Shu e 1200 - 1260 Slide Gain 366 Isola ed E. coli OMVs cul i a ed un il mid- loga i hmic, p es a iona y, s a iona y o dea h phase we e subjec ed o LC-MS/MS analysis o analyse he epe oi e o OMV p o eins which had been ob ained om di e en g ow h phases, mid-loga i hmic, p es a iona y and s a iona y o OMVs isola ed om glucose and mid- loga i hmic, s a iona y and dea h phase o OMVs isola ed om ace a e. Isola ed E. coli OMVs cul i a ed un il mid- loga i hmic, p es a iona y, s a iona y o dea h phase we e subjec ed o LC-MS/MS analysis o analyse he epe oi e o OMV p o eins which had been ob ained om di e en g ow h phases, mid-loga i hmic, p es a iona y and s a iona y o OMVs isola ed om glucose and mid- loga i hmic, s a iona y and dea h phase o OMVs isola ed om ace a e. Isola ed E. coli OMVs cul i a ed un il mid- loga i hmic, p es a iona y, s a iona y o dea h phase we e subjec ed o LC-MS/MS analysis o analyse he epe oi e o OMV p o eins which had been ob ained om di e en g ow h phases, mid-loga i hmic, p es a iona y and s a iona y o OMVs isola ed om glucose and mid- loga i hmic, s a iona y and dea h phase o OMVs isola ed om ace a e. Isola ed E. coli OMVs cul i a ed un il mid- loga i hmic, p es a iona y, s a iona y o dea h phase we e subjec ed o LC-MS/MS analysis o analyse he epe oi e o OMV p o eins which had been ob ained om di e en g ow h phases, mid-loga i hmic, p es a iona y and s a iona y o F ames/Sec 25 F ames 1498 Tempe a u e 25.0 ºC Viscosi y Wa e 0.889 cP Sy inge Pump Speed 40 De ec Th eshold 5 Blu Size Au o Max Jump Dis ance Au o 2.3 Caco-2 Wound Healing Assay Human Caco-2 cells we e cul u ed in collabo a ion wi h B i a S ee (Sys ema ic P o eome Resea ch & Bioanaly ics, Ins i u e o Expe imen al Medicine, Uni e si y o Kiel; chai : P o . D . And eas Tholey). Cells we e cul u ed in Roswell Pa k Memo ial Ins i u e (RPMI) medium (Gibco-In i ogen) supplemen ed wi h 10% e al cal se um (FCS; Gibco-In i ogen) and 1% penicillin/s ep omycin in a 5% CO2, 95% humidi y en i onmen a 37°C. Cells we e passaged weekly upon eaching 80% con luence. Wound healing assays we e conduc ed using µ-dishes wi h inse s (Ibidi GmbM) a a densi y o 4×105 cells/cm2. A e eaching a con luence o 70- 80%, cells we e se um-s a ed o e nigh using se um-dep i ed medium (0.1% FCS), he inse was emo ed, and cell laye s we e washed wice wi h phospha e-bu e ed saline (PBS). Cells we e hen incuba ed wi h 2 ml o 0.1% FCS se um-dep i ed medium con aining di e en concen a ions o E. coli OMVs (10, 50, and 100 µg/ml), OMV elu ion bu e , LPS E. coli O55:B5 (1 µg/mL, Sigma-Ald ich), ans o ming g ow h ac o β (TGFβ) (5 ng/mL, Sigma- Ald ich) and medium only. The mig a ion p ocess in o he cell- ee gap o app oxima ely 500 µm was measu ed by aking mic oscopic pho og aphs a 0, 12, and 30 hou s using a digi al came a on an in e ed mic oscope a 10x magni ica ion. The wound a ea was quan i ied using a wound healing plugin o ImageJ (Sua ez-A nedo e al., 2020). The pe cen age o wound closu e was calcula ed ela i e o he medium con ol (a bi a ily assigned as 100%) based on he a ea measu ed immedia ely a e inse emo al (A =0h), as well as 12 and 30 hou s a e incuba ion (A =∆h) (eq. 1). Wound'closu e% = /!!"#$"!!"∆$ !!"#$ 0×100% (eq. 1) GENERAL METHODS | II P A G E | 23 3 Sample P epa a ion o LC-MS Analysis De ails ega ding he quan i ies o p o ein and pep ide used, he numbe o echnical eplicas, and di e en MS pa ame e s o speci ic expe imen s a e desc ibed in he expe imen al p ocedu e sec ions o he co esponding chap e s. P o ein concen a ions we e de e mined in a leas iplica e using he Pie ce Coomassie o BCA P o ein Assay Ki acco ding o he manu ac u e 's ins uc ions. 3.1 P o eome Clean-up and Diges ion Human Gu Bac e ia Samples – Cell lysis o human gu bac e ia was pe o med by Ka h in Schä e (Depa men o In ec ious Diseases and Mic obiology, Uni e si y o Lübeck, UKSH Lübeck; Chai : P o . D . Jan Rupp). Cul u e media was emo ed by cen i uga ion a 2,100 g o 10 min a 4°C, washed wice wi h wa e , and cen i uged again. Bac e ial cells we e suspended in lysis bu e (6 M GndHCl (guanidine hyd ochlo ide), 100 mM HEPES (4-2- hyd oxye hyl-1-pipe azinee hanesul onic acid), 20 mM NaCl and 1x cOmple e p o ease inhibi o , pH 7.5). Cells we e lysed using en cycles o eeze- hawing (30 s, -80°C in an e hanol ba h ollowed by hawing in a sonica ion ba h o 30 s). A e cen i uga ion a 21,000 g o 20 min a 4°C, supe na an s we e collec ed and cell deb is was washed wice wi h lysis bu e , cen i uged and supe na an s we e pooled. Disul ide b idges we e educed and alkyla ed using 12 mM is(2-ca boxye hyl)phosphine (TCEP) and 40 mM 2-chlo oace amide (CAA) o 1 hou a 25°C and 800 pm. The samples we e hen p ecipi a ed using 9x olume e hanol a -20°C. A e 16 hou s o incuba ion a -20°C, he p ecipi a es we e cen i uged a 21,000 g o 10 min a 4°C and washed wice wi h cold e hanol. Residual e hanol was e apo a ed in a ume hood. P ecipi a es we e suspended in 0.5 M GndHCl, 12.5 mM HEPES, o 100 mM ie hylamoniumbica bona (TEAB) (all pH 8.5), diges ed by adding ypsin a a 1:40 enzyme o subs a e a io, and incuba ed o e nigh a 37°C on a shake a 800 pm. E. coli samples – 1 mg o E. coli cells we e p ocessed using he sample p epa a ion by simple ex ac ion and diges ion (SPEED) me hod (Doellinge e al., 2020), wi h TFA added a a 1:4 ( / ) a io o sample o TFA. Samples we e incuba ed o 5 minu es a 20°C and neu alized wi h 2 M T is base using 8- old he olume o TFA used o lysis. Aliquo s o 50 μg p o ein we e educed and alkyla ed by incuba ion in 10 mM TCEP and 40 mM CAA a 95°C o 5 minu es. Samples we e dilu ed 1:5 wi h wa e and p o eins we e diges ed wi h ypsin a a 1:50 enzyme- o-subs a e a io o 20 hou s a 37°C. II | GENERAL METHODS P A G E | 24 OMV samples – 40 µg o OMV samples we e p ocessed using di e en sample p epa a ion p o ocols, including in-solu ion, on-bead SP3 (Hughes e al., 2019), and a modi ied e sion o he on-memb ane il e -assis ed sample p epa a ion (FASP) me hod (Manza e al., 2005; Wiśniewski e al., 2009). In-solu ion Diges ion – OMV samples we e lysed by en cycles o eeze- haw (30 s, -80°C in an e hanol ba h ollowed by hawing in a sonica ion ba h o 30 s) ollowed by adjus men o 100 mM TEAB using 1 M TEAB. Al e na i ely, 40 µg OMV samples we e lyophilized be o e he addi ion o 50 µl o lysis bu e (2% (w/ ) sodium deoxychola e (SDC), 0.1 M TEAB, pH 8.5). Samples we e hen incuba ed a 95°C o 5 minu es o inac i a e p o eases, educed wi h 10 mM di hio h ei ol (DTT) a 56°C o 1 hou , and alkyla ed wi h 50 mM iodoace amide (IAA) in he da k a 20°C o 30 minu es. Fo samples con aining SDC, he concen a ion was adjus ed o 0.5% SDC wi h 0.1 M TEAB (pH 8.5) and ypsin was added a a 1:40 (w/w) enzyme- o-subs a e a io. The diges ion eac ion was pe o med o e nigh a 37°C on an o bi al shake a 800 pm. On-bead Diges ion – Fo OMV lysis, sodium dodecyl sul a e (SDS) o SDC was added o a inal concen a ion o 1% o 2% (w/ ), espec i ely. Subsequen ly, samples we e incuba ed a 95°C o 5 minu es be o e being educed using 10 mM DTT o 1 hou a 56°C and alkyla ed wi h 50 mM IAA in he da k o 30 minu es a 20°C. SP3 beads we e p epa ed by mixing equal amoun s o hyd ophilic and hyd ophobic beads, we e washed wice wi h wa e , and esuspended in wa e o a inal concen a ion o 20 μg/µl. Fo each sample, 20 µl o beads we e added, and p o ein binding was induced by adding e hanol o a inal concen a ion o 50% ( / ) and incuba ing o 5 minu es a 25°C a 800 pm. The beads we e immobilized using a magne ic ack, he supe na an was emo ed, and he beads we e washed wice wi h 200 µl o 80% e hanol. Beads we e esuspended in diges ion bu e (100 mM TEAB, pH 8.5) con aining ypsin a a 1:40 (w/w) enzyme- o-subs a e a io and incuba ed o e nigh a 37°C on a shake a 800 pm. Enhanced diges ion was pe o med by adding 0.5% (w/ ) SDC o 0.001% (w/ ) dodecyl-β-D-mal osid (DDM) o he diges ion bu e . The diges s we e acidi ied o pH 2 o 3 by adding i luo oace ic acid (TFA), and he beads we e pelle ed by cen i uga ion. The supe na an s we e s o ed on ice un il sample cleanup. Fo SDC diges s, samples we e addi ionally p ocessed using a modi ied phase ans e p o ocol. On-memb ane Diges ion – Fo OMV lysis, a inal concen a ion o 1% o 2% (w/ ) o SDS o SDC, espec i ely, was added. The samples we e incuba ed a 95°C o 5 minu es, educed wi h 10 mM DTT o 1 hou a 56°C, and loaded on o Amicon cen i ugal il e uni s (30K molecula weigh cu o , Millipo e). A e cen i uga ion a 12,000 g o 15 min a 20°C (all bu e exchanges we e pe o med by cen i uga ion unde iden ical condi ions), SDS-lysed samples GENERAL METHODS | II P A G E | 31 adjus ed acco ding o g adien leng h (20 o 40 s), wi h ions o unassigned, +1, and >+8 cha ge s a es excluded, and lock mass (445.12003 m/z) enabled. 4.2 Top-down LC-MS Measu emen s Nano low LC-ESI-MS measu emen s we e conduc ed using a Dionex Ul ima e 3000 HPLC sys em online coupled o a Fusion Lumos T ib id (Lumos) mass spec ome e (bo h The mo Fishe Scien i ic). Depending on he applied me hod, he mass spec ome e was equipped wi h he ield asymme ic ion mobili y spec ome y (FAIMS) p o in e ace. Liquid Ch oma og aphy – Gene ally, 1 o 1.5 µg p o ein p epa a ions we e concen a ed and washed on o a ap column (5 mM x 0.33 mm, 5 μm C4 esin, 300 Å; PepMap300, The mo Fishe Scien i ic) o 5 min wi h 2% ACN and 0.05% aqueous TFA a a low a e o 30 µl/min. Subsequen ly, p o eins we e sepa a ed on an analy ical column (75 μm x 50 cm, 2.6 μm C4 esin, 150 Å; Accuco e, The mo Fishe Scien i ic) a 300 nl/min and sepa a ed wi hin a 60-, 90- o 140-min linea g adien o inc easing LC sol en B (80% ACN, 0.1% FA) in LC sol en A (0.1% aqueous FA). Addi ionally, GELF EE samples we e sepa a ed on a sel -packed PLRP- S p e-column (150 µm x 4 cm, 5 µm PLRP-S esin, 1000 Å), be o e being sepa a ed on an analy ical column (75 µm x 17 cm, 5 µm PLRP-S esin, 1000 Å). LMWP samples, GELF EE ac ions, and SCX ac ions o B. he aio aomic on we e sepa a ed using a 90-minu e g adien om 15% o 55% B. In con as , he LMWP samples o B. p oduc a we e sepa a ed using a 60-minu e g adien om 15% o 60% B. The linea g adien was ollowed by a sha p inc ease o 98% sol en B o 2 min, an isoc a ic wash s ep o 13 min, and inally a column equilib a ion wi h 5% B o 15 min. Mass Spec ome y – A 15 Vol sou ce-induced dissocia ion was applied o a o p o ein ion desol a ion and he RF Lens was se a 30%. The acquisi ion was pe o med in “pep ide mode”, acco ding o he ecommenda ions o endo s o he mass ange below ca. 20−30 kDa. Dual CV FAIMS Me hod – Acquisi ion o MS spec a we e ob ained using wo me hods including wo di e en FAIMS compensa ion ol ages (CVs). The i s me hod in ol ed combining -60 V and -50 V, while he second me hod used -40 V and -30 V. Di e en mic oscan se ings we e used o each me hod, wi h only 2 mic oscans being u ilized o -60 V, and 4 mic oscans being used o all o he CVs. MS1 spec a we e eco ded in he O bi ap om 500 o 1800 m/z, a a esolu ion o 120K, using an AGC a ge alue o 200% and a maxIT o 246 ms. Bo h me hods ob ained MS2 spec a wi h a 60K esolu ion (3 s cycle ime) a e collision-induced dissocia ion (CID) wi h an NCE o 25%. MS2 spec a we e acqui ed wi hin 4 II | GENERAL METHODS P A G E | 32 mic oscans in a scan ange o 500 m/z o 2000 m/z, using a no malized AGC a ge o 400% and a maxIT o 250 ms. Only p ecu so s wi h a cha ge s a e be ween 4 and 50 o unde e mined cha ge s a es we e selec ed wi h enabled dynamic exclusion (exclude a e 2 imes, 60 s du a ion) plus and minus 2.5 m/z. Mu li-CV FAIMS Me hod – Fou di e en CVs (-60, -50, -40, -20 V) wi h di e en MS1 and MS2 se ings we e applied, based on (Kaulich e al., 2022a). Wi hin a cycle ime o 3 s MS2 spec a we e acqui ed wi h an isola ion window o 5 m/z, 50K esolu ion, 400% AGC a ge , 250 ms injec ion ime, and o agmen a ion CID wi h an NCE o 30% was u ilized. Only p ecu so s wi h a cha ge s a e be ween 4 and 50 o unde e mined cha ge s a es we e selec ed wi h enabled dynamic exclusion (n= 2, 60 s). Se ings o CVs -60 and -50 V: esolu ion 60K/50K (MS1/MS2), maximum injec ion ime 118/125 ms, mic oscans 2/2, AGC a ge 200%/400%. Se ings o CVs -40, -20 V: esolu ion 120k/60k, maximum injec ion ime: 246/250 ms, 4/4 mic oscans. 4.3 MALDI-TOF Measu emen s Ma ix-assis ed lase deso p ion/ioniza ion ime o ligh (MALDI-TOF) mass spec ome y was pe o med using a TOF/TOF 5800 mass spec ome e equipped wi h he 4000 se ies explo e so wa e (bo h AB Sciex). The mass spec ome e accumula ed 400 lase pulses a an in ensi y o 65%. Spec a we e acqui ed wi hin a mass ange o 100 m/z o 1200 m/z. P io o analysis, he ins umen was calib a ed using a six-pep ide solu ion o calib a e he a ge m/z ange. Samples we e p epa ed by mixing wi h a ying olumes ( / ) o a CHCA ma ix solu ion (3 mg/ml α-cyano-4-hyd oxycinnamic acid in 70% ace oni ile, 0.1% TFA) and spo ed as 1 µL d ops on o a 384-well Op i-TOF MALDI Inse (AB Sciex). 5 Da a P ocessing and Analysis De ails on he speci ic sea ch pa ame e s o each expe imen and he e sion o P o eome Disco e e (PD) used a e a ailable in he expe imen al p ocedu es sec ion o each chap e . 5.1 Da abase Sea ch Depending on he sample ype, andem mass spec a we e sea ched and compa ed o he UniP o e e ence p o eome o Bac e oides he aio aomic on pi-5482, Blau ia p oduc a ATCC 27340, Bi idobac e ium longum NCC 2705 o Esche ichia coli K12 (UP000001414 / 4,782 p o eins; UP000515789 / 5,372 p o eins; UP000000439 / 1,725 p o eins; UP000000625 / 4,450 p o eins, accessed be ween Sep embe 2020 and Augus 2022) o he in eg a ed GENERAL METHODS | II P A G E | 33 p o eogenomics da abase (iP gxDB) o Blau ia p oduc a ATCC 27340 (accessed No embe 2021) (Omasi s e al., 2017). In addi ion, common eposi o ies o ad en i ious p o eins we e included in he sea ches. Bo om-Up Sea ches – Pep ide iden i ica ion and quan i ica ion expe imen s we e pe o med using PD ( .2.2, .2.5, o .3.0; The mo Fishe Scien i ic) u ilizing i s buil -in sea ch engine Seques HT, INFERYS esco ing, o CHIMERYS algo i hm. These included ypsin as he p o eoly ic enzyme wi h up o wo o ou missed clea age si es allowed, ca bamidome hyla ion o cys eine (57.021 Da) as ixed modi ica ion, oxida ion o me hionine (15.995 Da) as a iable modi ica ions, a p ecu so ole ance o 10 ppm and a agmen ion ole ance o 0.02 Da. Fo TMT expe imen s, TMT6 was employed as he ixed modi ica ion on lysine and pep ide N- e mini. To assess unde /o e labeling, a iable TMT6 modi ica ions we e added as sea ch pa ame e s o lysine and pep ide N- e mini, as well as ixed modi ica ions o his idine, se ine, h eonine, and y osine. Addi ional sea ches, including phospho yla ion as a iable modi ica ions on se ine, his idine, h eonine, y osine, o a ginine (79.966 Da), and semi- yp ic sea ches, we e cus omized based on he sample ype. Re en ion- ime alignmen was applied o samples necessi a ing MS1 in ensi y-based quan i ica ion. All esul s we e adjus ed o 1% PSM, pep ide, and p o ein FDR, employing a a ge -decoy app oach using e e sed p o ein sequences and pos e io e o calcula ion by he pe cola o algo i hm (Käll e al., 2008). Top-Down Sea ches – P o eo o m iden i ica ion and quan i ica ion we e pe o med using PD ( .2.5.0.400; The mo Fishe Scien i ic) u ilizing i s buil -in P oSigh PD 4.1 o 4.2 nodes (P o einaceous Inc.). These included he high/high cRAWle node in combina ion wi h X ac o decon olu ion. The anno a ed p o eo o m sea ch, wi h a maximum o h ee p o eo o m spec um ma ches (P SMs) pe p ecu so , a minimum o h ee ma ched agmen s, and no del a M mode, was u ilized o iden i y ull-leng h p o eo o ms. T unca ed p o eo o ms we e de ec ed using he subsequence sea ch node, wi h a maximum o one P SM pe p ecu so and a equi emen o a leas six ma ched agmen s. Sea ches, u ilized a p ecu so and agmen mass ole ance o 10 ppm, wi h a iable modi ica ions ace yla ion (42.011 Da) and o myla ion (27.995 Da) a he p o eo o m N- e minus. Addi ional sea ches, speci ically designed o sea ch o ixed dehyd o-modi ica ions on cys eine (-1.008 Da) o iden i y po en ial disul ide b idges and open-modi ica ion sea ches wi h a 500 Da p ecu so window, we e cus omized based on he sample ype. Fo label- ee quan i ica ion, aw da a om mul i-CV measu emen s we e il e ed using F ees yle .1.6 based on FAIMS CVs, and he esul ing il e ed da a was sa ed as dis inc . aw iles, e ec i ely di iding hem in o ou ac ions. Quan i ica ion was ca ied ou employing he High-Resolu ion Fea u e De ec o node wi h he sliding window decon olu ion algo i hm, u ilizing an a e age e en ion ime wid h o 0.33 min II | GENERAL METHODS P A G E | 34 wi h mass ea u es needed o be p esen in only 1% o he o al da a iles. All esul s we e subjec ed o an FDR co ec ion o bo h P SMs and p o eo o ms, wi h a h eshold o 1%. 5.2 Genome and P o eome Sequence - based P edic ions P o ein sequences we e e ie ed om UniP o and genomic sequences we e ob ained om he Eu opean Nucleo ide A chi e (Leinonen e al., 2011). Unless o he wise no ed, de aul pa ame e s we e used o all p edic ion ools. P o ein sequences we e anno a ed using WebMGA (Web Se ices o me agenomic analysis) (Wu e al., 2011) ha used he COG (Clus e s o O hologous Genes) da abase o unc ional anno a ion (Galpe in e al., 2021). Fo ha , RPSBLAST was un on he p oka yo ic NCBI COG da abase wi h an E- alue cu -o o 0.001. Kyo o Encyclopedia o Genes and Genomes (KEGG) o hology and he p edic ion o pa hway-speci ic me abolic unc ions, as well as he econs uc ion o KEGG pa hways, we e acili a ed h ough he u iliza ion o Blas KOALA (KEGG O hology And Links Anno a ion) (Kanehisa e al., 2016). This analysis was pe o med a he genus le el using BLASTp o sea ch and compa e he da a wi h a non- edundan da ase o pangenome sequences. Genomic sequences we e subjec ed o subsys em anno a ion using he Rapid Anno a ion using Subsys em Technology (RAST) se e and analyzed using he SEED iewe (O e beek e al., 2014). A classic RAST ( . 2.0) anno a ion sea ch along wi h FIG am ( elease 70) was used o cu a ion o he genomic da a. As he au oma ic anno a ion p ocess may un in o p oblems, such as o e lapping gene pai s o o e lapping RNAs hese e o s and ameshi s we e ixed au oma ically (e en i ha equi es dele ing some gene candida es). Debug s a emen s we e u ned on and gaps we e back illed, allowing RAST o blas la ge gaps o missing genes. RAST acili a ed he classi ica ion o genes in o p ede ined subsys ems, which ep esen g oups o genes in ol ed in speci ic biological p ocesses o pa hways. Po en ial p o ein-coding genes we e c oss- e e enced o coding sequences (CDS) om he NCBI P oka yo ic Genome Anno a ion Pipeline (PGAP) (Ta uso a e al., 2016) and linked back o UniP o accession. The in eg a ion o PATRIC (Pa hosys ems Resou ce In eg a ion Cen e ) was used o display and analyze RAST-anno a ed genomes o u he in es iga e genome p ope ies. 5.3 Func ional in silico Analysis Physicochemical p ope ies such as he isoelec ic poin (pI) and g and a e age o hyd opa hy (GRAVY) sco e we e calcula ed using P o Pa am wi h de aul se ings o pK alues (Gas eige e al., 2005). Phobius was used o he p edic ion o p o ein localiza ion using a pos e io p obabili y ≥ 0.5 (Käll e al., 2004). Po en ial an imic obial pep ide (AMP) ac i i y and hei GENERAL METHODS | II P A G E | 35 unc ional a ge s we e assessed using AMP un (a p obabili y sco e o >0.5 indica es po en ial AMP ac i i y, while a sco e <0.5 indica es non-AMP ac i i y) (Chung e al., 2020). Disul ide b idges we e p edic ed using SCRATCH (Cheng e al., 2005) and unc ional domains and mo i s we e p edic ed using NCBI´s Conse ed Domains sea ch ( . 3.20) (Wang e al., 2023a). Polysaccha ide u iliza ion loci (PULs) we e assigned using he Polysaccha ide U iliza ion Loci Da aBase (PULDB) (Te apon e al., 2015). The di ec ion o enzyma ic eac ions was checked using he Explo Enz enzyme da abase (h ps://www.enzyme-da abase.o g/) (McDonald e al., 2007) and he MACiE da abase (Mechanism, Anno a ion, and Classi ica ion in Enzymes) (Holliday e al., 2005). 5.4 Func ional and S a is ical Da a Analyses LFQ Da a No maliza ion – To al p o ein and pep ide concen a ions we e de e mined and no malized by BCA assay p io o LC-MS/MS. The da a no maliza ion pipeline consis ed o he ollowing s eps: (I) Da a cleanup by emo ing p o eins om po en ial con aminan s and hose wi h low o medium con idence le els. (II) To al in ensi y no maliza ion was pe o med by median in ensi y no maliza ion. (III) The aw and no malized in ensi y da a we e es ed o no mal dis ibu ion and Pea son co ela ion using Ma plo lib in Py hon. (IV) Remo al o p o eins iden i ied in only one ou o h ee o wo ou o i e biological eplica es. (V) Calcula e he median o all biological eplica es. TMT Da a No maliza ion – Repo e ion in ensi ies we e co ec ed o signal in e e ence by sub ac ing he pe cen age o in e e ence om he measu ed epo e ion in ensi ies (Sa i ski e al., 2013). Fo each PSM o he same TMT channel epo e ion in ensi ies we e no malized o one and he no malized median in ensi ies we e sub ac ed by he co esponding isola ion in e e ence. Occu ing nega i e alues due o ecalcula ion we e eplaced by he minimum posi i e alue in each channel. Only spec a wi h >50% isola ion in e e ence we e used o ela i e quan i ica ion and subjec ed o no maliza ion as desc ibed o LFQ. Di e en ial Analyses and S a is ical Ra ionale – The Pe seus so wa e sui e ( .1.6.14.0) was u ilized o pe o m unc ional 1D en ichmen analyses, Fishe ’s exac es , and wo ails Welch's o S uden 's - es s (Tyano a e al., 2016). S a is ical es s we e co ec ed o mul iple es ing applying a pe mu a ion-based o Benjamini-Hochbe g FDR calcula ion based on he p- alue dis ibu ion a 1 o 5% (Benjamini and Hochbe g, 1995). Di e en ially abundan p o eins we e iden i ied wi h Log2 old change ± 0.485. Signi ican di e ences we e assessed using ANOVA wi h Dunne 's mul iple compa ison es o iden i y speci ic g oups o condi ions ha we e signi ican ly di e en om a con ol g oup. S a is ically signi ican di e ences we e conside ed when * (p < 0.05); ** (p < 0.01); **** (p < 0.0001). Di ec ion pa hway analysis (DPA) II | GENERAL METHODS P A G E | 36 was pe o med using he di ec PA package in R ( .4.2.2) o pe o m es s a is ics in wo- dimensional space wi h a modi ied Pea son co ela ion es , o iden i y conco dan ly highe , lowe , and disco dan ly abundan p o eins (Yang e al., 2014). By speci ying eigh di e en di ec ions, DPA was used o pe o m COG and KEGG pa hway analysis on he selec ed di ec ions, wi h a p- alue ≤ 0.05 conside ed signi ican . Clea age si e speci ici y analysis was pe o med using iceLogo mo i s ia he s andalone e sion o iceLogo ( .1.2) (Colae e al., 2009). P io o analysis, ull yp ic pep ides, pep ides in which ini ia o N- e minal me hionine excision, sha ed pep ides, iden ical pep ide sequences wi h mul iple modi ica ions, and pep ides wi h canonical C- e minus we e emo ed o elimina e alse-posi i e clea age si es. The calcula ed clea age speci ici ies we e co ec ed o he na u al abundance o he co esponding amino acids in he o ganism's p o eome. Venn diag ams we e c ea ed using Venny (Oli e os, 2007). The sequence co e age o op-down da a was calcula ed using he p o i package (Quas e al., 2022) in R ( .4.2.2). UpSe plo s we e gene a ed using he UpSe plo and Ma plo lib package (Lex e al., 2014) in Py hon ( . 3.11.1). Anno a ion o sho gun p o eomics mass spec ome y da a was pe o med using he In e ac i e Pep ide Spec al Anno a o (B ademan e al., 2019). P o ein c ys al s uc u e p edic ions we e gene a ed using AlphaFold Colab 2.3.0 (Jumpe e al., 2021) and isualized using PyMOL (Sch ödinge , 2002). Expe imen al design wo k lows we e c ea ed using BioRende .com. iBAQ Calcula ion – iBAQ alues we e ob ained by di iding a p o ein’s o al non-no malized in ensi y by he numbe o heo e ically obse able yp ic pep ides be ween 7 and 30 amino acids wi h up o 2 missed clea ages (eq. 4). iBAQ =∑'in ensi y/# heo e ical'pep ides' (eq. 4) To ob ain he ela i e iBAQ alue ( iBAQ) o each p o ein, he iBAQ alue was no malized by di iding by he sum in ensi y o all iBAQ alues (eq. 5). iBAQ =iBAQ/∑iBAQ (eq. 5) To es ima e he ela i e abundance o his idine-con aining p o eins, he iBAQ alues we e scaled o 100% and mul iplied by he absolu e numbe o his idine esidues in each p o ein (eq. 6). iBAQ(*+,)= iBAQ×#His idine' esidue' (eq. 6) P A G E | 37 III PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON 1 In oduc ion and Summa y ............................................................................................. 39 2 Expe imen al Design ....................................................................................................... 41 2.1 Compa ison o Label-F ee and TMT-Based Quan i ica ion ..................................... 41 2.2 P o eo o m-Di ec ed Analysis .................................................................................. 42 3 Resul s .............................................................................................................................. 43 3.1 E alua ion o TMT Labeling E iciency and Sample Consis ency ........................... 43 3.2 Compa ison o LFQ and TMT P o eomic Analyses ................................................. 45 3.3 Ca bohyd a e-Dependen P o ein Abundance ........................................................ 47 3.4 P o eo o m-Di ec ed Top-Down Analysis ................................................................ 50 3.5 Analysis o P o eo o m Te mini ............................................................................... 51 3.6 Disco e y-Based Open Modi ica ion Sea ch ........................................................... 54 4 Discussion and Conclusion ............................................................................................ 57 4.1 Fu u e Di ec ion o Quan i a i e Analysis ............................................................... 57 4.2 P o eo o m-Di ec ed Analysis o B. he aio aomic on´s P o eome .......................... 59 III | PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON P A G E | 38 Pa s o he ollowing chap e ha e been published in “The in acellula p o eome o he gu bac e ium Bac e oides he aio aomic on is widely una ec ed by a swi ch om glucose o suc ose as main ca bohyd a e sou ce” Gen h e al., P o eomics, 22(22), 1–6, (2022). Supplemen a y ma e ial o (Gen h e al., 2022) Addi ional supplemen a y in o ma ion’s is eely a ailable o download a he publishe ’s websi e h ps://www.doi.o g/10.1002/pmic.202200189. The MS p o eomics aw da a and comple e P o eome Disco e sea ch esul s ha e been deposi ed o he P o eomeXchange Conso ium (h p://www.p o eomexchange.o g/) ia he PRIDE (Vizcaíno e al., 2014) pa ne eposi o y wi h he da a se iden i ie PXD033704. PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON | III P A G E | 39 1 In oduc ion and Summa y The human gu mic obio a signi ican ly enhances he nu i ional alue o human die s by b eaking down mac omolecules and ac i a ing key in es inal genes o acili a e nu ien abso p ion (Ecklu-Mensah e al., 2022). S udies in ge m- ee mice ha e demons a ed ha mic obial coloniza ion educes he equi ed calo ic in ake o weigh main enance by 30% and induces apid changes in body a composi ion (Wos mann e al., 1983; Bäckhed e al., 2004). Die a y choices can in luence he composi ion o he gu mic obio a and he immune esponse, bo h o which a e key ac o s in he de elopmen and p og ession o in lamma o y bowel disease (IBD) (Dolan and Chang, 2017). Indi iduals wi h IBD commonly de elop inc eased sensi i i y o in ole ance o ce ain oods, leading hem o a oid speci ic die a y componen s (Ballegaa d e al., 1997; Zallo e al., 2013). No ably, he adop ion o Wes e n die a y habi s, o en associa ed wi h high le els o p ocessed oods and e ined ca bohyd a es, signi ican ly inc eases he isk o gas oin es inal in lamma o y diso de s, including IBD (Ng, 2014; Khademi e al., 2021). Howe e , i is impo an o acknowledge ha die a y in e en ions can also be u ilized o p omo e heal hy gu mic obial unc ions (Ecklu-Mensah e al., 2022). Wi hin he human gu , Bac e oides ep esen s one o he mos abundan gene a o bac e ia (A umugam e al., 2011). Bac e oides he aio aomic on, cons i u ing app oxima ely 6% o he o al gu mic obio a, plays a c ucial ole in ein o cing he mucosal ba ie , main aining immune esponse homeos asis, and p ocessing nu ien s (Zocco e al., 2007). I s complex epe oi e o glycosylhyd olases enables he me abolism o a wide ange o o he wise indiges ible die a y polysaccha ides and hos -de i ed glycans in he human gu (Xu and Go don, 2003). The impac o die a y suga s on he compe i i e dynamics wi hin he human gu mic obio a has g ea consequences. P olonged consump ion o a high-suga die can signi ican ly al e gu mic obial di e si y (Do e al., 2018; Alasma e al., 2023), leading o a displacemen cha ac e ized by educed le els o Bac e oide es, simila o he dysbiosis obse ed in IBD (F ank e al., 2007). This die a y shi p omp s bac e ia o employ a ious adap i e mechanisms, which can include he applica ion o speci ic ca bohyd a e anspo and u iliza ion sys ems o i ulence genes ha media e oxin p oduc ion o immune e asion (Ponce e al., 2009). While monosaccha ides, such as glucose and uc ose, may a es he coloniza ion o B. he aio aomic on in he gu by supp essing he exp ession o coloniza ion- ela ed p o eins (Townsend e al., 2019), inc eased glucose in ake in mice p omo es mucoly ic bac e ia, including B. agilis (Khan e al., 2020). This p omo ion could po en ially comp omise he in eg i y o he p o ec i e in es inal mucosal ba ie , a c i ical ac o in ini ia ing in es inal in lamma ion (Png e al., 2010). The con adic o y indings highligh he need o u he esea ch on his p ominen genus and i s suga -mic obio a in e ac ions. III | PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON P A G E | 40 This chap e , p omp ed by he obse ed co ela ion be ween inc eased suga consump ion and he po en ial de elopmen o IBD (Khademi e al., 2021), aims o e alua e p o eomic changes in B. he aio aomic on in esponse o di e en suga sou ces. Aim o his s udy: - U ilize wo quan i a i e bo om-up p o eomics analyses – label- ee quan i ica ion (LFQ) and isoba ic labeling-based quan i ica ion using TMT – o e alua e p o eomic changes induced by he p esence o suc ose and glucose. - Pe o m a compa a i e analysis o quan i a i e esul s o de e mine he op imal me hodology o u u e p o eomic quan i a i e analysis. - Iden i y p o eins whose abundance le els a e in luenced by he ype o ca bohyd a e. - Apply a ious sample p epa a ion echniques o deple e he high-molecula -weigh p o eome and inc ease he co e age o he low-molecula -weigh p o eome. - Conduc a p o eo o m-di ec ed op-down analysis and employ a disco e y-based open modi ica ion sea ch o iden i y pos - ansla ional modi ica ions and po en ial p o eoly ic clea age e en s. PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON | III P A G E | 47 FIGURE III-7 | Analysis o he P o eomic Response be ween LFQ and TMT. (A) Compa ison o log2 a ios o suc ose e sus glucose o LFQ and TMT app oaches. P o eins exhibi ing highe abundance in he p esence o ei he suc ose (32 p o eins, ed do s) o glucose (6 p o eins, blue do s) a e highligh ed. The signi icance (p ≤ 0.05) was de e mined using a modi ied Pea son's co ela ion es (Yang e al., 2014). The ed line ep esen s he Pea son co ela ion (P ), excluding one ou lie . Di ec ion pa hway analysis o p o eins o highe abundance in he p esence o (B) suc ose o (C) glucose. Bo h g aphs display he numbe o selec ed p o eins in each COG ca ego y, he pe cen age o p o eins in he speci ied ca ego y, and he co esponding p- alues. 3.3 Ca bohyd a e-Dependen P o ein Abundance S a is ical analysis ( wo-sided Welch's - es wi h Benjamini-Hochbe g FDR co ec ion o mul iple es ing, q ≤ 0.05 and Log2 old change o ±0.485) iden i ied 37 di e en ially abundan p o eins o LFQ (FIGURE III-5A) and 32 o TMT (FIGURE III-5B). Visualiza ion o he LFQ esul s e ealed di e en ial abundance o p o eins associa ed wi h ca bohyd a e me abolism ( ed do s), amino acid anspo and me abolism (yellow do s), and ino ganic ion anspo and -2 -1 0123456 -3 -2 -1 0 1 2 3 105 124 120 62 150 43 6 334 108 1.9E-26 8.3E-6 1.0E-5 7.5E-5 3.6E-4 1.1E-3 2.8E-3 2.1E-2 3.6E-2 134 83 29 334 15 75 64 48 2.0E-21 3.8E-5 1.2E-4 6.6E-4 1.2E-3 2.8E-3 3.4E-3 1.1E-2 TMT log2 (Suc ose/Glucose) LFQ log2 (Suc ose/Glucose) P = 0.81 B A C Ca bohyd a e anspo / me abolism Amino acid anspo / me abolism Ene gy p oduc ion / con e sion Ino ganic ion anspo / me abolism Cell wall/memb ane/en elope biogenesis Lipid anspo / me abolism Cell mo ili y Func ion unknown Coenzyme anspo / me abolism 020 40 60 80 100 P o ein g oups (%) p- alue T ansla ion, ibosomal, biogenesis Nucleo ide anspo / me abolism Signal ansduc ion mechanisms Func ion unknown Sec e ion, esicula anspo Replica ion, ecombina ion, epai T ansc ip ion PTM, p o ein u no e , chape ones 020 40 60 80 100 P o ein g oups (%) p- alue III | PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON P A G E | 48 me abolism (blue do s) (FIGURE III-8A, B). No ably, ce ain p o eins such as aldolase 1- epime ase (Q8AAU2), py u a e-phospha e dikinase (Q8AA21), uc okinase (Q8A6W9), glycine clea age sys em H p o ein (Q8A4S8), and ROK amily ansc ip ional ep esso (Q8A4V4) consis en ly exhibi ed high in ensi y-based absolu e quan i ica ion (iBAQ) alues, ega dless o he ca bon sou ce used in cul i a ion (FIGURE III-8C-D). These alues, calcula ed by di iding a p o ein's o al non-no malized in ensi y by he numbe o heo e ically obse able yp ic pep ides o ha p o ein, indica e he ela i e absolu e abundance o p o eins. These esul s sugges consis en abundance le els o ce ain p o eins and may indica e hei po en ial ele ance in di e se cellula p ocesses unde di e en cul u e condi ions. FIGURE III-8 | O e iew o LFQ Quan i a i e Resul s. (A) Volcano plo wi h dashed e ical lines ep esen s Log2 cu o s and he dashed ho izon al line ep esen s a q- alue o 0.05 ( wo-sided Welch's - es wi h Benjamini-Hochbe g FDR co ec ion o mul iple es ing). (B) P o eins in ol ed in amino acid, ca bohyd a e, o ino ganic ion anspo and me abolism acco ding o COG anno a ions. iBAQ in ensi ies o p o eins iden i ied in he p esence o (C) glucose o (D) suc ose. Addi ionally, an inc eased abundance o p o eins associa ed wi h uc an me abolism was iden i ied, sugges ing a specialized adap a ion o he u iliza ion o uc ose-based oligosaccha ides. This adap a ion was acili a ed by he s a ch u iliza ion sys em (Sus), encoded wi hin he polysaccha ide u iliza ion locus (PUL) 22, which includes eigh open eading ames (FIGURE III-9B) (Ma ens e al., 2008; Sonnenbu g e al., 2010). The egula ion o his ope on is media ed by a hyb id wo-componen sys em, posi ioned adjacen o he PUL, enhancing he e iciency o uc ose-based ca bohyd a e u iliza ion (Sonnenbu g e al., 2010). 0500 1000 1500 2000 2 4 6 8 10 12 -2 0 2 4 6 8 0 1 2 3 4 5 6 7 0500 1000 1500 2000 2 4 6 8 10 12 12214 17 20 1 4 24 3 7 10 816 21 19 6 22 18 13 15 9 5 23 11 1 2 34 5 67 89 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 12 4 20 213 8 14 17 3 1 24 16 7 21 10 22 6 19 18 23 15 95 11 Glucose Log10 iBAQ Ranked p o eins C -Log10 p- alue LFQ Log2 (Glucose / Suc ose) A B Amino acid anspo / me abolism 1) Aspa a e amino ans e ase 2) Glycine clea age sys em H p o ein Ca bohyd a e anspo / me abolism 3) Alpha-1,2-mannosidase 4) F uc okinase 5) Alpha-xylosidase 6) Glycoside hyd olase amily 92 7) L- ucose isome ase 8) Le anase 9) Alpha-mannosidase 10) Be a-galac osidase 11) Alpha-1,2-mannosidase 12) ROK amily ansc ip ional ep esso 13) Glycoside hyd olase amily 32 14) Aldose 1-epime ase 15) Be a-galac osidase 16) Alpha-1,2-mannosidase 17) Py u a e-phospha e dikinase 18) Glucan 1,4-alpha-glucosidase SusB 19) 4-alpha-glucano ans e ase Ino ganic ion anspo / me abolism 20) o 24) SusC homolog Suc ose Log10 iBAQ Ranked p o eins D PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON | III P A G E | 49 Pe iplasmic hyd olysis o suc ose b eaks i in o i s cons i uen monosaccha ides, glucose, and uc ose, which a e hen impo ed in o he cy oplasm and di ec ed owa d cen al me abolic pa hways (FIGURE III-9A). Glucose, a p e e ed ene gy sou ce in nume ous mic oo ganisms, can access cen al me abolic pa hways like glycolysis h ough he Embden-Meye ho pa hway. This pa hway gene a es py u a e, ATP, and p ecu so me aboli es o he ica boxylic acid (TCA) cycle. Unlike o he ca bohyd a es, uc ose could also di ec ly en e he Embden- Meye ho pa hway o ene gy gene a ion, wi hou he need o addi ional ene gy-consuming con e sion s eps (FIGURE III-9A). This me abolic pa hway con e s an ad an age, enhancing he e iciency o uc ose u iliza ion ac oss a ious bac e ia, including B. he aio aomic on. FIGURE III-9 | Suc ose U iliza ion Pa hway in B. he aio aomic on. (A) Suc ose up ake occu s ia he Sus sys em and upon he clea age o he O-glycosidic bond, glucose and uc ose molecules a e channeled in o he majo glycoly ic pa hways. The gene a ed py u a e en e s he ica boxylic acid (TCA) cycle o u he p ocessing. (B) Polysaccha ide u iliza ion locus (PUL) 22 encodes o he Sus sys em. Enzymes essen ial o his ca abolic p ocess a e highligh ed in g ay and ep esen ed by he ollowing abb e ia ions: HK (hexokinase), GPI (glucose-6-phospha e isome ase), FRK ( uc okinase), and PFK (phospho uc okinase). O O O Suc ose Sus OO Glucose F uc ose HK ATP ADP O P FRK ATP ADP O P O P GPI PFK 2 ATP 2 ADP F uc ose-6-P Glucose- 6-P O P P O P PF uc ose- 1,6-bP Glycolysis TCA cycle Py u a e PUL 22 1754 Hyb id wo componen sys em F uc okinase Monosaccha ide impo Glycoside hyd olases 32 SusE SusD homolog SusC homolog Glycoside hyd olase 32 1757 1758 1760 1762 17631761 17651759 A B III | PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON P A G E | 50 3.4 P o eo o m-Di ec ed Top-Down Analysis To inc ease he co e age o he low-molecula - weigh p o eome, h ee dis inc me hods we e employed. These me hods included wo HMWP deple ion echniques, GELF EE ac iona ion, and SCX ac iona ion. The numbe o iden i ica ions pe me hod anged om 360 o 540 p o ein g oups (FIGURE III-10A) and 1,298 o 1,534 p o eo o ms (FIGURE III- 10B), espec i ely. No ably, all me hods displayed compa able iden i ica ion me ics, as demons a ed by he mean P SMs pe p o eo o m (FIGURE III-10D), esidue clea age (FIGURE III-10D), -Log (E- alue) (FIGURE III- 10E), and he size dis ibu ion o he iden i ied p o eo o ms (FIGURE III-10F). The o e lap be ween he di e en me hods anged om 3% o 9%, and he majo i y o p o eo o ms we e uniquely iden i ied by indi idual me hods, cons i u ing app oxima ely 25% each (FIGURE III-10G). Combining all h ee da abase sea ches esul ed in he iden i ica ion o 865 p o ein g oups FIGURE III- 10A), ep esen ed by a o al o 3,117 p o eo o ms (FIGURE III-10B). De ailed in o ma ion on he exac iden i ica ion me ics o each me hod is p o ided in Appendix 1.2. In summa y, he numbe o iden i ica ions pe SCX ac ion anged om 125 o 403 p o ein g oups (FIGURE A-12A) and 185 o 1,280 p o eo o ms (FIGURE A-12B). The highes numbe o p o ein g oups and p o eo o m iden i ica ions was achie ed in ac ion 3, which mos ly con ains highe cha ged species (FIGURE A-12A-B). Compa ison o he acidic and basic HMWP deple ion showed ha compa able numbe s o FIGURE III-10 | Top-down Me hodology Compa ison. Numbe o (A) iso o ms, (B) p o eo o ms, (C) mean P SMs pe p o eo o m. Dis ibu ion o (D) esidue clea age, (E) –Log (E- alue) and (F) heo e ical p o eo o m mass. (G) P o eo o m o e lap be ween di e en me hods. 360 409 540 865 25 33 19 34 1298 1321 1534 3117 HMWP deple ion GELF EE SCX Combined 0 200 400 600 800 1000 ∑ Iso o ms A 0 10 20 30 40 Mean P SMs pe p o eo o m D 0 1000 2000 3000 4000 ∑ P o eo o ms B C 0 20 40 60 80 100 Residue clea age (%) 35 27 39 33 1.4 1.3 1.3 1.3 3.9 4.6 3.5 3.9 0.0 0.5 1.0 1.5 2.0 2.5 -Log10 (E- alue) E 0 5 10 15 20 25 Theo. mass (kDa) F 710 (23%) 94 (3%) 779 (25%) 880 (28%) 288 (9%) 160 (5%) 206 (7%) SCX GELF EE HMWP deple ion G PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON | III P A G E | 51 p o ein g oups and p o eo o m iden i ica ions we e ob ained (FIGURE A-12A-B), wi h each deple ion me hod con ibu ing a compa able numbe o unique iden i ica ions (FIGURE III-10A- B). A compa ison o GELF EE ac ions analyzed using di e en s a iona y phases (C4 and PLRP-S), indica ed a gene ally highe numbe o p o ein g oups (FIGURE A-12G) and p o eo o m iden i ica ions o he C4 column (FIGURE A-12H). The obse ed inc ease in he a e age numbe o P SMs pe p o eo o m iden i ica ion o he PLRP-S column (FIGURE A-12I) can be a ibu ed o dec eased esolu ion due o wide elu ion p o iles, esul ing in peak b oadening and poo sepa a ion o adjacen peaks compa ed o he C4 column (FIGURE A-12J- K). These de ia ions a e due o di e ences in column cha ac e is ics, including he la ge pa icle size (5.0 µm, 1000 Å), sho e column leng h (17 cm), and po en ial dead olume in oduced du ing c imping wi h ZIRCOFIT UHPLC i ings o he PLRP-S column, as opposed o he comme cially pu chased C4 column (2.6 µm, 150 Å, 50 cm). These di e ences may lead o ewe p o eo o ms being e ec i ely sepa a ed in a gi en ime ame o he PLRP-S column. I is impo an o no e ha bo h columns ope a ed a iden ical low a es, g adien s, and eluen s ( o de ailed in o ma ion, e e o chap e II.4.2). The e o e, op imiza ion o g adien pa ame e s, including he design o a nonlinea g adien o PLRP-S columns, p o ides an oppo uni y o imp o e ch oma og aphic sepa a ion and inc ease he numbe o p o eo o ms (T udgian e al., 2014). 3.5 Analysis o P o eo o m Te mini Ou o 3,117 iden i ied p o eo o ms, 2,942 (94%) had molecula weigh s be ween 2-10 kDa and consis ed mos ly o unca ed p o eo o ms (FIGURE III-11A). N- e minal unca ion accoun ed o 42% o he p o eo o ms, excluding N- e minal me hionine excisions (NME), while C- e minal unca ion accoun ed o 25% o he p o eo o ms (FIGURE III-11B). No ably, he hea shock p o ein (Q8AAA0) and he 60 kDa chape onin (Q8A6P8) we e p e alen in bo h TDP and BUP analyses, exhibi ing nume ous N- and C- e minal unca ed p o eo o ms (38 and 36 espec i ely, TABLE A-2) and mul iple pep ides ha ing high PSM coun s (TABLE III-2). FIGURE III-11 | Neo- e mini Analysis o Iden i ied P o eo o ms. (A) Size dis ibu ion and (B) pe cen age dis ibu ion o iden i ied p o eo o ms, including N- and/o C- e minal unca ed p o eo o ms wi h N- e minal ini ia o me hionine excision and in ac ini ia o me hionine. 2 4 6 8 10 12 14 16 18 20 0 100 200 300 400 500 600 700 N-Te m C-Te m In e n Canonical ∑ P o eo o ms Theo mass (kDa) C- e m 25% In e n 28% N- e m 42% Canonical 5% NME: In ac 65% 35% Excised A B III | PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON P A G E | 52 In e nal p o eo o ms esul ing om bo h N- and C- e minal unca ion accoun ed o an addi ional 28%. Only 5% o p o eo o ms we e classi ied as ull-leng h canonical p o eins, wi h 65% e aining and 35% lacking hei N- e minal me hionine (FIGURE III-11B). App oxima ely 15% o he p o eo o ms wi h alanine, glycine o se ine posi ioned a he P1´ si e ollowing he ini ia o me hionine e ained he ini ia o me hionine, esul ing in 85% lacking he ini ia o me hionine (FIGURE III-12A). Me hionine clea age was de ec ed in all p o eo o ms exhibi ing a p oline a P1´ (FIGURE III-12B). Al hough lysine is he mos common amino acid a P1´ (30.8%, FIGURE III-12C), and NME ypically a o s amino acids wi h smalle side chains a P1´(F o in e al., 2006), a p o eo o m wi h lysine a P1´ was s ill exhibi ed NME clea age (FIGURE III-12B). In o al, 23 p o eins we e iden i ied as bo h NME and non-NME p o eo o ms (FIGURE III-12D). Among hem, he 60 kDa chape onin (Q8A6P8) was iden i ied wi h 15 p o eo o ms e aining he ini ial me hionine and 13 lacking i (FIGURE III-12E). These p o eo o ms exhibi ed mino C- e minal amino acid unca ions, possibly esul ing om po en ial exopep idase ac i i y. This obse a ion shows how a p o eoly ic e en , such as NME, in conjunc ion wi h po en ial exopep idase ac i i y, can lead o he gene a ion o mul iple p o eo o ms (FIGURE III-12E). FIGURE III-12 | Analysis o Me hionine Clea age. (A) Numbe o iden i ied p o eo o ms wi h N- e minal ini ia o me hionine excisions (wi h NME) and in ac ini ia o me hionine (wi hou NME). (B) In luence o he gy a ion adius o he P1´ amino acid esidue on me hionine clea age. (C) Amino acid composi ion o he P1´ posi ion ollowing ini ia o me hionine. (D) O e lap o p o eins wi h and wi hou he NME. (E) Iden i ied p o eo o ms o 60 kDa chape onin (Q8A6P8). G A S C T P V D N L I Q E H M F K Y W R 0 20 40 60 80 100 0% 10% 20% 30% G A S C T P V D N L I Q E H M F K Y W R 0 50 100 150 200 Wi h NME Wi hou NME Numbe o p o eo o ms Amino acid (P1´) A Me clea age e iciency (%) Amino acid (P1′) 0.20 0.93 1.65 2.38 Side-chain gy a ion adius (Å) 0.00 C G I L V A P F H W Y C M K R D E N Q S T B 57 (26%) 23 (11%) 139 (63%) Wi hou NME 162 p o eins Wi h NME 80 p o eins [-].MA...KK.[G] [-].MA...GV.[D] [-].MA...AN.[A] [-].MA...AN.[A] [-].MA...NA.[V] [-].MA...VK.[V] [-].MA...KV.[T] [-].MA...VT.[L] [-].MA...TL.[G] [-].MA...LG.[P] [-].MA...GP.[K] [-].MA...PK.[G] [-].MA...KG.[R] [-].MA...GR.[N] [-].MA...AP.[H] [M].AK...VD.[A] [M].AK...VD.[A] [M].AK...DA.[L] [M].AK...AL.[A] [M].AK...LA.[N] [M].AK...AN.[A] [M].AK...NA.[V] [M].AK...AV.[K] [M].AK...KV.[T] [M].AK...LG.[P] [M].AK...RN.[V] [M].AK...KD.[G] [M].AK...QN.[T] [L].FN...VD.[A] [Y].FV...EA.[L] [V].RE...HA.[A] [V].RE...AA.[G] [V].RE...RV.[A] [N].AR...TE.[C] [C].VI...MM.[-] 0100 200 300 400 500 Sequence leng h Iden i ied p o eo o ms Ini ia o me hionine clea ed Ini ia o me hionine no clea ed D E Amino acid (P1′) Numbe o p o eo o ms Amino acid (P1′) B A C Me hionine clea age (%) PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON | III P A G E | 53 Analysis o he N- e minal esidues p eceding he clea age si e (P1), and he C- e minal amino acids ollowing i (P1´), e ealed a uni o m dis ibu ion o abundance o he majo i y o he de ec ed neo- e mini (FIGURE III-13). Howe e , he de ec ion o p o eo o ms wi h ei he aspa a e a P1 o p oline a P1´ led o he de ec ion o a p ominen Asp-P o sequence logo in he acidic HMWP deple ion, GELF EE ac iona ion, and SCX ac iona ion expe imen s (FIGURE III-13 B-D). Con e sely, in he HMWP dele ion pe o med unde basic condi ions (pH 8.5), his speci ic sequence pa e n was no obse ed (FIGURE III-13A). Ins ead, he clea age da a showed an inc eased abundance o wo dis inc p o eo o ms: one ea u ing aspa agine a P1, and he o he ea u ing ei he glycine o se ine a P1´, esul ing in no iceable sequence logos, Asn-Gly and Asn-Se , espec i ely (FIGURE III-13A). The speci ic clea age pa e ns, which a ied depending on he me hod and pH o he deple ion used, sugges ha a i icial clea age e en s may ha e been in oduced du ing sample p epa a ion o LC-MS/MS analysis. FIGURE III-13 | Analysis o P o eo o m Neo-Te mini. P o eo o m analysis o (A) basic HMWP deple ion, (B) acidic HMWP deple ion, (C) GELF EE ac iona ion, and (D) SCX ac iona ion. Hea maps illus a e he dis ibu ion and in ensi y o a ious p o eo o ms cha ac e ized by hei N- e minal (P1) and C- e minal (P1´) esidues, while ba plo s summa ize he o al numbe s o p o eo o ms iden i ied. Coun Coun Amino acid (P1) Amino acid (P1' ) Coun Coun Amino acid (P1) Amino acid (P1' ) Coun Coun Amino acid (P1) Amino acid (P1' ) AB C D Coun Coun Amino acid (P1) Amino acid (P1' ) III | PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON P A G E | 54 3.6 Disco e y-Based Open Modi ica ion Sea ch Du ing he p epa a ion o biological samples and conduc ing LC-MS/MS analysis, a i icial modi ica ions, non-co alen adduc o ma ion, and a i icial unca ion e en s may occu (Scha e e al., 2021). To add ess his issue and o iden i y bo h known and no el modi ica ions, a disco e y-based open modi ica ion sea ch was pe o med. This allowed o he iden i ica ion o p ecu so mass shi s wi hou a p ede ined lis o PTMs. While se e al mass shi s hin ed owa ds po en ial in e es ing PTMs on p o eo o ms o B. he aio aomic on, i 's c ucial o emphasize ha hese iden i ica ions a e solely based on MS1 in ensi ies, and mass shi s may also be a ibu ed o inaccu acies in p ecu so mass decon olu ion (Jeong e al., 2020). The e o e, he ollowing esul s se e as p elimina y indica ions and wa an u he alida ion. Thousands o pep ide spec um ma ches (P SMs) pe mass shi we e de ec ed, each exhibi ing a ying deg ees o mass accu acy (p ecu so mass ole ance: 10 ppm). This esul ed in a b oad dis ibu ion o del a p ecu so mass shi s. Following manual inspec ion o p ecu so iso ope dis ibu ion and agmen a ion spec a o po en ial a PTM, he monoiso opic mass co esponding o en ies in he PSI-MOD (Mon ecchi- Palazzi e al., 2008) o UniMod da abase (C easy and Co ell, 2004) will be epo ed. The de ec ed mass shi s included me hionine oxida ion (+15.994 Da), cys eine dioxida ion (+31.988 Da), and combina ions o mul iple oxida ions (FIGURE III-14). These modi ica ions can occu spon aneously du ing sample p epa a ion and s o age (Kaulich e al., 2022b). Addi ionally, ce ain mass shi s indica e he absence o speci ic amino acid esidues, such as cys einyl (+103.009 Da), phenylalaninyl (+147.068 Da), and y osinyl (+163.063 Da). F equen misassignmen s we e o en iden i ied as ei he inco ec anno a ion (-131.040 Da) o absence (+131.040 Da) o N- e minal ini ia o me hionine esidues. Inco ec mass shi s, a ising om mul iple PTMs o inco ec ly assigned modi ica ions, such as he absence o ini ia o me hionine and misassignmen s o ace yla ion (-42.010 Da), con ibu ed o p e alen mass shi s o +89.03 Da (FIGURE III-14 and FIGURE A-13A). FIGURE III-14 | Dis ibu ion o P ecu so Del a Mass Shi s. A ows highligh po en ial PTMs wi h ma ched monoiso opic masses acco ding o he PSI-MOD (Mon ecchi-Palazzi e al., 2008) o UniMod (C easy and Co ell, 2004) da abase. -200 -150 -100 -50 050 100 150 200 0 2500 5000 7500 10000 60000 P SMs Δ P ecu so mass shi (Da) Me hyl- (14 Da) Oxida ion (16 Da) Me hionyl- (131 Da) Ace yl (-42 Da) (89 Da) Me hionyl- (131 Da) Ty osinyl- (163 Da) Phenylalanyl- (147 Da) Cys einyl- (103 Da) Dioxida ion (32 Da) Fo myl- (-28 Da) Ace yl- (-42 Da) Me hionyl- (-131 Da) T ioxida ion (48 Da) PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON | III P A G E | 55 The o ma ion o disul ide bonds in cys eine-con aining p o eo o ms can be indica ed by small mass shi s, such as –2 Da and –4 Da. Se e al po en ial disul ide bonds ha e been iden i ied in p o eins such as 50S ibosomal p o ein L32 (Q8A138), which displays a zinc inge mo i (Cys-Xaa2-Cys-Xaa9-Cys-Xaa2-Cys) (FIGURE A-13B), and pep idyl-p olyl cis- ans isome ase (Q8A607), ea u ing an eigh -cys eine mo i (8CM) (FIGURE A-13C). Al hough 8CM mo i s a e p esen in a la ge numbe o ungal ex acellula memb ane p o eins (Kulka ni e al., 2003) and plan de ensins (José-Es anyol e al., 2004), hei ole in bac e ial p o eins is unclea . A mass shi o 45.987 Da was de ec ed on ibosomal p o ein S12 (Q8A472), sugges ing a po en ial β-me hyl hio-aspa ic acid modi ica ion (FIGURE III-15A). This PTM has been epo ed in o he bac e ial ibosomal p o ein S12 p o eins (Kowalak and Walsh, 1996; S ade e al., 2004, 2011). Fu he mo e, he po en ial modi ica ion was de ec ed in a BUP sea ch, on he same aspa ic acid wi hin a pep ide spanning he same sequence (FIGURE A-13D). P o eo o ms o he glycine clea age sys em H p o ein (Q8A4S8) exhibi ed a mass shi o 188.033 Da, po en ially indica ing an N6-lipoyllysine modi ica ion (FIGURE III-15B). This PTM is essen ial o ans e ing a me hylamine g oup om he P p o ein o he T p o ein in he glycine clea age sys em, gene a ing CO2, NH3, and N5,N10-me hylene- e ahyd o ola e (THF) (McCa hy and Booke , 2020). FIGURE III-15 | Pu a i e PTMs in B. he aio aomic on p o eins. (A) β-me hyl hio-aspa ic acid on ibosomal p o ein S12 (Q8A472). (B) N6-lipoyllysine on glycine clea age sys em H p o ein (Q8A4S8). (C) O-(pan e heine 4'-phospho yl)se ine on acyl ca ie p o ein (Q8A2E6). Dis al hiol Ribosomal p o ein S12 β-me hyl hio-aspa ic acid (Mass Di e ence: 0.005 Da & 0.34 ppm, P-Sco e: 2.3E-58, Residue clea age: 23%) Glycine clea age sys em H p o ein N6-lipoyllysine (Mass Di e ence: 0.058 Da & 3.96 ppm, P-Sco e: 1.0E-35, Residue clea age: 16%) Acyl ca ie p o ein O-(pan e heine 4'-phospho yl)se ine (Mass Di e ence: 0.005 Da & 0.56 ppm, P-Sco e: 1.3E-90, Residue clea age: 61%) NPTIQQLVRKGREVLVEKSKSPALD 25 S 26 CPQRRGVCVRVYTTTPKKPNSAMR 50 K 51 VARVRLTNQKEVNSYIPGEGHNLQ 75 E 76 HSIVLVRGGRVK LPGVRYHIVRG 100 T 101 LDTAGVAGRTQRRSKYGAKRPKPG 125 Q 126 AAPAKKKC D NMNFPQNLKYTNEHEWIRVEGDIAY 25 V 26 GITDYAQEQLGDIVFVDIPTVGET 50 L 51 EAGETFGTIEVV TISDLFLPLAG 75 E 76 ILEQNEALEENPELVNKDPYGEGW 100 L 101 IKMKPADASAAEDLLDAEAYKAVV 125 N 126 GC K NSEIASRVKAIIVDKLGVEESEVTN 25 E 26 ASFTNDLGAD LDTVELIMEFEKE 50 F 51 GISIPDDQAEKIGTVGDAVSYIEE 75 H 76 A K C S HN ab S g OOH O CH3 H N O S S O HN HN OP O OH O CH3 H3C OH HN O N H HS O O A B C III | PROTEOMIC ANALYSIS OF B. THETAIOTAOMICRON P A G E | 56 The la ges and mos abundan po en ial PTM disco e ed was O-(pan e heine 4'- phospho yl)se ine (340,085 Da) on he acyl ca ie p o ein (ACP) (Q8A2E6) (FIGURE III-15C). The p os he ic moie y, 4'-phosphopan e heinyl (Ppan ), has a highly eac i e dis al hiol g oup ha acili a es he co alen anspo o a ious chemical g oups, including acyl g oups, du ing a y acid biosyn hesis (Chan and Vogel, 2010). In e es ingly, ACP p o eo o ms exhibi ing he Ppan modi ica ion showed a wide ange o mass shi s ha may be caused by di e en PTMs (FIGURE III-16). Some o hese mass shi s may be a ibu ed o common acyl in e media es du ing a y acid biosyn hesis, such as ace yl (+42,010 Da) and malonyl (+86,000 Da) (Chan and Vogel, 2010). Since a y acid biosyn hesis in ol es mul iple s eps o p oduce ull-leng h chains ( ypically C16 o C18) (C onan and Thomas, 2009), he p esence o la ge mass shi s may indica e longe chain-leng h in e media es. Fu he mo e, se e al mass shi s may indica e hiol- ela ed modi ica ions, such as cys einyla ion (119.004 Da) and sul u dioxide (SO2) addi ion (+63.961 Da) (FIGURE III-16). FIGURE III-16 | Dis ibu ion o P ecu so Mass Shi s on Acyl Ca ie P o ein (Q8A2E6). All indica ed mass shi s addi ionally exhibi he O-(pan e heine 4'-phospho yl)se ine mass shi o 340.085 Da. A ows highligh po en ial PTMs wi h ma ched monoiso opic masses acco ding o PSI-MOD (Mon ecchi- Palazzi e al., 2008) o UniMod (C easy and Co ell, 2004) da abases. 050 100 150 200 250 300 0 500 1000 2500 P SMs Δ P ecu so mass shi (Da) 16 Da SOH 42 Da SCH3 O 64 Da SSH OO S O OH O 86 Da 119 Da SS NH2 OH O O O NH P O OH O H3CCH3 HO NHO N H SH O Po en ial in e media es o a y acid syn hesis 4’-Phosphopan e heinyl P A G E | 63 IV INFLUENCE OF PH ON BACTERIAL PROTEOMES 1 In oduc ion and Summa y ............................................................................................. 64 2 Expe imen al Design ....................................................................................................... 66 2.1 Bo om-up LFQ Analysis o HGM P o eomes .......................................................... 66 2.2 Top-down LFQ Analysis o B. p oduc a ................................................................... 66 3 Resul s .............................................................................................................................. 68 3.1 P o eomic Adap a ions o Acidic and Alkaline pH in B. longum .............................. 68 3.2 Analysis o he Acidic Response o h ee HGM Membe s ....................................... 71 3.3 Compa ison o LMWP-Top-Down and Full-P o eome Bo om-Up Analysis o B. p oduc a ........................................................................................................ 79 3.4 Po en ial pH-induced Asp-P o Clea age ................................................................. 84 3.5 Phospho yla ion o HP p o eins .............................................................................. 87 4 Discussion and Conclusion ............................................................................................ 89 4.1 Quan i a i e analysis o he B. longum p o eome .................................................... 89 4.2 Quan i a i e analysis o he B. he aio aomic on p o eome ..................................... 92 4.3 Quan i a i e analysis o he B. p oduc a p o eome .................................................. 93 4.4 Asp-P o pep ide bond hyd olysis ............................................................................. 98 IV | INFLUENCE OF PH ON BACTERIAL PROTEOMES P A G E | 64 1 In oduc ion and Summa y The human gu mic obiome (HGM), comp ising di e se mic obial communi ies, plays a c ucial ole in main aining heal h. I s composi ion and balance a e in luenced by a ious ac o s, including g ow h ac o s, mic onu ien s, an imic obial compounds, and gu pH (Rodiono e al., 2019; Beam e al., 2021; Fi man e al., 2022). While some a iabili y in he pH o he gas oin es inal ac is no mal due o ac o s like die and mic obial ac i i y, signi ican de ia ions om ypical anges can al e he mic obiome’s composi ion and unc ion, po en ially impac ing heal h (Fi man e al., 2022). Gene ally, he p oximal small bowel has lowe pH le els (pH 5.5 o pH 7.0) compa ed o he descending and ec osigmoid colon, which main ains sligh ly highe pH le els (pH 6.6 o pH 7.5) (Nugen e al., 2001). No ably, inc eased colonic acidi y has been associa ed wi h a ious gas oin es inal diseases, including i i able bowel synd ome (IBS) and in lamma o y bowel disease (IBD) (Nugen e al., 2001; Ringel-Kulka e al., 2015). Mic obial esponses o a ying pH condi ions in ol e physiological and molecula adap a ions aimed a main aining in acellula pH (pHi) homeos asis. These adap a ions can include p o on ansloca ion by specialized pumps such as ATP syn hase and small ion (Na⁺, K⁺, o Ca²⁺)/H⁺ an ipo e s (K ulwich e al., 2011). Addi ionally, enzyme-ca alyzed eac ions, including p ocesses such as deca boxyla ion, consume p o ons, whe eas deaminase-ca alyzed eac ions inc ease he concen a ion o alkaline compounds (K ulwich e al., 2011). P o ec i e mechanisms also include changes in lipid composi ion o educe p o on pe meabili y o he cell, p omo ion o bio ilm o ma ion, adjus men o cell densi y, and implemen a ion o epai mechanisms o coun e ac inc eased damage o mac omolecules (Guan and Liu, 2020). Despi e ex ensi e in es iga ions in o mic obial acid s ess esponses, knowledge ega ding he p o eomic adap a ions o speci ic HGM membe s o acidic s ess emains limi ed. Unde s anding hese p o eomic changes could p o ide aluable insigh s in o how hese mic obes main ain esilience and adap o acidic en i onmen s. Add essing his esea ch gap could ad ance unde s anding o gu mic obial dynamics and hei po en ial implica ions o human heal h, as well as acili a e he de elopmen o a ge ed he apeu ic s a egies o gas oin es inal diseases. INFLUENCE OF PH ON BACTERIAL PROTEOMES | IV P A G E | 65 Aim o his s udy: - To iden i y p o eomic al e a ions in esponse o di e se pH condi ions (pH 6.0, pH 7.0, and pH 8.0), label- ee quan i ica ion analyses using bo om-up p o eomics we e conduc ed on h ee HGM species: Bac e oides he aio aomic on, Blau ia p oduc a, and Bi idobac e ium longum. - Compa e p o ein abundances o iden i y p o eins exhibi ing co-abundance in bo h acidic and alkaline esponses, as well as hose exhibi ing speci ic abundance unde acidic o alkaline pH condi ions ela i e o g ow h a pH 7.0. - Iden i y concu en and dis inc p o eomic al e a ions in pa hway- ela ed o s ess-associa ed p o eins among he h ee bac e ial species. - U ilize a op-down p o eomics label- ee quan i ica ion analysis o quan i y p o eo o ms. - Pe o m a compa a i e analysis o bo om-up and op-down quan i a i e esul s o e alua e hei capaci y o quan i y he same p o eins, whe he di e en ially abundan o no . - Conduc a disco e y-based open modi ica ion sea ch o iden i y po en ial pos - ansla ional modi ica ions. IV | INFLUENCE OF PH ON BACTERIAL PROTEOMES P A G E | 66 2 Expe imen al Design 2.1 Bo om-up LFQ Analysis o HGM P o eomes To analyze he p o eomic esponse o cul i a ion a di e en pH alues in B. he aio aomic on, B. p oduc a, and B. longum, he bac e ia we e cul u ed in YCFA medium a acidic (pH 6.0) and neu al (pH 7.0) condi ions in i e biological eplica es. B. longum was addi ionally cul i a ed unde alkaline (pH 8.0) condi ions (FIGURE IV-1, chap e II.2.1). A mid-s a iona y phase, cells we e ha es ed and lysed using eeze- hawing and p o eins we e cleaned up using e hanol p ecipi a ion (chap e II.3.1). In acellula p o eomes we e isola ed, diges ed using ypsin wi h a 1:40 enzyme- o-subs a e a io and subjec ed o solid-phase ex ac ion (chap e II.3.4). All samples we e sepa a ed online by e e sed-phase ch oma og aphy wi h a g adien o 120 minu es and measu ed in iplica e on he Q-Exac i e Plus mass spec ome e (chap e II.4.1). The acqui ed aw da a we e sea ched agains he espec i e e e ence p o eomes using PD 2.5 (chap e II.5.1). Median no maliza ion was used o cen e he da a dis ibu ion and compensa e o gene al concen a ion di e ences ha could be caused by mino a ia ions in sample concen a ion (chap e II. 5.4). The aw and no malized in ensi y da a we e es ed o no mal dis ibu ion and Pea son co ela ion (chap e II. 5.4). S a is ical analysis ( wo-sided Welch's - es wi h Benjamini-Hochbe g FDR co ec ion o mul iple es ing, q ≤ 0.05) was pe o med on high-con idence p o ein iden i ica ions (1% FDR) wi h a leas h ee quan i a i e alues ou o he i e biological eplica es. FIGURE IV-1 | Expe imen al Design o Analyze he pH-Induced P o eomic Response o HGM Bac e ia. Bo om-up p o eomic LFQ analysis o B. he aio aomic on, B. p oduc a, and B. longum cul i a ed in YCFA medium a pH 6.0 and pH 7.0, wi h addi ional cul i a ion o B. longum a pH 8.0. 2.2 Top-down LFQ Analysis o B. p oduc a To pe o m label- ee quan i ica ion o p o eo o ms in B. p oduc a, h ee o he i e biological eplica es g own in YCFA medium unde di e en pH condi ions (pH 6.0 and pH 7.0) we e p ocessed using acidic and basic deple ion o he high molecula -weigh p o eome (FIGURE IV- 2) (Cassidy e al., 2019). This app oach aimed o enhance he iden i ica ion o low-molecula - weigh p o eo o ms. De ails abou he sample p epa a ion a e desc ibed in chap e II.3.2. Samples we e sepa a ed by e e sed-phase ch oma og aphy using a 60 min g adien and 3x LC-MS/MS 120 min In ensi y-based quan. MS1 PD 2.5 Da a analysisHGM membe lysis Diges ion SPE pH 8.0 pH 6.0 pH 7.0 Only B. longum INFLUENCE OF PH ON BACTERIAL PROTEOMES | IV P A G E | 67 analyzed in duplica es on he Fusion Lumos mass spec ome e , employing he mul i-CV FAIMS me hod which ou dis inc CVs (-60, -50, -40 and -20, chap e II.4.2) (Kaulich e al., 2022a). The acqui ed aw da a we e il e ed using F ees yle .1.6 based on he applied CVs, di iding each LC-FAIMS-MS/MS analysis in o ou dis inc aw iles. Subsequen ly, hese iles we e sea ched agains he B. p oduc a e e ence p o eome using PD 2.5 (chap e II.5.1). Quan i ica ion was pe o med u ilizing he high- esolu ion ea u e de ec o node wi h he sliding window decon olu ion algo i hm wi h an a e age e en ion ime wid h o 0.33 min. All esul s we e subjec ed o an FDR co ec ion o bo h P SMs and p o eo o ms, wi h a h eshold o 1%. Iden i ied p o eo o ms we e equi ed o ha e a minimum C-Sco e o 40 (Leduc e al., 2014). Top-down da a p ocessing s eps included CV summa ion, median calcula ion, and median no maliza ion. The aw and no malized in ensi y da a we e es ed o no mal dis ibu ion and Pea son co ela ion (chap e II. 5.4). S a is ical analysis ( wo-sided S uden 's - es wi h pe mu a ion-based FDR co ec ion o mul iple es ing, q ≤ 0.05) equi ed a leas wo quan i a i e alues ou o h ee biological eplica es. FIGURE IV-2 | Expe imen al Design o Top-down LFQ Analysis o he B. p oduc a P o eome. B. p oduc a cul i a ed in YCFA medium a pH 6.0 and pH 7.0 we e subjec ed o acidic and basic HMWP deple ion o op-down label- ee quan i ica ion. 2x LC-MS/MS 60 min In ensi y-based quan. MS1 PD 2.5 Da a analysisB. p oduc a lysis HMWP deple ion pH 6.0 pH 7.0 Mul i CV FAIMS IV | INFLUENCE OF PH ON BACTERIAL PROTEOMES P A G E | 68 3 Resul s 3.1 P o eomic Adap a ions o Acidic and Alkaline pH in B. longum Da a P ocessing – P o eomic changes o B. longum a e cul i a ion a pH 6.0, pH 7.0, and pH 8.0 we e analyzed by BUP-LFQ analysis. A o al o 991 p o eins, co e ing 57% o he encoded B. longum p o eome, we e iden i ied. P o ein abundance p o iles o bo h biological and echnical eplica es (FIGURE A-14), as well as cul u e pH compa isons (pH 7.0/pH 6.0, pH 7.0/pH 8.0, and pH 8.0/pH 6.0), we e no malized o a median alue o ze o (FIGURE IV-3A- B). The da ase s showed low a iabili y, wi h median coe icien s o a ia ion anging om 3.8% o 6.7% (FIGURE IV-3C). S ong conco dance among biological and echnical eplica es is demons a ed by high a e age Pea son co ela ion coe icien s o 0.95 (FIGURE A-14E). P incipal-componen analysis (PCA) e ealed pH-dependen sepa a ion and clus e ing o biological eplica es, wi h he i s wo p incipal componen s accoun ing o 78% o he a ia ion (FIGURE IV-3D). FIGURE IV-3 | E alua ion o Da a P ocessing o he B. longum P o eome. Dis ibu ion o Log2 a ios o (A) aw da a and (B) median no malized da a. Each his og am is o e laid wi h a Gaussian dis ibu ion cu e. The median (M) o he da ase s is displayed in he uppe le co ne o each his og am. (C) Box-and-whiske plo s o he coe icien o a ia ion o he iden i ied p o eomes. Boxes cap u e he lowe and uppe qua iles wi h he median displayed as a ho izon al line in he middle; whiske s ep esen he 1–99 pe cen ile. (D) P incipal componen (PC) analysis wi h each ci cle ep esen s a biological eplica e cul i a ed a he indica ed pH. Quan i a i e da a – This s udy aimed o examine he p o eomic esponse o B. longum o acidic (pH 6.0) and alkaline (pH 8.0) cul u e condi ions by compa ing p o ein abundance a ios pH 7.0 s. pH 6.0 (indica ing he acidic esponse) and pH 7.0 s. pH 8.0 (indica ing he alkaline esponse). A o al o 933 and 935 p o eins we e quan i ied o he acidic and alkaline esponses, espec i ely. Subsequen s a is ical analysis, employing a wo-sided Welch's - es -6 -4 -2 0 2 4 6 0 100 200 300 pH 7.0/pH 6.0 ∑ P o eins Log2 a io M= -0.19 M= 0.00 M= -0.22 -6 -4 -2 0 2 4 6 0 100 200 300 pH 7.0/pH 8.0 ∑ P o eins -6 -4 -2 0 2 4 6 0 100 200 300 pH 8.0/pH 6.0 ∑ P o eins -6 -4 -2 0 2 4 6 0 100 200 300 pH 7.0/pH 6.0 ∑ P o eins Log2 a io M= -0.46 M= 1.36 M= -1.85 -6 -4 -2 0 2 4 6 0 100 200 300 pH 7.0/pH 8.0 ∑ P o eins -6 -4 -2 0 2 4 6 0 100 200 300 pH 8.0/pH 6.0 ∑ P o eins BA C pH 6.0 pH 7.0 pH 8.0 0 5 10 15 20 60 80 Coe icien s o a ia ion (%) D -10 010 20 -10 0 10 20 pH 6.0 pH 7.0 pH 8.0 PC 2 (32% a iance) PC 1 (46% a iance) INFLUENCE OF PH ON BACTERIAL PROTEOMES | IV P A G E | 69 wi h Benjamini-Hochbe g FDR co ec ion (FDR ≤ 0.05) and a Log2 old change h eshold o ±0.485, iden i ied 444 di e en ially abundan p o eins o he acidic esponse and 332 o he alkaline esponse. Changes in p o ein abundance we e de ec ed o p o eins associa ed wi h cellula p ocesses such as p o ein expo , olding, deg ada ion, DNA p o ec ion, and epai , as well as p o eins o he ansla ional machine y such as ibosomal subuni s and aminoacyl- RNA syn he ases (FIGURE IV-4A-B). Addi ionally, se e al p o eins in ol ed in amino acid me abolism, a y acid syn hesis, and pep idoglycan biosyn hesis we e de ec ed as di e en ially abundan (FIGURE IV-4C-D). The acidic p o eomic esponse will be la e desc ibed and discussed (see chap e IV.3.2). The e o e, he ollowing esul s ocus on he alkaline esponse and unc ional classes o p o eins ha had di e en ial abundances in bo h he acidic and alkaline esponse and hose ha had di e en ial abundance a e he acid o alkaline pH compa ed o g ow h a pH 7.0. A pH 8.0, a highe abundance o p o eins in ol ed in me hionine and cys eine biosyn hesis o in e con e sion was obse ed, including homose ine O-ace yl ans e ase (Me A; Q8G7A5), cys a hionine γ-syn hase (Me B; Q8G565), cys a hionine β-syn hase (Cbs; Q8G564), and me hionine syn hase (Me E; Q8G651). FIGURE IV-4 | B. longum Acidic and Alkaline P o eomic Response. (A-B) Volcano plo s o he acidic (pH 7.0/pH 6.0) and (C-D) alkaline (pH 7.0/pH 8.0) esponse. P o eins a e labeled by hei espec i e gene names and a e colo -coded acco ding o hei in ol emen in cellula p ocesses, wi h "PUF" deno ing p o eins o unknown unc ion. Dashed e ical lines ep esen Log2 cu o s, while he dashed ho izon al line co esponds o a q- alue o 0.05 (Two-sided Welch's - es , co ec ed o mul iple es ing by Benjamini-Hochbe g FDR calcula ion). q=0.05 D A q=0.05 Aminoacyl RNA syn he ases P o ein olding and deg ada ion Me hyl ans e ases P o ec ion and epai o DNA Ribosomal subuni s Miscellaneous Cys & Me me abolism Fa y acid biosyn hesis BCAA, Gly, Se and Th me abolism His, Phe, Ty and T p me abolism P o biosyn hesis Pep idoglycan biosyn hesis B q=0.05 q=0.05 C ClpB G oS G pE U D XseA XseB P sH SigH PUF PUF PUF Cah Impa 0 1 2 3 4 5 6 7 8 9 -6 -4 -2 0 2 4 6 -Log p- alue Log2(pH 7.0/pH 6.0) PUFs P o ein expo Th S T mB LepB LigA RpmF RpsL PUF Lacl PUF PUFs Gc H Cah PUF Impa 0 1 2 3 4 5 6 7 8 9 -6 -4 -2 0 2 4 6 -Log10 p- alue Log2(pH 7.0/pH 8.0) Me A Cbs Hu H HisI HisH HisE A oA A oB FabG 0 1 2 3 4 5 6 7 8 9 -6 -4 -2 0246 -Log10 p- alue Log2 a io (pH 7.0/pH 6.0) Cbs Me B Me A MiaB T mB MiaA FabG FabG Z A oA A oE A oG Ty A Z Z Hu H HisH 0 1 2 3 4 5 6 7 8 9 -6 -4 -2 0 2 4 6 -Log10 p- alue Log2(pH 7.0/pH 8.0) IV | INFLUENCE OF PH ON BACTERIAL PROTEOMES P A G E | 70 Addi ionally, wo S-adenosyl-L-me hionine-dependen enzymes, RNA-me hyl ans e ases (T mB; Q8G3T4) and RNA-me hyl hio ans e ase (MiaB; Q8G4H4), we e mo e abundan a pH 8.0. A pH 7.0, p o eins o highe abundance included ibosomal subuni s, p o eins o unknown unc ion (PUFs; Q8G7V3, Q8G449, Q8G6I8, and Q8G6N5), and his idine syn hesis enzymes such as imidazole glyce ol phospha e syn hase (HisH; Q8G4S6), phospho ibosyl- ATP py ophospha ase (HisE; Q8G694), and phospho ibosyl-AMP cyclohyd olase (HisI; Q8G6F6) (FIGURE IV-4). Con e sely, his idine ammonia-lyase (Hu H), which is in ol ed in his idine deg ada ion, and se e al aminoacyl- RNA syn he ases exhibi ed highe abundance a bo h pH 6.0 and pH 8.0 (FIGURE IV-4). By di ec ional analysis (Yang e al., 2014) o he 932 sha ed quan i ied p o eins be ween he acidic and alkaline esponses signi ican a ia ions in p o ein abundance and pa hway changes ac oss di e en pH en i onmen s could be iden i ied. Di e en ial changes in p o ein abundance (p ≤ 0.05) we e ca ego ized in o ou g oups (FIGURE IV-5A). FIGURE IV-5 | Compa ison o he Acidic and Alkaline Response o he B. longum P o eome. (A) Di ec ional analysis wi h di e en ially abundan p o eins colo ed based on hei change in abundance (p ≤ 0.05), using a modi ied Pea son’s co ela ion es (Yang e al., 2014). (B) Di ec ional pa hway analysis on p o eins wi h sha ed abundance a pH 7.0 o (C) sha ed abundance a pH 6.0 and pH 8.0 using COG anno a ions. The numbe and pe cen age o p o eins and he co esponding p- alues o each ca ego y a e shown. -6 -4 -2 0 2 4 6 -4 -2 0 2 4 6 125 110 72 48 23 8.6E-11 1.7E-2 1.7E-2 2.6E-2 5.0E-2 68 119 80 45 51 9.0E-9 4.3E-4 6.1E-4 7.4E-4 2.3E-2 T ansla ion, ibosomal s uc u e and biogenesis Func ion unknown T ansc ip ion Cell wall/memb ane/en elope biogenesis De ense mechanisms 020 40 60 80 100 P o ein g oups (%) p- alue Nucleo ide anspo and me abolism Amino acid anspo and me abolism Ca bohyd a e anspo and me abolism Ene gy p oduc ion and con e sion Coenzyme anspo and me abolism 020 40 60 80 100 P o ein g oups (%) p- alue pH speci ic esponse Opposi e esponse Sha ed espsone pH 7.0 Sha ed espsone pH 6.0 & pH 8.0 B Log2 (pH 7.0/pH 6.0) Log2 (pH 7.0/pH 8.0) C A INFLUENCE OF PH ON BACTERIAL PROTEOMES | IV P A G E | 71 Fi s ly, 65 p o eins (30%) had pH-speci ic esponses, meaning hey had di e en ial changes in abundance only a a speci ic pH condi ion, ei he pH 6.0 o pH 8.0. Second, 14 p o eins (6%) exhibi ed unspeci ic esponses, inc easing hei abundance in one condi ion bu dec easing in ano he . Examples o hese p o eins include cys a hionine β-syn hase (Cbs; Q8G564), ni ogen egula o y p o ein N-II (GlnB; Q8G738), and 30S ibosomal p o ein S12 (RpsL; P59162), which inc eased in abundance unde acidic condi ions and dec eased unde alkaline condi ions. Thi d, 71 p o eins (32%) main ained a consis en inc ease in abundance speci ically a pH 7.0. Las ly, ano he 71 p o eins (32%) exhibi ed a sha ed esponse a bo h pH 6.0 and pH 8.0, main aining a consis en inc ease in abundance unde bo h acidic and alkaline condi ions. The Pea son co ela ion coe icien be ween he acidic and alkaline esponse o 0.59 sugges s a mode a ely complemen a y p o eomic esponse o acidic and alkaline condi ions (FIGURE IV-5A). Di ec ional pa hway analysis on p o eins wi h consis en inc ease in abundance speci ically a pH 7.0 e ealed signi ican en ichmen (p < 0.01) in ansla ional and ansc ip ional p ocesses, as well as cell wall biogenesis, de ense mechanisms, and p o eins o unknown unc ion (FIGURE IV-5B). P o eins wi h a consis en inc ease in abundance a pH 6.0 and pH 8.0 we e en iched in COG ca ego ies in ol ed in he anspo and me abolism o nucleo ides, amino acids, ca bohyd a es, and coenzymes, as well as p o eins associa ed wi h ene gy p oduc ion and con e sion (FIGURE IV-5C). These include 5-enolpy u ylshikima e-3-phospha e (EPSP) syn hase (A oA; Q8G5N6) (TABLE A-3), which is essen ial o ca alyzing he o ma ion o EPSP and ino ganic phospha e om shikima e-3- phospha e and phosphoenolpy u a e in he shikima e pa hway. 3.2 Analysis o he Acidic Response o h ee HGM Membe s Da a P ocessing – A o al o 1,497, 1,772, and 927 p o eins we e iden i ied in he B. he aio aomic on, B. p oduc a, and B. longum da ase s, espec i ely (FIGURE IV-6A-C). An o e iew o he da ase s be o e and a e median no maliza ion and he Pea son co ela ion analysis is p o ided in he Appendix (FIGURE A-15). P o eins quan i ied in h ee ou o i e eplica es o a leas one cul u e condi ion accoun ed o 30% (1449 ou o 4782) o all encoded p o eins o B. he aio aomic on, 31% (1663 o 5365) o B. p oduc a, and 52% (889 o 1725) o B. longum (FIGURE IV-6A-C). FIGURE IV-6 | P o ein Iden i ica ion O e iew. The o al numbe o p o eins encoded in he genome, iden i ied p o eins, iden i ied p o eins, quan i ied p o eins (de ec ed in h ee o i e eplica es) and di e en ially abundan p o eins o (A) B. he aio aomic on, (B) B p oduc a and (C) B longum. 528 1449 1737 4782 Di . abundan Quan i ied Iden i ied To al p o eins 020 40 60 80 100 P o eins il e ed (%) A 502 1663 1772 5365 Di . abundan Quan i ied Iden i ied To al p o eins 020 40 60 80 100 P o eins il e ed (%) B C 446 889 927 1725 Di . abundan Quan i ied Iden i ied To al p o eins 020 40 60 80 100 P o eins il e ed (%) IV | INFLUENCE OF PH ON BACTERIAL PROTEOMES P A G E | 72 S a is ical analysis o he acid pH esponse (pH 7.0 s. pH 6.0) o he h ee bac e ia iden i ied 528, 501, and 446 p o eins as di e en ially abundan o B. he aio aomic on, B. p oduc a, and B. longum, espec i ely (FIGURE IV-6A-C). Quan i a i e Da a – Signi ican p o eomic adap a ions o acidic cul u e condi ions we e obse ed ac oss all HGM bac e ia (FIGURE IV-7A-C). Al hough a FDR o 5% was chosen as he h eshold o s a is ical signi icance, he majo i y o p o eins wi h di e en ial abundance (Log2 old change h eshold o ±0.485) we e iden i ied wi h an FDR o 1% (FIGURE IV-7D). In B. he aio aomic on, an equal numbe o di e en ially abundan p o eins we e de ec ed wi h inc eased abundance a bo h pH 6.0 and pH 7.0 (FIGURE IV-7D). In con as , B. p oduc a and B. longum exhibi ed a highe numbe o p o eins wi h inc eased abundance a pH 6.0 (FIGURE IV-7D). Mos signi ican changes in ol ed p o eins in ol ed in me abolic pa hways such as ca bohyd a e u iliza ion, amino acid biosyn hesis and deg ada ion, pu ine and py imidine biosyn hesis, and pep idoglycan syn hesis. Addi ionally, p o eins in ol ed in cellula espi a ion, ansc ip ional and ansla ional p ocesses, and he p o ec ion and epai o mac omolecules exhibi ed a ia ions in abundance. A summa y o di e en ially abundan p o eins and hei espec i e old changes in he h ee HGM bac e ia is p o ided in TABLE A-4, and explained in de ail in he ollowing sec ions. FIGURE IV-7 | Quan i a i e P o eome Analysis o Human Gu Bac e ia. Volcano plo o all quan i ied p o eins o (A) B. he aio aomic on, (B) B. p oduc a, and (C) B. longum a pH 7.0 s. pH 6.0. The dashed e ical lines ep esen Log2 a io h esholds and he dashed ho izon al lines ep esen a q- alue o 0.05 o 0.01 (Two-sided Welch's - es , Benjamini-Hochbe g FDR co ec ed). (D) The o al numbe o di e en ially abundan p o eins quan i ied wi h inc eased abundance a pH 7.0 and pH 6.0 wi h a q- alue o 0.05 o 0.01. 0 1 2 3 4 5 6 7 8 -6 -4 -2 0 2 4 6 -Log10 p- alue Log2 a io (pH 7.0/pH 6.0) 0 1 2 3 4 5 6 7 8 -6 -4 -2 0 2 4 6 -Log10 p- alue Log2 a io (pH 7.0/pH 6.0) 0 1 2 3 4 5 6 7 8 -6 -4 -2 0 2 4 6 -Log10 p- alue Log2 a io (pH 7.0/pH 6.0) q=0.05 D q=0.01 q=0.05 q=0.01 q=0.05 q=0.01 A CB 273 201 191 255 301 255 -400 -200 0 200 400 B. he aio aomic on B. p oduc a B. longum Numbe o di e en ially abundan p o eins Inc eased pH 7.0 (5% FDR) Inc eased pH 7.0 (1% FDR) Inc eased pH 6.0 (1% FDR) Inc eased pH 6.0 (5% FDR) pH 6.0 pH 7.0 pH 6.0 pH 7.0 pH 6.0 pH 7.0 INFLUENCE OF PH ON BACTERIAL PROTEOMES | IV P A G E | 79 had an inc eased abundance o he cell shape-de e mining p o ein (M eB; A0A2S4GRY8), capsule biosyn hesis p o ein (CapA; A0A7G5MP92) (TABLE A-4), and se e al s age 0 and III spo ula ion p o eins a pH 6.0 (TABLE A-5). CapA acili a es he addi ion o cell su ace capsula polysaccha ide ha can p o ec bac e ia om immune esponses and al e hos physiology (Po e and Ma ens, 2017). While he eichoic acid biosyn hesis p o ein (A0A7G5MS69) was mo e abundan a pH 7.0, he D-alanyl-lipo eichoic acid biosyn hesis p o ein (Dl D; A0A7G5MQG2) inc eased nine old a pH 6.0 (TABLE A-4). Fu he mo e, se e al F s p o eins in ol ed in cell elonga ion, cell cycle con ol, and cell di ision we e mo e abundan a pH 6.0 (TABLE A-4). No ably, he cell di ision p o ein F sL (A0A4P6M6W0) showed he highes change in abundance in he da ase , wi h a 16- old inc ease a pH 6.0. Acidic condi ions ha e been epo ed o educe he cell leng h o E. coli by modula ing he di ision machine y, speci ically he e minal cell di ision p o ein F sN (Muelle e al., 2020). Al hough hese indings o igina e om a G am-nega i e bac e ium and may no di ec ly apply o B. p oduc a, a G am-posi i e bac e ium lacking genomic da a o F sN, s udies on o he G am-posi i e bac e ia like S. au eus and S. pneumoniae, which also lack iden i iable F sN homologs, ha e demons a ed signi ican size al e a ions in esponse o pH a ia ions (Pe ez e al., 2019; Muelle e al., 2020). These obse a ions sugges he hypo hesis ha he cellula mo phology o B. p oduc a migh also change in esponse o pH a ia ions. Howe e , ini ial elec on mic oscope expe imen s conduc ed by Ka h in Schä e (Depa men o In ec ious Diseases and Mic obiology, Uni e si y o Lübeck, UKSH Lübeck; chai ed by P o . D . Jan Rupp) on B. p oduc a in esponse o a ious cul u e pH alues did no e eal signi ican changes in cell mo phology. Al hough hese p elimina y indings do no conclusi ely ule ou he possibili y ha he cellula mo phology o B. p oduc a changes unde hese speci ic condi ions, u he esea ch is equi ed o explo e his hypo hesis. 3.3 Compa ison o LMWP-Top-Down and Full-P o eome Bo om-Up Analysis o B. p oduc a Da a P ocessing – Top-down p o eomic analysis o acidic and basic HMWP deple ions o B. p oduc a iden i ied 923 and 818 p o eo o ms (FIGURE IV-11A), co esponding o 211 and 190 p o ein g oups (FIGURE IV-11B), espec i ely. The majo i y o iden i ied p o eo o ms we e be ween 2-10 kDa in molecula weigh and we e unca ed e sions o he canonical p o eins (FIGURE IV-11C and D). N- e minal unca ion accoun ed o 31% ± 4% o p o eo o ms, excluding N- e minal me hionine excisions (NME) (FIGURE IV-11B and D). Addi ionally, C- e minal unca ion ep esen ed 28% ± 3%, while in e nal p o eo o ms esul ing om bo h N- and C- e minal unca ion con ibu ed an addi ional 30 ± 8%. This le 12% o iden i ied IV | INFLUENCE OF PH ON BACTERIAL PROTEOMES P A G E | 80 p o eo o ms as ull-leng h canonical p o eins, wi h 66% ± 3% e aining hei N- e minal me hionine and 34% ± 3% lacking i (FIGURE IV-11E and F). In e es ingly, only 404 and 356 p o eo o ms had a leas one quan i a ion alue a e acidic and basic HMWP deple ion, espec i ely (FIGURE IV-11A). Bo h da ase s had a high pe cen age o missing alues, wi h 38% and 42% missing alues a e acidic and basic HMWP deple ion, espec i ely. A e da a p ocessing s eps including CV summa ion, median no maliza ion, and il e ing o a leas wo quan i a i e alues pe pH condi ion, he da ase s we e educed o 175 p o eo o ms a e acidic and 135 a e basic deple ion, lea ing many p o eo o ms unquan i ied (FIGURE IV-11A). FIGURE IV-11 | Iden i ied P o eo o ms using he Acidic and Basic HMWP Deple ion Me hod. Numbe o iden i ied, quan i ied, and di e en ially abundan (A) p o eo o ms and (B) p o ein g oups. Dis ibu ion o iden i ied neo- e mini by p o eo o m size o (C) he acidic and (D) basic HMWP deple ion me hod, and dis ibu ion by pe cen age o (E) he acidic and (F) basic HMWP deple ion me hod. An o e iew o he da a dis ibu ion be o e and a e median no maliza ion and Pea son co ela ion analysis o biological and echnical eplica es is p o ided in he Appendix (FIGURE A-16 and FIGURE A-17). S a is ical analysis ( wo-sided S uden 's - es , pe mu a ion- based FDR co ec ion, FDR ≤ 0.05, and a Log2 old change o ±0.485) iden i ied 26 and 30 di e en ially abundan p o eo o ms in acidic and basic HMWP deple ion expe imen s, espec i ely (FIGURE IV-11A and TABLE A-6). In o al, 21 p o eins we e di e en ially abundan in he acidic HMWP deple ion and 24 p o eins in he basic HMWP deple ion (FIGURE IV-11B). No ably, six p o eins had di e en ial abundance changes in bo h HMWP deple ions, wi h simila abundance changes obse ed o iden ical p o eo o ms o unca ed p o eo o ms o he same p o ein (TABLE A-7). Quan i a i e da a – The ollowing analysis ocused on compa ing p o eo o m-based quan i ica ions om bo h acidic and basic HMWP deple ion me hods wi h he ull-p o eome pep ide-based BUP analysis. While bo h p o eomics-based quan i ica ion echniques o e 923 402 175 26 818 350 135 30 211 144 91 21 190 155 78 24 In e n 37% C- e m 25%N- e m 27% > In ac excised 63% excised > In ac 69% 37% 31% NME: NME: Iden i ied A leas one quan i ica ion alue Quan i ied Di e en ially abundan 0 200 400 600 800 1000 ∑ P o eo o ms Acidic deple ion Basic deple ion A Iden i ied A leas one quan i ica ion alue Quan i ied Di e en ially abundan 0 50 100 150 200 250 ∑ P o ein g oups B 2 4 6 8 10 12 14 16 18 20 0 50 100 150 200 ∑ P o eo o ms Theo. mass (kDa) C E D F Canonical 12% C- e m 31% In e n 22% N- e m 35% 11% 2 4 6 8 10 12 14 16 18 20 0 50 100 150 200 C-Te m N-Te m In e nal ∑ P o eo o ms Theo. mass (kDa) INFLUENCE OF PH ON BACTERIAL PROTEOMES | IV P A G E | 81 aluable insigh s, hey ha e di e en capabili ies and limi a ions. The TDP analysis in his s udy p ima ily ocused on iden i ying smalle p o eins (<30 kDa), whe eas he applied BUP analysis a ge ed he en i e p o eome, hus quan i ying a b oade ange o p o eins, including la ge ones, bu i does no iden i y p o eo o ms. De ailed a ia ions in quan i ica ion, encompassing coun s o p o eins and p o eo o ms, a e de ailed in TABLE A-8 and summa ized o bo h HMWP dele ion analysis in TABLE IV-1. TABLE IV-1 | Quan i a i e Resul s o Top-down P o eomics and Bo om-up P o eomics. Compa ison o p o eo o m- and p o ein-le el quan i a i e da a acqui ed by ull p o eome LFQ bo om- up and acidic and basic HMWP deple ion op-down LFQ analysis. TOP-DOWN QUANTITATIVE RESULTS DIFFERENTIALLY ABUNDANT NOT DIFFERENTIALLY ABUNDANT NOT QUANTIFIED B OTTOM - UP DIFFERENTIALLY ABUNDANT 13 p o eins 27 p o eins 469 p o eins 16 p o eo o ms 42 p o eo o ms N/A NOT DIFFERENTIALLY ABUNDANT 22 p o eins 67 p o eins 1,089 p o eins 33 p o eo o ms 147 p o eo o ms N/A NOT QUANTIFIED 3 p o eins 20 p o eins 3 p o eo o ms 33 p o eo o ms While 1,558 p o eins we e quan i ied in he BUP analysis bu no in he TDP analysis, he TDP uniquely quan i ied a o al o 36 p o eo o ms om 23 p o eins ha we e absen in he BUP analysis (TABLE IV-1). The majo i y o hese (33 p o eo o ms om 20 p o eins) we e no di e en ially abundan in he TDP analysis (TABLE IV-1). Howe e , h ee p o eo o ms om h ee di e en p o eins, an uncha ac e ized p o ein (A0A7G5MR36), an acyl ca ie p o ein (A0A7G5MSW4), and a ca bohyd a e ABC anspo e subs a e-binding p o ein (A0A7G5MNS4), showed di e en ial abundance in he TDP analysis (TABLE IV-1). Fu he di e ences be ween he wo quan i ica ion me hods included 27 p o eins ( ep esen ed by 42 p o eo o ms) which we e de ec ed as di e en ially abundan in BUP da ase bu in he TDP da ase (TABLE IV-1). Con e sely, 22 p o eins ( ep esen ed by 33 p o eo o ms) we e di e en ially abundan in he TDP da a bu no in he BUP da a. These included p o eo o ms o ecombina ion-p omo ing nuclease RpnA (RpnA; A0A7G5MZ14) and 50S ibosomal p o ein L29 (RpmC; A0A4P6LZK9), bo h o highe abundance a pH 7.0, and p o eo o ms o DNA- binding p o ein HU (Hup; A0A2S4GGS2) and ibosomal p o ein S16 (RpsP; A0A4P6M2Y5), which we e mo e abundan a pH 6.0 (FIGURE IV-12). No ably, all di e en ially abundan p o eo o ms o DNA-binding p o ein HU p o eo o ms exhibi ed a canonical N- e minal (TABLE A-6), which has been epo ed o be ele an o o ming a DNA–p o ein complex (Alma za e al., 2015). This complex p o ec s DNA om endonucleoly ic clea age and oxida i e s ess damage unde acidic pH (Alma za e al., 2015). Fo 67 p o eins, bo h quan i ica ion me hods de ec ed no signi ican di e ence be ween pH condi ions, while 13 p o eins we e classi ied as IV | INFLUENCE OF PH ON BACTERIAL PROTEOMES P A G E | 82 di e en ially abundan by bo h TDP and BUP (TABLE IV-1). O hese 13 p o eins, each TDP deple ion me hod uniquely quan i ied 5 p o eins, wi h 3 p o eins showing di e en ial abundance in bo h deple ion analyses, including he NlpC/P60 domain-con aining p o ein (NlpC; A0A7G5N0P5), D-alanyl-lipo eichoic acid biosyn hesis (Dl D; A0A7G5MQG2), and a Gn R amily ansc ip ional egula o (Gn R; A0A7G5MZW5) (FIGURE IV-12 and TABLE IV-2). No ably, bo h he BUP and he wo TDP p o eomics-based quan i ica ions e ealed iden ical abundance changes, wi h Gn R being mo e abundan a pH 7.0 and NlpC and Dl D being mo e abundan a pH 6.0 (FIGURE IV-12 and TABLE IV-2). No ably, e en he di e en p o eo o ms o Gn R exhibi ed consis en changes in abundance (TABLE IV-2). Mo eo e , se e al p o eo o ms, including he in e nal 36-amino acid-long p o eo o m o Dl D and he N- e minal unca ed 44- amino acid-long p o eo o m o Gn R, we e quan i ied as di e en ially abundan in bo h TDP da ase s (TABLE A-7). FIGURE IV-12 | Compa ison Top-down and Bo om-up Label- ee-quan i ica ion. (A) Bo om-up label- ee quan i a i e analysis. (B) Top-down label- ee quan i a i e analysis om acidic deple ion and (C) om basic deple ion. Highligh ed p o eins: D-alanyl-lipo eichoic acid biosyn hesis (Dl D; A0A7G5MQG2), Gn R amily ansc ip ional egula o (Gn R; A0A7G5MZW5), DNA-binding p o ein HU (Hup; A0A2S4GGS2), NlpC/P60 domain-con aining p o ein (NlpC; A0A7G5N0P5), ecombina ion- p omo ing nuclease RpnA (RpnA; A0A7G5MZ14), 50S ibosomal p o ein L29 (RpmC; A0A4P6LZK9) and 30S ibosomal p o ein S16 (RpsP; A0A4P6M2Y5). Gn R RpnA RpmC RpmC Hup Hup NlpC RpsP Dl D Hup Hup RpsP 0 1 2 3 4 -9 -6 -3 0 3 6 9 -Log10 p- alue Log2(pH 7.0/pH 6.0) ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! RpmC ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! RpnA ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D NlpC D D D D D D D D D D D D D D D D D Dl D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D Gn R D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D 0 1 2 3 4 5 6 7 8 -9 -6 -3 0 3 6 9 -Log10 p- alue Log2(pH 7.0/pH 6.0) RpsP Hup q=0.05 A RpmC Gn R RpmC RpnA Gn R Gn R NlpC Dl D 0 1 2 3 4 -9 -6 -3 0 3 6 9 -Log10 p- alue Log2(pH 7.0/pH 6.0) q=0.05 q=0.05 pH 7.0pH 6.0 B C INFLUENCE OF PH ON BACTERIAL PROTEOMES | IV P A G E | 83 TABLE IV-2 | O e lap o Di e en ially Abundan P o eins be ween Bo om-up and Top-down P o eome Analyses. Top-down Log2 a ios ep esen quan i ied p o eo o ms, wi h mul iple alues co esponding o di e en quan i ied p o eo o ms. Log2 a io (pH 7.0/pH 6.0) PROTEIN NAME ACCESSION TDP BUP ACIDIC BASIC NlpC/P60 domain-con aining p o ein A0A7G5N0P5 -6.4 -6.8 -1.3 D-alanyl-lipo eichoic acid biosyn hesis A0A7G5MQG2 -2.5 -1.9 -3.2 Gn R amily ansc ip ional egula o A0A7G5MZW5 2.3 3.9, 2.3, 1.9 1.6 ABC anspo e subs a e-binding A0A7G5MSE0 -2.9 - -0.5 Uncha ac e ized p o ein A0A7G5N1D2 -2.8 - 1.2 50S ibosomal p o ein L19 A0A4P6M0Y8 -1.7 - 0.8 XRE amily ansc ip ional egula o A0A7G5N1G9 -1.7 - 1.0 Uncha ac e ized p o ein A0A7G5N1C6 2.7 - 3.5 DUF1002 domain-con aining p o ein A0A7G5MNQ5 - -6.0 1.3 Recombinase RecT A0A7G5N1F5 - 0.9 -0.8 NADH pe oxidase A0A4P6LUJ8 - 1.0 0.9 Uncha ac e ized p o ein A0A7G5N144 - 1.8 2.3 DUF3502 domain-con aining p o ein A0A7G5MQI1 - 7.8 0.8 FIGURE IV-13 | Quan i ica ion o he C- e minal Region o he Gn R amily ansc ip ional egula o (A0A7G5MZW5). Dis ibu ion o iden i ied (A) pep ides and (B) p o eo o ms wi h highligh ed Log2 a ios (pH 7.0/pH 6.0). (C) NCBI-CDD and Alpha old s uc u e p edic ion e ealed an N- e minal HTH (helix- u n-helix) DNA-binding domain and C- e minal e ec o -binding and oligome iza ion (E-O) domain. 020 40 60 80 100 120 [R].IT...VR.[D] [R].DL...QK.[A] [K].AL...ER.[T] [R].TN...QR.[T] [R].FI...LK.[T] [K].TE...EK.[I] [K].TE...DK.[M] [K].IG...KK.[V] [K].IG...IK.[K] [K].ED...KK.[V] Iden i ied pep ides 3.93 2.27 & 2.29 1.85 020 40 60 80 100 120 [M].SW...KK.[-] [L].VY...KK.[-] [L].NM...KK.[-] [N].MI...KK.[-] [N].MI...EE.[K] [N].MI...EE.[E] [N].MI...SE.[E] [N].MI...GL.[S] [N].MI...IG.[L] [M].ID...KK.[-] [I].DN...KK.[-] [D].NL...KK.[-] [N].LK...KK.[-] [L].KT...KK.[-] [T].EL...KK.[-] [L].AS...KK.[-] [G].LS...KK.[-] Sequence leng h Iden i ied p o eo o ms HTH Domain NC E-O Domain N- e minal DNA-binding domain C- e minal e ec o -binding and oligome iza ion domain C A 1.6 1.0 1.6 1.8 1.4 1.4 B IV | INFLUENCE OF PH ON BACTERIAL PROTEOMES P A G E | 84 3.4 Po en ial pH-induced Asp-P o Clea age The analysis o neo- e mini in TDP da ase s e ealed mul iple p o eo o ms o he ibosomal p o ein S16 (A0A4P6M2Y5) wi h N- o C- e minal Asp-P o pep ide bond clea ages. Fu he sequence analysis o ibosomal p o ein S16 e ealed wo Asp-P o bonds wi hin a loop s uc u e, ha sepa a es he p ominen N- e minal β-shee s om he C- e minal α-helices (FIGURE IV-14A-B). O e all, i e p o eo o ms exhibi ing an Asp-P o clea age we e iden i ied (FIGURE IV-14C), wo o which we e di e en ially highe abundan a pH 6.0 in he acid HMWP deple ion TDP analysis (FIGURE IV-12B). Al hough he basic HMWP deple ion TDP analysis also de ec ed hese wo p o eo o ms, quan i a i e da a could no be acqui ed. A e-analysis o he BUP da a, inco po a ing semi- yp ic pep ides, iden i ied se e al pep ides wi h he same Asp-P o clea age, mos o which we e o highe abundance a pH 6.0 (FIGURE IV-14D). Fu he subcellula localiza ion p edic ions using Phobius sugges ed ha he esul ing p o eo o ms con aining C- e minal α-helices may be di ec ed ou side he cy oplasmic space (TABLE IV-3). Addi ionally, p edic ions o an imic obial pep ide (AMP) ac i i y using AMP un sugges ed ha hese p o eo o ms exhibi ac i i y agains bo h G am-posi i e and G am- nega i e bac e ia (TABLE IV-3). FIGURE IV-14 | Asp-P o Clea age in 30S Ribosomal P o ein S16 (A0A4P6M2Y5). (A) The p o ein sequence exhibi s wo Asp-P o clea age si es ( esidues 41-42 and 45-46). (B) The s uc u e p edic ion highligh s he posi ion o he Asp-P o pep ide bonds si ua ed in a s uc u al loop. (C) Iden i ied p o eo o ms and (D) iden i ied pep ides wi h highligh ed Log2 old changes (pH 7.0/pH 6.0). -0.48 -0.9 -0.7 -1.37 -1.05 -0.50 -0.14 -0.19 -0.22 , -1.04 -0.62 -0.27 -0.10 0.00 , -0.54 , 1.22 -0.38 -1.13 -1.23 -0.54 -1.46 -0.97 -0.31 -0.28 -0.26 -0.76 -1.30 -0.43 -0.84 -0.92 -0.46 -0.91 010 20 30 40 50 60 70 80 [R].MG...YR.[I] [K].KA...YR.[I] [R].II...SR.[S] [R].II...PR.[D] [R].DG...YD.[P] [R].DG...QD.[P] [R].DG...VY.[K] [R].DG...YK.[V] [R].DG...AK.[K] [G].KF...YK.[V] [K].FI...QD.[P] [K].FI...VY.[K] [K].FI...YK.[V] [K].FI...AK.[K] [K].FI...KK.[W] [I].GT...KK.[W] [G].TY...AK.[K] [G].TY...KK.[W] [Y].DP...AK.[K] [D].PN...YK.[V] [D].PN...AK.[K] [D].PN...KK.[W] [Q].DP...KK.[W] [D].PS...AK.[K] [D].PS...KK.[W] [K].VD...SK.[I] [K].KW...SK.[I] [K].WL...SK.[I] [W].LA...SK.[I] [L].AN...SK.[I] [A].NG...SK.[I] [N].GA...SK.[I] [G].AQ...SK.[I] [Q].PT...SK.[I] [K].IF...EK.[-] Sequence leng h Iden i ied pep ides 010 20 30 40 50 60 70 80 [-].MA...NQ.[D] [M].AV...EK.[-] [M].AV...VD.[E] [M].AV...VY.[K] [M].AV...QD.[P] [M].AV...NQ.[D] [M].AV...PN.[Q] [M].AV...YD.[P] [M].AV...TY.[D] [M].AV...GT.[Y] [M].AV...IG.[T] [M].AV...RD.[G] [M].AV...AD.[S] [M].GQ...EK.[-] [G].QK...EK.[-] [G].QK...QD.[P] [Q].KK...EK.[-] [K].KA...EK.[-] [D].SR...EK.[-] [R].DG...EK.[-] [D].GK...EK.[-] [G].KF...EK.[-] [G].TY...EK.[-] [D].PN...EK.[-] [D].PS...EK.[-] [Y].KV...EK.[-] [D].EE...EK.[-] [K].KW...EK.[-] [K].WL...EK.[-] Sequence leng h Iden i ied p o eo o ms 30S ibosomal p o ein S16(A0A4P6M2Y5) 1 MAVKIRLRRM GQKKAPFYRI IVADSRSPRD GKFIEEIGTY DPNQDPSVYK jjVDEEAAKKWL ANGAQPTEVV SKIFKAAGIE K 81 Clea age si es A C D N C B -1.54 -2.02 Loop INFLUENCE OF PH ON BACTERIAL PROTEOMES | IV P A G E | 85 To in es iga e he p edic ed AMP ac i i y, i e syn he ic pep ides, each consis ing o 22 amino acids, we e designed o co e bo h he N- and C- e minal Asp-P o clea age si es (TABLE A-9). Ini ial es ing o he po en ial AMP ac i i y was conduc ed a concen a ions anging om 0.01 o 1 µM agains a ious membe s o he human gu mic obio a (B. he aio aomic on, B. longum, A. caccae, C. buy icum, C. amosum and L. plan um). These expe imen s we e pe o med by Ka h in Schä e (Depa men o In ec ious Diseases and Mic obiology, Uni e si y o Lübeck, UKSH Lübeck; chai ed by P o . D . Jan Rupp) and did no e eal signi ican AMP ac i i y. TABLE IV-3 | Sequence and S uc u e P edic ions o 30S Ribosomal P o ein S16. Fu he in es iga ion was ocused on e alua ing he possibili y o a i icially gene a ed Asp-P o bond hyd olysis du ing sample p epa a ion, LC-MS measu emen , and hei po en ial o igin om acidic cul i a ion. Analysis o he amino acid equencies su ounding non- yp ic clea age si es in he BUP da ase s e ealed dis inc sequence logos among di e en bac e ial species (FIGURE IV-15). Speci ically, B. he aio aomic on and B. p oduc a showed an inc eased ela i e equency o Asp a he P1 posi ion and P o a he P1´ posi ion (FIGURE IV-15A). In e es ingly, his equency inc eased o pep ides iden i ied a pH 6.0, pa icula ly o B. he aio aomic on, sugges ing ha acidic condi ions may p omo e he po en ial hyd olysis o Asp-P o bonds. In con as , B. longum exhibi ed a signi ican ly inc eased ela i e equency only o P o a he P1´ posi ion. Fu he analysis ocusing on pep ides wi h Asp a he P1 posi ion (FIGURE IV-15C) o P o a he P1´ posi ion (FIGURE IV-15D) con i med he p esence o pep ides wi h Asp-P o clea age and also indica ed hei occu ence o B. longum. S EQUENCE FRAGMENT (#AA) AMP (P REDICTION ) AMP T ARGET (P REDICTION) L OCALIZATION (P REDICTION) S TRUCTURE P REDICTION Fi s DP mo i clea ed [M].AV...YD.[P] N - e m ( 40) AMP (0.757) G am -posi i e (0.550), G am -nega i e (0.578) Cy oplasmic ( 0.775) [D].PN...EK.[ -] C - e m (40) AMP (0.702) G am -nega i e (0.586) Non -cy oplasmic ( 0.681) Second DP mo i clea ed [M].AV...QD.[P] N -Te m ( 44) AMP (0.7181) G am -nega i e (0.527) Cy oplasmic ( 0.774) [D].PS...EK.[ -] C -Te m (36) AMP (0.788) G am -posi i e (0.575), G am -nega i e (0.566) Non -cy oplasmic ( 0.685) IV | INFLUENCE OF PH ON BACTERIAL PROTEOMES P A G E | 86 FIGURE IV-15 | Bo om-up Pep ide Clea age Analysis Highligh s Asp-P o Sequence Logos. IceLogo plo illus a ing he ela i e equency o P2-P2′ amino acids o iden i ied pep ides o B. he aio aomic on, B. p oduc a and B. longum. (A) Iden i ied pep ides a pH 7.0 and (B) a pH 6.0. (C) Iden i ied pep ides wi h Asp a he P1 posi ion and (D) wi h P o a he P1´ posi ion. Amino acids ha a e signi ican ly en iched ( op) o deple ed (bo om) (p ≤ 0.05) a e colo ed black, wi h Asp and P o esidues highligh ed in ed. Mo eo e , signi ican ly highe abundance a pH 6.0 was obse ed o pep ides wi h clea ed N- o C- e minal Asp-P o and Asp-Xaa bonds in bo h B. he aio aomic on (FIGURE IV-16A) and B. p oduc a (FIGURE IV-16B). Con e sely, pep ides wi h in ac Asp-P o showed no signi ican changes in abundance and we e dis ibu ed a ound ze o (FIGURE IV-16A-B). Addi ionally, a pH 7.0, pep ides wi h clea ed N- o C- e minal Asp-Xaa bonds exhibi ed signi ican ly highe abundance in B. longum (FIGURE IV-16C). FIGURE IV-16 | Pep ide Abundance Dis ibu ion. Dis ibu ion o all iden i ied pep ides, semi yp ic pep ides (excluding hose wi h in ac Asp-P o, clea ed Asp-P o and Asp-Xaa bonds), pep ides wi h in ac Asp-P o bonds, pep ides wi h clea ed Asp-P o and clea ed Asp-Xaa bonds. (A) B. he aio aomic on, (B) B p oduc a, and (C) B longum. Signi ican di e ences we e calcula ed ia one- way ANOVA wi h Dunne ’s co ec ion. * (p < 0.05); ** (p < 0.01); **** (p < 0.0001). The numbe on he op indica es he numbe o iden i ied pep ides. P2 P1 P1' P2' % Di e ence -45 -22.5 22.5 45 ∑ Analyzed si es = 1910 P2 P1 P1'P2' % Di e ence -30 -15 15 30 ∑ Analyzed si es = 2778 A P2 P1 P1'P2' % Di e ence -45 -22.5 22.5 45 ∑ Analyzed si es = 2088 pH 7.0 pH 7.0 pH 7.0 B. longum B. he aio aomic on B. p oduc a P2 P1 P1' P2' % Di e ence -100 -50 50 100 ∑ Analyzed si es = 377 C P2 P1 P1' P2' % Di e ence -100 -50 50 100 ∑ Analyzed si es = 220 P2 P1 P1' P2' % Di e ence -100 -50 50 100 ∑ Analyzed si es = 131 Asp P1 Asp P1 Asp P1 B. longum B. he aio aomic on B. p oduc a P2 P1 P1' P2' % Di e ence -45 -22.5 22.5 45 ∑ Analyzed si es = 1924 P2 P1 P1'P2' % Di e ence -30 -15 15 30 ∑ Analyzed si es = 2795 B P2 P1 P1'P2' % Di e ence -45 -22.5 22.5 45 ∑ Analyzed si es = 2500 pH 6.0 pH 6.0 pH 6.0 B. longum B. he aio aomic on B. p oduc a P2 P1 P1' P2' % Di e ence -100 -50 50 100 ∑ Analyzed si es = 223 D P2 P1 P1' P2' % Di e ence -100 -50 50 100 ∑ Analyzed si es = 172 P2 P1 P1' P2' % Di e ence -100 -50 50 100 ∑ Analyzed si es = 169 P o P1' P o P1' P o P1' B. longum B. he aio aomic on B. p oduc a 25490 1976 752 127 257 -3 -2 -1 0 1 2 3 4 Log2 (pH 7.0/pH 6.0) * **** A ** 18164 2126 574 101 99 -3 -2 -1 0 1 2 3 4 Log2 (pH 7.0/pH 6.0) **** B **** **** ** **** 9247 1753 457 58 29 -3 -2 -1 0 1 2 3 4 Asp-Xaa bonds clea ed Asp-P o bonds clea ed All iden i ied pep ides Asp-P o bonds in ac Semi yp ic pep ides Log2 (pH 7.0/pH 6.0) C INFLUENCE OF PH ON BACTERIAL PROTEOMES | IV P A G E | 87 3.5 Phospho yla ion o HP p o eins An open-modi ica ion sea ch o he TDP da ase iden i ied se e al po en ial se yl- phospho yla ions on his idine-con aining phosphoca ie p o eins (HP ). O e all, a high numbe o ma ching agmen ions co e ing he phospho yla ion si e, along wi h a ma ching dis ibu ion o heo e ical and expe imen ally obse ed peaks o unmodi ied and phospho yla ed HP p o eo o ms, was obse ed (FIGURE IV-17 and FIGURE A-19A-D). Using P oSigh Anno a o , po en ial phospho yla ion si es we e inco po a ed in o HP p o ein en ies, acili a ing a a iable sea ch o HP phospho yla ion. Combined wi h an addi ional da abase sea ch o he bo om-up p o eomic da a ha included phospho yla ion as a a iable modi ica ion a se ine, his idine, h eonine, y osine, and a ginine esidues, se e al phospho yla ed pep ides and p o eo o ms we e iden i ied. These indings sugges ed Se -41 phospho yla ion o HP (A0A2S4GRU0 and A0A7G5MYS7), Se -46 phospho yla ion o HP (A0A4V0Z7D2), and A g-46 phospho yla ion o HP (A0A7G5MP53) (FIGURE IV-18). Al hough pep ides spanning he espec i e phospho yla ion si es o HP (A0A7G5MP53 and A0A4V0Z7D2) we e de ec ed by he bo om-up p o eomics analysis, phosphopep ides could only be iden i ied o wo HP a ian s (A0A2S4GRU0 and A0A7G5MYS7) (FIGURE A-19E-H). FIGURE IV-17 | Phosphose ine Modi ica ion o HP (A0A2S4GRU0). Dis ibu ion o expe imen ally obse ed and heo e ical peaks o iden i ied p o eo o ms o (A) unmodi ied HP and (B) phospho yla ed HP . (C) Iden i ied p o eo o m sequence and agmen ion spec um, highligh ing he de ec ed phospho yla ion si e a Se -41 in yellow. The iden i ied b- and y-ions a e highligh ed in blue and ed, espec i ely. Do plo illus a es he mass e o in ppm o each obse ed b- and y-ion, espec i ely. E o (ppm) Rela i e Abundance (%) m/z A B C IV | INFLUENCE OF PH ON BACTERIAL PROTEOMES P A G E | 88 FIGURE IV-18 | Sequence Alignmen o HP P o eins Highligh ing P oposed Phospho yla ion Si es. Posi ions o he wo conse ed amino acid esidues: a his idine (His-15) nea he N- e minus and a se ine (Se -46) in he cen al pa o he p o ein, a e highligh ed. The quan i a i e da a o he BUP analysis e ealed a di e en ial abundance o HP (A0A2S4GRU0) a pH 6.0. Mo eo e , analysis o pep ide-le el abundance e ealed a highe abundance o phosphopep ides a pH 6.0, while unmodi ied pep ides we e mo e abundan a pH 7.0 (FIGURE IV-19A-B). S a is ical analysis o he TDP da a, which inco po a ed he a iable HP phospho yla ion si es, quan i ied se e al phospho yla ed p o eo o ms as di e en ially abundan a pH 6.0, bo h by he acidic (FIGURE IV-19C) and he basic HMWP deple ion (FIGURE IV-19D). Con e sely, unmodi ied o N- e minal o myla ed p o eo o ms did no exhibi di e en ial abundance (FIGURE IV-19C-D). O e all, he obse ed inc ease in he abundance o phospho yla ed pep ides and p o eo o ms o HP (A0A2S4GRU0) a pH 6.0 sugges s ha HP phospho yla ion may ep esen a di ec esponse o cul u e acidi ica ion. FIGURE IV-19 | Quan i ica ion o Phospho yla ion on HP (A0A2S4GRU0). (A) Scaled pep ide abundance a pH 6.0 and pH 7.0. Pep ides wi h se ine phospho yla ion (P) o me hionine oxida ion (O) a e highligh ed. One boxplo is absen as he phosphopep ide was exclusi ely iden i ied a pH 6.0. P o eo o m-di ec ed label- ee analysis om (B) acidic and (C) basic deple ion. Phospho yla ed HP p o eo o ms wi h di e en ial abundance a pH 6.0 a e highligh ed in yellow (Two-sided S uden 's - es , Pe mu a ion-based FDR co ec ed, q- alue o 0.05). Iden i ied using A0A4P6M732 8 -VNNLIGLHLRPAG-20 42-ANAKSVLSV-50 A0A7G5MP53 8 -ITDPEGIHARPAG—20 42-GDCKRIFGI-50 Top -down p o eomics A0A4V0Z7D2 8 -IGISNGLEARPIA—20 42-VNAKSIMGM-50 Top -down p o eomics A0A7G5MYS7 8 -LNET-----GDVK—20 37-IDAKSILGV-45 Top -down & Bo om- up p o eomics A0A2S4GRU0 8 -LNSI-----DKVK—20 37-IDAKSIMGI-45 Top -down & Bo om- up p o eomics A0A4P6LXB3 8 -FSEI-----NEIK—20 37-IDAKSILGM-45 A0A4P6LTJ7 7 -FKEV-----DEIV—19 36-VDAKSIMGM-44 P P ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Unmodi ied N-Fo myl HP (Se -P) ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! HP (Se -P) 0 1 2 3 4 -9 -6 -3 0 3 6 9 -Log10 p- alue q=0.05 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Unmodi ied N-Fo myl HP (Se -P) ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! HP (Se -P) HP (Se -P) 0 1 2 3 4 -9 -6 -3 0 3 6 9 -Log10 p- alue q=0.05 [K].IS...VK.[S] [K].IS...DK.[V] [K].SF...GR.[Y] [K].SF...AK.[S] [K].SF...TK.[F] [K].FD...GR.[Y] [R].YV...SK.[A] [K].SI...SK.[A] [K].SI...SK.[A] [K].SI...SK.[A] [K].SI...SK.[A] [K].AI...VD.[-] 0 1 2 3 1 >50 PSMs 40 30 20 10 Scaled abundance pH 6.0 pH 7.0 A B0 20 40 60 80 O P Sequence posi ion P P OO D C Log2(pH 7.0/pH 6.0) Log2(pH 7.0/pH 6.0) INFLUENCE OF PH ON BACTERIAL PROTEOMES | IV P A G E | 95 syn hesis o his idine, which consumes o he amino acids and me aboli es, can be disad an ageous du ing acid s ess condi ions. The e o e, i can be hypo hesized ha B. p oduc a also possesses a his idine up ake sys em o assimila e ex acellula his idine, o a oid he high ene gy cos associa ed wi h amino acid biosyn hesis. Al hough his idine up ake in B. p oduc a has no been s udied o my knowledge, s udies in o he G am-posi i e bac e ia sugges he adap a ion o common ABC anspo e s o his idine up ake (Vi eschak e al., 2008). To expe imen ally alida e he impo ance o his idine biosyn hesis o he up ake o his idine in he acid s ess esponse o B. p oduc a, se e al ollow-up expe imen s can be pe o med. Fo ins ance, ansposon sequencing can be used o compa e he bac e ial i ness o wild- ype B. p oduc a agains a single-gene dis up ion lib a y g own in media wi h and wi hou his idine supplemen a ion unde acid s ess. This app oach can iden i y genes equi ed o de imen al o he g ow h o B. p oduc a unde acid s ess condi ions. While single knock- ou s udies in E. coli sugges ed ha mos amino acid anspo and me abolism genes, including his idine biosyn he ic genes, a e non-essen ial (Baba e al., 2006), ansposon sequencing s udies in S. au eus e ealed a c ucial gene (SAUSA300_0846) encoding a his idine anspo e (Bee ham e al., 2024). The his idine anspo e unc ion was con i med by measu ing he up ake o adio-labeled his idine in bo h wild- ype S. au eus and he mu an s ain, whe e his idine up ake was la gely los in he mu an . In he absence o exogenous his idine, he wild- ype s ain exhibi ed an addi ional 5-hou lag phase, indica ing i s eliance on his idine o g ow h and i s abili y o adap and syn hesize his idine. G ow h expe imen s conduc ed a pH 4.3 and pH 7.2, wi h o wi hou his idine, con i med he impo ance o his idine anspo ia he SAUSA300_0846 anspo e o S. au eus g ow h a acidic pH. No ably, he mu an s ain exhibi ed a mo e han 200- old inc ease in he exp ession o his idine biosyn hesis genes unde acid s ess condi ions. Cu en ly, o he bes o my knowledge, hei s udy s ands as he pionee ing explo a ion o a po en ial his idine-dependen acid ole ance sys em. Thei me iculous alida ion o he po en ial in ol emen o his idine in he acid esis ance o S. au eus se s an excellen example and should se e as a benchma k o u u e esea ch in o he po en ial his idine-dependen acid ole ance sys em o B. p oduc a. Top-down P o eomic Analysis – In addi ion o he con en ional p o eome analysis using a BUP app oach, a TDP app oach was employed o cha ac e ize and quan i y indi idual p o eo o ms. To enhance he de ec ion o he low-molecula -weigh p o eome, wo deple ion me hods and LC-FAIMS-MS2 analysis wi h in e nal CV s epping (Kaulich e al., 2022a) we e u ilized o boos sensi i i y and inc ease he numbe o p o eo o ms. Recen s udies ha e shown ha u ilizing FAIMS wi h in e nal CV s epping no only doubles he numbe o quan i ied p o eo o ms bu also main ains quan i ica ion accu acy (Kline e al., 2023). While a newe e sion o P o eome Disco e e ( .3.0) was used by Kline and colleagues, allowing o he IV | INFLUENCE OF PH ON BACTERIAL PROTEOMES P A G E | 96 speci ica ion o mul iple CVs pe ile in he spec um selec o node, da abase sea ches in his s udy we e conduc ed using P o eome Disco e e ( .2.5.0.400). This e sion does no suppo mul iple CVs pe ile. Consequen ly, he esul ing da a iles had o be sliced in o subse s il e ed by he applied CVs be o e da abase sea ching (Leipe e al., 2023). The TDP analysis o acidic and basic HMWP deple ions o B. p oduc a quan i ied 175 p o eo o ms (19% o he 923 iden i ied p o eo o ms) and 135 p o eo o ms (17% o he 818 iden i ied p o eo o ms), espec i ely (FIGURE IV-11A and B). The low numbe o quan i ied p o eo o ms can be a ibu ed o he signi ican numbe o missing alues obse ed (38% and 42%), which is compa able o o he TDP-LFQ s udies ha epo ed 43% (Leipe e al., 2023) o 54.6% (N ai e al., 2016). In e es ingly, he numbe o missing alues can depend on he applied CVs, which anged in he p o eo o m analysis o Caeno habdi is elegans om 23% (CV -20) o 43% (CV -50) (Leipe e al., 2023). No ably, Kline and colleagues quan i ied 499 E. coli p o eo o ms, which ep esen s only 29% o he 1,719 iden i ied p o eo o ms, po en ially indica ing a simila occu ence o missing alues in hei analysis (Kline e al., 2023). The highe numbe o missing alues in a p o eo o m-cen ic analysis can be a ibu ed o b oade iso opic en elopes and wide cha ge s a e anges compa ed o a pep ide-cen ic analysis (Basha a e al., 2023). Consequen ly, co-elu ing p o eo o ms o di e en cha ge s a es can sha e o e lapping m/z anges e en when hey ha e highly dis inc masses. This phenomenon can in e e e wi h esol ing and accu a ely quan i ying p o eo o ms by spec al decon olu ion algo i hms, such as X ac decon olu ion employed by he P oSigh PD node in P o eome Disco e e . While mul i-dimensional p o ein ac iona ion s a egies can mi iga e p o eo o m co-elu ion (Cassidy e al., 2021a; Kaulich e al., 2024), he inc eased complexi y can pose challenges o LFQ analysis in co ec ly de e mining p o eo o m abundances ac oss nume ous ac ions. The e o e, u u e ad ancemen s in decon olu ion algo i hms a e essen ial, pa icula ly hose ha possess he capabili y o esol e and quan i y co-elu ing p o eo o ms o acqui e p ecu so s wi h minimal in e e ence. Such ad ancemen s a e c ucial o mi iga ing da a loss and ensu ing he comp ehensi e cha ac e iza ion o p o eomic samples, ul ima ely ad ancing TDP-LFQ analysis (Jeong e al., 2020, 2022; Basha a e al., 2023). Al hough he TDP analysis p ima ily a ge ed he LMWP (<30 kDa), i de ec ed di e en ial abundance o 38 p o eins (TABLE IV-1). Speci ically, 21 p o eins exhibi ed di e en ial abundance in he acidic deple ion analysis and 24 in he basic deple ion analysis (FIGURE IV- 11B). Six p o eins showed di e en ial abundance in bo h deple ion me hods (TABLE A-7). Impo an ly, bo h deple ion me hods allowed compa able quan i ica ion o abundance changes o iden ical p o eo o ms o he same p o ein (TABLE A-7). A compa ison o he quan i ica ion esul s o BUP and TDP e ealed he non-di e en ial abundance o 67 p o eins and he di e en ial abundance o 13 p o eins (TABLE IV-1). Among hese 13 p o eins, h ee (Gn R; A0A7G5MZW5, NlpC; A0A7G5N0P5, and Dl D; A0A7G5MQG2) exhibi ed di e en ial abundance in bo h TDP deple ion analyses and he BUP INFLUENCE OF PH ON BACTERIAL PROTEOMES | IV P A G E | 97 analysis (FIGURE IV-12 and TABLE IV-2). No ably, a pH 6.0, he D-alanyl-lipo eichoic acid biosyn hesis p o ein (Dl D; A0A7G5MQG2), esponsible o ca alyzing he D-alanyla ion o lipo eichoic acid, exhibi ed inc eased abundance. D-alanyla ion o eichoic acids can mask he nega i e cha ge o he cell memb ane, enhancing g ow h and su i al in low pH en i onmen s (Boyd e al., 2000; Wu e al., 2022). While BUP quan i ica ion aims o measu e he abundance o pep ides de i ed om a gi en p o ein, i o en ails o cap u e p o eo o m a ia ions due o limi a ions in pep ide- o-p o ein in e ence. Es ima ions by N ai and colleagues sugges ha a ound 40% o p o eo o m-le el dynamics in abundan , low-molecula -weigh (<30 kDa) p o eins emain unde ec ed by BUP (N ai e al., 2016). Despi e hese limi a ions, bo h pep ide- and p o eo o m-le el quan i ica ion o Gn R indica e a highe abundance a pH 7.0 in he C- e minal p o ein egion (FIGURE IV-13). Simila ly, BUP analysis o he DNA-binding p o ein HU (Hup; A0A2S4GGS2) indica ed highe abundance o se e al pep ides om he N- e minal egion a pH 6.0, po en ially c ucial o DNA p o ec ion unde acidic s ess (Alma za e al., 2015). Howe e , no all pep ides we e quan i ied, and some also showed inc eased abundance a pH 7.0. In con as , p o eo o m analysis e ealed a di e en ially inc ease in abundance o DNA-binding p o ein HU p o eo o ms spanning he N- e minal egion a pH 6.0 (TABLE A-6). This u he emphasizes he impo ance o in eg a ing TDP analyses o iden i y po en ial biologically p o eo o m abundances esul ing om a ian exp ession, p o eoly ic unca ion, o changes in PTM s oichiome y. In e es ingly, many o he di e en ially abundan p o eo o ms om he same p o ein had abundance changes p ima ily cha ac e ized by unca ion, a he han o he PTMs (TABLE A- 6). Howe e , he p o ein sequence da abase o B. p oduc a used o his analysis con ained only a ew au oma ically UniP o -anno a ed PTMs, wi h mos p o eins lacking in o ma ion on po en ial o alida ed modi ica ions. Consequen ly, du ing he P o eome Disco e e da abase sea ch, p o eo o ms wi h po en ial PTMs ha we e no anno a ed in he UniP o da abase emained elusi e. The e o e, an addi ional disco e y-open modi ica ion sea ch was u ilized o iden i y po en ial PTM-ca ying p o eo o ms. This app oach success ully iden i ied and quan i ied mul iple se yl-phospho yla ions on HP p o eins (FIGURE IV-18 and FIGURE IV-19). Al hough hese indings equi e u he alida ion, he applica ion o a disco e y-open modi ica ion sea ch may be conside ed o complemen u u e analysis, especially o bac e ial species wi h limi ed UniP o -anno a ed PTMs. O e all, he combina ion o BUP and TDP analysis iden i ied po en ial se yl-phospho yla ions on HP (A0A2S4GRU0, A0A7G5MYS7, and A0A4V0Z7D2), along wi h one a ginyl- phospho yla ion on HP (A0A7G5MP53) (FIGURE IV-18). The p esence o phosphopep ides wi h missed clea ages nea he p o eoly ic si e (P1´) (FIGURE A-19E and G) could po en ially s eng hen he iden i ica ion o phospho yla ion si es, as nea by phospho yla ion si es (P1´, P2', and P3') can in e e e wi h yp ic clea age (Dickhu e al., 2014; Ge shon, 2014). Al hough he a ginyl-phospho yla ion was de ec ed wi h 68 P SMs, he absence o agmen ions a ound IV | INFLUENCE OF PH ON BACTERIAL PROTEOMES P A G E | 98 he modi ica ion si e, he lack o de ec ed phosphopep ides, and he limi ed li e a u e on a ginyl-phospho yla ion in HP p o eins make i s PTM localiza ion unce ain wi h he cu en ly a ailable da a. Mo eo e , sequence analysis o B. p oduc a's HP p o eins e ealed no able di e ences om o he membe s o his p o ein amily. While ypical HP p o eins ea u e wo highly conse ed amino acid esidues, a his idine (His-15) nea he N- e minus and a se ine (Se -46) in he cen al pa o he p o ein, se ing as phospho yl g oup accep o s (Meadow e al., 1990; B ochu and Vadeboncoeu , 1999; Casabon e al., 2006), he majo i y o B. p oduc a's HP p o eins lack i e amino acids nea he N- e minus, including he conse ed His-15. This de iciency po en ially makes hem incapable o o ming doubly phospho yla ed HP (His∼P)(Se ∼P) (FIGURE IV-18). Typically, HP phosphoca ie p o eins a e in ol ed in ca bohyd a e phospho yla ion du ing anspo in o bac e ial cells ia he phospho ans e ase sys em (Meadow e al., 1990). Depending on hei phospho yla ion s a e, HP p o eins can also ac as co egula o s o he ca aboli e global egula o CcpA (Homeye e al., 2007), which con ols he ca aboli e ep ession/ac i a ion o up o 10% o o al genes (Ponce e al., 2004). This can p o ide a di ec link o he me abolic s a e o he bac e ial cell and a egula o y ole in he quo um sensing o he cell (Ha e al., 2018). The combined quan i a i e da a o he BUP and he TDP analysis showed ha he HP p o ein (A0A2S4GRU0), hei phospho yla ed pep ides as well as hei phospho yla ed p o eo o ms we e di e en ially mo e abundan a pH 6.0 (FIGURE IV-19). In con as , unmodi ied pep ides co e ing he same p o ein sequence and unmodi ied o o myla ed p o eo o ms we e highe abundan a pH 7.0 (FIGURE IV-19). No ably, his obse a ion was independen o he applied deple ion me hod o he TDP analysis (FIGURE IV- 19C-D). The obse ed inc ease in HP abundance and se yl-phospho yla ion unde acidic g ow h condi ions aligns wi h p io esea ch demons a ing a simila end o inc eased HP p o ein abundance and HP (Se -P) o ma ion in esponse o cul u e acidi ica ion (Casabon e al., 2006; Heunis e al., 2014). Hence, he esul s o his s udy u he emphasize he signi ican in luence o cul u e pH on he phospho yla ion s a us o HP p o eins. 4.4 Asp-P o pep ide bond hyd olysis No ably, pep ides wi h N- o C- e minal Asp-P o bond clea ages we e de ec ed o all h ee bac e ia (FIGURE IV-15C-D). Speci ically, o B. he aio aomic on, he e was an inc eased ela i e equency o Asp a he P1 posi ion and P o a he P1´ posi ion o pep ides iden i ied a pH 6.0 (FIGURE IV-15B), sugges ing ha acidic condi ions may p omo e he hyd olysis o Asp-P o bonds. This po en ial biological esponse is u he suppo ed by a signi ican inc ease in pep ides exhibi ing N- o C- e minal Asp-P o and Asp-Xaa clea ages o B. he aio aomic on (FIGURE IV-16A) and B. p oduc a (FIGURE IV-16B). In con as , he p o eome o B. longum did INFLUENCE OF PH ON BACTERIAL PROTEOMES | IV P A G E | 99 no show an Asp-P o sequence logo (FIGURE IV-15A-B) and nei he signi ican changes in he abundance o pep ides wi h N- o C- e minal Asp-P o clea ages (FIGURE IV-16C). Se e al ac o s, such as he pep ide sequence, p o ein olding, empe a u e, and pH alue, can a ec he hyd olysis o pep ide bonds (Ma cus, 1985; Li e al., 2009). The Asp-P o bond, in pa icula , exhibi s inc eased suscep ibili y o pep ide bond hyd olysis unde acidic condi ions and ele a ed empe a u es (Ma cus, 1985; Li e al., 2009). The p oposed acidolysis o Asp-P o bonds sugges s ha he β-ca boxyl g oup o Asp ini ia es a nucleophilic a ack on he ca bonyl ca bon o he amide bond, acili a ing in a esidue cycliza ion, o ming an uns able cyclic anhyd ide in e media e, which may cause a b eak in he polypep ide chain (Piszkiewicz e al., 1970). This eac ion equi es he p esence o an adjacen p o ona ed amide ni ogen. Gene ally, he enhanced a e o Asp-P o clea age can be a ibu ed o he g ea e basici y o he ni ogen a om as pa o p oline's cyclic s uc u e, which inc eases i s basici y (pKa o 10.6) compa ed o he p ima y amine g oups o o he amino acids (pKa o 8.7 o 9.9), hus inc easing i s nucleophilici y and acili a ing as e hyd olysis o Asp-P o bonds unde acidic condi ions (Piszkiewicz e al., 1970). The impo ance o such a labile pep ide bond can be c ucial o planning and pe o ming p o eomic sample p epa a ion and subsequen LC-MS measu emen , especially o N- and C- e minomics expe imen s. Fo example, acidi ica ion using pu e TFA o cell lysis and high concen a ions o T is o neu aliza ion such as applied in he SPEED p o ocol, should be a oided (Doellinge e al., 2020). Fu he mo e, T is-based bu e s exhibi signi ican pH changes upon empe a u e change compa ed o o he bu e sys ems such as sodium phospha e bu e solu ions, which a e gene ally mo e esis an o acidi ica ion upon empe a u e change (Kolhe e al., 2010). The choice o educing agen can also be c i ical, as TCEP can cause a signi ican d op in pH compa ed o di hio h ei ol (DTT) (Schee linck e al., 2015). Addi ionally, di e en bu e s a e equi ed depending on he p o ease used o diges ion. Fo example, pepsin o neop osin necessi a es highly acidic condi ions (pH 1.5 o pH 2.5) o op imal enzyma ic ac i i y, a condi ion ha has been demons a ed o signi ican ly inc ease he hyd olysis o Asp-P o bonds (Sch äde e al., 2017). Mo eo e , mos in-solu ion wo k lows in ol e SPE o sample clean-up, ypically in ol ing o ganic sol en s wi h TFA o FA, which can also inc ease Asp-P o bond clea age (Winkels e al., 2022). Gene ally, p olonged exposu e o acidic condi ions o hea ing should be a oided (Kaulich e al., 2024), and gen le me hods such as eeze-d ying o desicca ion o e apo a e o ganic sol en s a e SPE a e p e e able. Al e na i ely, di e en sample and clean-up p o ocols, such as SP3 (Hughes e al., 2019) o FASP (Manza e al., 2005; Wiśniewski e al., 2009), can be applied, which o en do no equi e an addi ional SPE s ep. Typically, p o eomic LC-MS analysis usually employs an acidic mobile phase and a he mos a -con olled column o en wi h ele a ed empe a u es o op imal ch oma og aphic sepa a ion, con olled e en ion imes, and educed p essu e o he LC sys em (Ga cía, 2005; Lenčo e al., 2022). Howe e , hese condi ions, in combina ion wi h IV | INFLUENCE OF PH ON BACTERIAL PROTEOMES P A G E | 100 p olonged in-column esidence ime, can induce in-column pep ide hyd olysis o Asp-P o bonds (Lenčo e al., 2021). Despi e he signi ican en ichmen and abundance o pep ides wi h Asp-P o and Asp-Xaa clea age a pH 6.0 o B. he aio aomic on and B. p oduc a, he possibili y o a i icial pep ide bond hyd olysis du ing sample p epa a ion and LC-MS analysis canno be uled ou . While employing he same sample p epa a ion s eps and LC-MS se up o he ull p o eome analysis o he h ee HGM bac e ia, sligh a ia ions, such as he o al sample olume a e SPE and ex ended acuum cen i uge e apo a ion imes a e SPE sample clean-up, may ha e occu ed. Addi ionally, he po en ial o he obse ed esul s o be gene a ed in i o, e lec ing a biological e ec o acidic cul i a ion, canno be dis ega ded. Fu he expe imen s using a p o eomic sample p epa a ion wo k low designed o limi he a i icial gene a ion o Asp-P o clea age in combina ion wi h an N- e minomics app oach may cla i y he o igin o hese pep ide o ma ions (Winkels e al., 2022). While Asp-P o clea age is epo ed o be a i icial in mos s udies, selec i e clea age o Asp- P o bonds has been epo ed o be equi ed o co ec olding (Pa ick and Egland, 2019), in e molecula p o ein c oss-linking o ming an Asp-Lys isopep ide bond (Osička e al., 2004) o co alen ly linking chond oi in sul a e o ming a p o ein-glycosaminoglycan-p o ein complex (Zhuo e al., 2004). Mo eo e , se e al s udies ha e epo ed in i o cell-compa men and pH- speci ic au oca aly ic clea age be ween Asp-P o bonds o se e al p o eins exhibi ing a Gly- Asp-P o-His (GDPH) sequence, which is commonly ound in on Willeb and ac o ype D domains. Fo example, au oca aly ic clea age a body empe a u e has been epo ed o se e al human p o eins be o e being sec e ed in o he in es inal mucus. This includes GDPH clea age o IgGFc-binding p o ein (FCGBP) o MUC2 mucin, which is igge ed by he lowe pH o he endoplasmic e iculum o he Golgi appa a us, espec i ely (Lidell e al., 2003; Eh enc ona e al., 2021). Simila ly, au oca aly ic GDPH clea age o p oH3 p ecu so s occu s du ing passage h ough he low pH o he Golgi complex (Thu eson and F ies, 2000). While he exac unc ion o GDPH clea age is s ill a opic o ongoing in es iga ions, i may be an impo an ac o o co alen p o ein o ca bohyd a e c oss-linking, especially in he mucus (Lidell e al., 2003). Fo ins ance, al e a ions in he in e ac ome o p o eins wi hin he a e ial ex acellula ma ix, po en ially con ibu ing o p o ein agg ega ion cascades, a e sugges ed by esul s om he en ichmen o Asp-P o clea age p oduc s o NOTCH3, associa ed wi h disease-a ec ed b ain issue (Lee e al., 2023). The esul s o his s udy indica e a po en ial acidic-induced Asp-P o clea age o 30S ibosomal p o ein S16 (A0A4P6M2Y5; FIGURE IV-14), wi h C- and N- e minal agmen s exhibi ing po en ial AMP ac i i ies (TABLE A-9). Al hough p elimina y AMP es ing agains membe s o he HGM did no con i m such ac i i y, u he expe imen s wi h o he membe s o he gu mic obio a may be necessa y o de e mine he p esence o po en ial AMP ac i i y. INFLUENCE OF PH ON BACTERIAL PROTEOMES | IV P A G E | 101 Simila o he p o eo o m concep , which sugges s ha di e en p o ein species ob ained h ough p ocesses like unca ion o o he pos - ansla ional modi ica ions can ha e dis inc unc ions (Jungblu e al., 2016), se e al ibosomal p o eins ha e been iden i ied wi h "moonligh ing" unc ions, whe ein a single p o ein esul ing om one gene se es mul iple oles in a cell o o ganism, including exhibi ing an imic obial ac i i ies (Hu ado-Rios e al., 2022). Fo example, Bacillus equilensis isola ed om heal hy human eces can sec e e ibosomal p o ein L1 as an an imic obial molecule (Gho eishi e al., 2023), while Lac obacillus sali a ius, isola ed om he eces o ou -mon h-old human in an s, sec e es ibosomal p o eins L27 and L30 wi h an imic obial ac i i ies (Pidu i e al., 2018). No ably, se e al an imic obial pep ides om ibosomal p o eins L30, L39, S19, and S30 ha e been iden i ied in cellula ex ac s o human non-in lamed colonic mucosa, indica ing a di e se p esence o ibosomal an imic obial pep ides in human colonic mucus (Howell e al., 2003; Tollin e al., 2003; An oni e al., 2013). Whe he B. p oduc a ibosomal p o ein S16 may unc ion as a moonligh ing p o ein wi h an imic obial ac i i y emains o be elucida ed. P A G E | 102 P A G E | 103 V PROTEOGENOMIC ANALYSIS OF B. PRODUCTA 1 In oduc ion and Summa y ........................................................................................... 105 2 Expe imen al Design ..................................................................................................... 107 2.1 Cul u e Condi ion E ec s on SEP P oduc ion ....................................................... 107 2.2 Pep ide and P o eo o m Valida ion ........................................................................ 108 3 Resul s ............................................................................................................................ 110 3.1 Iden i ica ion and Valida ion o T ansla ed SEP .................................................... 110 3.2 SEP P o eo o m Di e si y ...................................................................................... 113 3.3 Impac o Cul i a ion Condi ions on SEP T ansla ion ............................................ 115 3.4 Biochemical P edic ions o Iden i ied SEP ............................................................ 116 4 Discussion and Conclusion .......................................................................................... 117 4.1 Challenges and Ad ances in SEP Iden i ica ion ................................................... 117 4.2 Fu u e Di ec ions o SEP Resea ch ...................................................................... 119 V | PROTEOGENOMIC ANALYSIS OF B. PRODUCTA P A G E | 104 Pa s o he ollowing chap e ha e been published in “Iden i ica ion o p o eo o ms o sho open eading ame-encoded pep ides in Blau ia p oduc a unde di e en cul i a ion condi ions.” Gen h e al., Mic obiology Spec um, 11(6), e0252823, (2023). Supplemen a y ma e ial o (Gen h e al., 2023) Addi ional supplemen a y in o ma ion’s is eely a ailable o download a he publishe ’s websi e h ps://doi.o g/10.1128/spec um.02528-23. The MS p o eomics aw da a and comple e P o eome Disco e sea ch esul s ha e been deposi ed o he P o eomeXchange Conso ium (h p://www.p o eomexchange.o g/) ia he PRIDE (Vizcaíno e al., 2014) pa ne eposi o y wi h he da a se iden i ie PXD041979. PROTEOGENOMIC ANALYSIS OF B. PRODUCTA | V P A G E | 111 Pep ide Valida ion – The applied BUP app oach iden i ied 2,379 p o eins ac oss all se en cul i a ion condi ions, o which 261 p o eins (9.1%) had less han 100 amino acids (FIGURE V- 2). Among hese, 55 p o eins passed he PSM il e ing c i e ia, ep esen ed by 142 pep ides, which we e subsequen ly subjec ed o he e i ica ion p ocess (FIGURE V-2). The alida ion o hese non-canonical pep ides using PepQue y led o 107 pep ides passing he s ic e i ica ion c i e ia (FIGURE V-3). Ou o he 35 pep ides ha did no pass he PepQue y e i ica ion p ocess, 12 we e no ma ched by PepQue y o any MS/MS spec a wi h su icien quali y sco es, 4 had supe io ma ches o pep ides in he e e ence da abase, and 19 we e ei he be e ma ched wi h e e ence pep ides ca ying po en ial PTMs o did no mee s a is ical h esholds (p- alue < 0.01) ma ching he non-canonical sequence (FIGURE V-3). Subsequen BLASTp analysis o he emaining pep ides ha success ully passed PepQue y iden i ied a single amino acid a ia ion (SAV) in one pep ide compa ed o a Re Seq p o ein. Conside ing he po en ial occu ence o single nucleo ide polymo phisms in gene ic sequences, which can a ise h ough a ious mechanisms such as DNA eplica ion e o s, he e was an inc eased likelihood ha he de ec ed sequence a ian may no necessa ily be linked o a no el SEP. To ensu e he accu acy and eliabili y o he esul s, bo h he pep ide and i s co esponding SEP we e consequen ly excluded as a p ecau iona y measu e. Compa ison wi h di e en in silico diges ions, e en when ea ing leucine and isoleucine as equi alen in pep ide sequence analysis (FIGURE A-20), led o he exclusion o wo addi ional pep ides, bu no hei espec i e SEP. The e o e, po en ial pep ide con amina ion due o he use o p o ein-con aining yeas ex ac o BHI could be la gely excluded. In conclusion, 104 pep ides we e alida ed (FIGURE V-3), which led o he iden i ica ion o 44 SEP (FIGURE V-2). FIGURE V-3 | Valida ion o Non-canonical Pep ides. O e iew o s ingen il e ing and PepQue y pep ide alida ion. Abb e ia ion: SAV (single amino acid a ia ion). V | PROTEOGENOMIC ANALYSIS OF B. PRODUCTA P A G E | 112 P o eo o m Valida ion – A o al o 1,572 p o eo o ms we e iden i ied and subjec ed o a igo ous il e ing and alida ion p ocess, esul ing in he iden i ica ion o 177 po en ial non- canonical p o eo o ms (FIGURE V-4). Among hese, 60 p o eo o ms me he c i e ia o bo h p o ein size and numbe o P SMs. Addi ional il e ing, which included speci ic c i e ia such as C-Sco e, E- alue, and he numbe o agmen ions, led o he exclusion o 5 p o eo o ms ha did no mee he es ablished c i e ia (FIGURE V-4). As a esul , a o al o 55 alida ed p o eo o ms (FIGURE V-4), de i ed om 19 SEP, success ully passed he il e ing and alida ion p ocess (FIGURE V-2). FIGURE V-4 | Valida ion o Non-canonical P o eo o ms. S ingen il e ing and p o eo o m alida ion, including he dis ibu ion o iden i ied N- and C- e mini and pos - ansla ional modi ica ions (PTMs) on SEP p o eo o ms. The a e age numbe o P SMs iden i ied o each SEP p o eo o m was 24, wi h some exhibi ing conside ably highe numbe s, up o 2,360 (FIGURE V-5A). The iden i ied SEP p o eo o ms had a median -Log E- alue o 17 (FIGURE V-5B), 26 agmen ions (FIGURE V-5C), and a esidue clea age a e o 44% (FIGURE V-5D). These esul s, combined wi h a median sequence co e age o 98% (FIGURE V-5E), collec i ely p o ide s ong e idence o a high con idence le el associa ed wi h he iden i ied p o eo o ms. FIGURE V-5| Me ics o P o eo o m Iden i ica ions. (A) Numbe o P SMs, (B) -Log E- alues, (C) ma ching agmen ions, (D) esidue clea age, and (E) sequence co e age o p o eo o m iden i ica ions. The box-and-whiske plo s illus a e he lowe qua ile and uppe qua ile, wi h he median displayed as a ho izon al line and he mean depic ed as a c oss. Whiske s ep esen he minimum and maximum alues ha all wi hin 1.5 imes he in e qua ile ange 0 50 100 150 200 250 1500 3000 Numbe o P SMs A 0 20 40 60 80 100 Ma ching agmen ions C 0 20 40 60 80 100 % Residue clea age D 0 20 40 60 80 100 % Sequence co e age E 0 5 10 15 20 25 -Log E- alue B PROTEOGENOMIC ANALYSIS OF B. PRODUCTA | V P A G E | 113 3.2 SEP P o eo o m Di e si y Neo- e mini analysis o he iden i ied p o eo o ms iden i ied 26 canonical p o eo o ms o which 16 e ained hei N- e minal me hionine and 10 lacked hei N- e minal me hionine, indica ing N- e minal me hionine excision (NME) (FIGURE V-4). The emaining p o eo o ms exhibi ed ei he N- o C- e minal unca ions, o unca ions a bo h ends (FIGURE V-4). Analysis o he posi ions su ounding neo- e mini si es (P2-P2') indica ed an inc eased speci ici y o me hionine a he P1 posi ion (FIGURE V-6A). While p o eo o ms esul ing om NME we e excluded om his analysis, hese esul s may s ill indica e po en ial NME by me hionine aminopep idase. The iden i ica ion o N- e minally me hionine- unca ed p o eo o ms, in conjunc ion wi h he p esence o smalle amino acid esidues, such as se ine o alanine in he P1´ posi ion – known o hei inc eased e iciency in me hionine emo al (Meinnel e al., 1993) – sugges s he possibili y o al e na i e ansla ion ini ia ion. Such p o eo o m a ian s we e obse ed o BP4 and BP7, wi h N- e minal unca ion ini ia ing a me hionine posi ion 5, as well as canonical a ian s wi h o wi hou N- o mylme hionine (FIGURE V-7). Al e na i e ini ia ion seems likely, gi en he sho N- e minal po ion, which is insu icien o a signal pep ide (Peng e al., 2019). Simila ly, BP14 p o eo o ms lacked he encoded N- e minal po ion o 24 amino acids, including a second me hionine a posi ion 24 (FIGURE V-6B). The missing N- e minal po ion ell wi hin he ypical ange o a po en ial signal pep ide (Peng e al., 2019). Phobius analysis (Käll e al., 2004) e ealed a po en ial signal pep ide o he missing N- e minal segmen (pos e io p obabili y: 0.87) and a non-cy oplasmic egion o he emaining C- e minal segmen (pos e io p obabili y: 0.96) (FIGURE V-6B). Whe he his ep esen s al e na i e ini ia ion, signal pep idase clea age, o ano he unca ion e en emains o be elucida ed. Unde s anding hese mechanisms and explo ing whe he hese a ian s play specialized oles in esponse o speci ic en i onmen al condi ions o s imuli could p o ide insigh s in o he egula o y p ocesses con olling hei exp ession. FIGURE V-6 | Neo- e mini Analysis o Iden i ied SEP P o eo o ms. (A) IceLogo analysis o neo- e mini si es (P2-P2'), showing signi ican ly en iched ( op) o deple ed (bo om) amino acids (p ≤ 0.05). P o eo o ms esul ing om N- e minal me hionine excision we e excluded p io o he analysis. (B) BP14 signal pep ide p edic ion by Phobius, highligh ing ini ia o and al e na i e ini ia o me hionine in yellow. A B MVDGIGLFVMQFFSLSHAKGDGVMATKSIIKDVNIRDNKLCRTFASAIENASGRRGKDVQLSRSFKEISGEKIRELFGDKA 020 40 60 81 0 Signal pep ide Iden i ied p o eo o m 0.5 1.0 Signal pep ide Non-cy oplasmic P2 P1 P1'P2' % Di e ence 30 15 -15 -30 ∑ Analyzed si es = 25 V | PROTEOGENOMIC ANALYSIS OF B. PRODUCTA P A G E | 114 FIGURE V-7 | Po en ial Al e na i e Ini ia ion in SEP P o eo o ms. P o eo o ms o (A) BP4 and (B) BP7. Fo each p o eo o m, b-ions and y-ions a e displayed in blue, while c-ions and z-ions a e displayed in ed, along wi h co esponding E- alue, P-Sco e, and esidue clea age in o ma ion. O e all, mul iple SEP p o eo o ms we e iden i ied, some po en ially ea u ing N- e minal o myla ion, ace yla ion, and disul ide bonds (FIGURE V-4). Speci ically, p o eo o ms o BP3, BP4, BP7, BP10, and BP12 exhibi ed N- e minal o myla ion, while a p o eo o m o BP3 showed N- e minal ace yla ion. In addi ion o i s ole in p o ein syn hesis, he N- o myl g oup may also se e as an indica o o co ansla ional memb ane inse ion, wi h he N- e minal o myl g oup equen ly e ained (Bien enu e al., 2015). Disul ide b idges we e de ec ed o p o eo o ms o BP12, BP24, and BP46, bu he p ecise assignmen o hese linkages o speci ic cys eine esidues in BP12 was no easible wi h he a ailable da a (FIGURE A-21A-B). Seconda y s uc u e p edic ion using Alpha old e ealed he p esence o α-helix and β-shee o ma ions, as well as po en ial disul ide bonds be ween Cys35-Cys51 and Cys38-Cys54 o BP12 (FIGURE V-8 and FIGURE A-21C-E). FIGURE V-8 | Alpha old S uc u e P edic ion o BP12. The p edic ed disul ide b idges be ween Cys35- Cys51 and Cys38-Cys54 a e indica ed by lines connec ing he o ange-colo ed cys eine esidues. A Canonical (E- alue: 3.7E-21, P-Sco e: 5.4E-117, Residue clea age: 87%) Fo myla ed canonical (E- alue: 6.5E-20, P-Sco e: 1.7E-89, Residue clea age: 67%) Canonical (E- alue: 8.7E-22, P-Sco e: 2.6E-135, Residue clea age: 90%) Fo myla ed canonical (E- alue: 1.9E-22, P-Sco e: 6.4E-81, Residue clea age: 57%) B Al e na i e ini ia ion - Me 5 (E- alue: 3.3E-20, P-Sco e: 1.9E-95, Residue clea age: 71%) Missing Al e na i e ini ia ion - Me 5 (E- alue: 3.2E-21, P-Sco e: 1.4E-118, Residue clea age: 81%) Missing C N Loop 1 Loop 2 1 MGIKVKVNFD KRKLESAIKD QARESLRNRS YDAKC35PFC38HT TFSAHPGPNVC51 PHC54RKTVDL NLNIKL 66 PROTEOGENOMIC ANALYSIS OF B. PRODUCTA | V P A G E | 115 3.3 Impac o Cul i a ion Condi ions on SEP T ansla ion Among he 44 SEP iden i ied by BUP, 11 we e consis en ly de ec able ac oss all se en g ow h condi ions (FIGURE V-9A). Fi e o hese SEP we e exclusi ely iden i ied unde acidic g ow h condi ions, while se en SEPs we e consis en ly p esen ac oss all condi ions excep o he acidic YCFA condi ion (pH 6.0 wi h he p esence o SCFAs). Addi ionally, speci ic SEP we e exclusi ely iden i ied in he p esence o pa icula componen s, such as yeas ex ac o LPS. These obse a ions poin owa ds he signi ican impac o en i onmen al a iables on SEP p oduc ion dynamics. Howe e , no all SEPs we e in luenced by hese ex e nal ac o s. Fo example, h ee SEP (BP26, BP36, and BP42) we e consis en ly p oduced in he YCFA medium (pH 7.0), ega dless o he p esence o absence o SCFAs, sugges ing hei independence om hese speci ic ac o s. Addi ionally, all p e iously desc ibed SEP excep BP15 we e iden i ied, including i e SEPs (BP3, BP5, BP8, BP11, and BP12) p e iously de ec ed only in co-cul u e wi h o he bac e ia (SIHUMIx) (Pe uschke e al., 2021) (FIGURE V-9B). Thei iden i ica ion in single bac e ial cul i a ion da a sugges s ha hei biosyn hesis may no be solely elian on in e species in e ac ions o communica ion wi hin he mic obiome. Fu he mo e, i sugges s ha he p oduc ion o mul iple SEP migh ha e been in luenced by speci ic bac e ial g ow h and s ess ac o s (FIGURE V-9B). Fo ins ance, BP11 could be linked o he p esence o yeas ex ac in he medium a pH 7.0, while BP5 was only de ec ed a pH 6.0 (FIGURE V-9B), indica ing a po en ial in ol emen in he acid s ess esponse. FIGURE V-9 | P esence o SEP ac oss Di e se Cul u e Condi ions. Bo om-up iden i ica ions ac oss he se en di e en cul u e condi ions o (A) all de ec ed SEP (44 in o al) and (B) p e iously desc ibed SEP (14 in o al). Highligh ed SEP a e epo ed o be exclusi ely p oduced wi hin he mic obiome communi y. Se size indica es he o al numbe o SEP iden i ied in each condi ion, wi h in e sec ion size ep esen ing sha ed SEP ac oss di e en condi ions. Wi h pe mission om (Gen h e al., 2023). 11 7 7 5 33221111 0 5 10 15 BP3, BP8, BP12 21BP11 1BP5 0 5 10 e a b d c g e a b g d c In e sec ion size In e sec ion size 17 30 19 30 34 34 37 10 12 10 11 12 12 13 A B Se size Se size BHI medium (pH 7.0 +SCFAs) a BHI medium (pH 7.0 -SCFAs) b BHI medium (pH 7.0 +Yeas ) c BHI medium (pH 7.0 +LPS) d YCFA medium (pH 6.0 +SCFAs) e YCFA medium (pH 7.0 +SCFAs) YCFA medium (pH 7.0 -SCFAs) g V | PROTEOGENOMIC ANALYSIS OF B. PRODUCTA P A G E | 116 3.4 Biochemical P edic ions o Iden i ied SEP The po en ial unc ions o he iden i ied SEP we e analyzed by p edic ing physicochemical p ope ies and by examining sequence homologies based on e e ence domains, amilies, speci ic si es, o mo i s. A dis inc bimodal dis ibu ion o isoelec ic poin (pI) alues was obse ed, indica ing a no able shi o he SEP owa ds mo e basic pI alues compa ed o he B. p oduc a e e ence p o eome (FIGURE V-10A). Fu he mo e, examina ion o he g and a e age o hyd opa hy (GRAVY) sco es e ealed a b oade spec um o hyd ophilic p ope ies (FIGURE V-10B), which aligned wi h he amino acid composi ion o he SEP cha ac e ized by a educed equency o hyd ophobic amino acids like alanine, leucine, and isoleucine, alongside a highe occu ence o lysine and a ginine esidues (FIGURE V-10C). One pa icula ly no ewo hy inding was he po en ial an imic obial pep ide (AMP) ac i i y exhibi ed by 25 SEP, as p edic ed by AMP un (Chung e al., 2020) o AMP scanne .2 (Vel i e al., 2018) (FIGURE V- 10D). Among hese, BP16, BP34, and BP40 exhibi ed consis en AMP p edic ions (FIGURE V- 10A). No ably, BP40 also exhibi ed he po en ial o a ge bo h G am-posi i e and G am- nega i e bac e ia. Addi ionally, app oxima ely 71% o he iden i ied SEP we e p edic ed o be non-cy oplasmic (FIGURE V-10E). Fu he mo e, h ee non-cy oplasmic SEP (BP14, BP37, BP45) we e p edic ed o ha e signal pep ides, hin ing a hei po en ial in ol emen in cell-cell and cell-hos communica ion (Hayes e al., 2010). FIGURE V-10 | Biochemical P edic ions o Iden i ied SEP. Dis ibu ion o (A) isoelec ic poin s (pI), (B) g and a e age o hyd opa hy (GRAVY), and (C) amino acid composi ion be ween he B. p oduc a e e ence p o eome and he iden i ied SEP. (D) O e lap o an imic obial pep ide (AMP) p edic ions. (E) Dis ibu ion o cellula localiza ions by Phobius. 71.1% 64.4% 6.7% 2.2% 26.7% Signal pep ide p edic ion Cy oplasmic Non-cy oplasmic T ansmemb ane Signal pep ide Non-signal Pep ide D E 14 (31%) 3 (7%) 8 (17%) AMP Scanne 11 SEP AMP un 17 SEP B A C 0% 2% 4% 6% 8% 10% G I L V A P F H W Y C M K R D E N Q S T 2 4 6 8 10 12 14 0 50 100 Dis ibu ion (%) pI -2 -1 0 1 2 0 50 100 Dis ibu ion (%) GRAVY Re e ence p o eome Iden i ied SEP PROTEOGENOMIC ANALYSIS OF B. PRODUCTA | V P A G E | 117 4 Discussion and Conclusion 4.1 Challenges and Ad ances in SEP Iden i ica ion The p esence o unanno a ed p o ein-coding sORFs we e analyzed by alida ing hei espec i e ansla ed SEP p oduc s a he p o ein le el. U ilizing a ious g ow h condi ions, alongside he applica ion o bo h BUP and TDP me hodologies, a o al o 45 SEP we e success ully iden i ied (FIGURE V-4). While BUP p edominan ly de ec ed he majo i y o SEP p ima ily due o i s highe sensi i i y compa ed o TDP (Cassidy e al., 2023), TDP exclusi ely iden i ied one SEP (BP46) (FIGURE V-2). Among he 45 SEP iden i ied, 31 SEP (BP16-BP46) we e iden i ied o he i s ime, while 14 SEP (BP1-BP14) had been p e iously desc ibed (Pe uschke e al., 2021). Bo h BUP and TDP app oaches complemen ed each o he in he iden i ica ion o SEP, indica ing he s eng hs o each me hodology (FIGURE V-2). To ensu e high-con idence iden i ica ion o SEP, s ic il e ing c i e ia we e implemen ed, aligning wi h ecen ecommenda ions in he ield (Chen e al., 2023). Despi e 17 ou o 44 BUP iden i ica ions elying on a single pep ide o suppo he p o ein-coding po en ial o he sORFs, hese pep ides exhibi ed an a e age o 69 PSMs. Al hough single-pep ide iden i ica ions can be suscep ible o inc eased alse disco e y a es (Nes izhskii, 2010; Hadje as e al., 2023), he majo i y o MS-based SEP iden i ica ions ypically ely on a single unique pep ide (Sla o e al., 2013; Cassidy e al., 2019). This is due o he small p o ein size o SEP, esul ing in a limi ed gene a ion o yp ic pep ides sui able o iden i ica ion. To add ess his limi a ion, he use o mul iple p o eases (Ba el e al., 2020; Kaulich e al., 2021) o he in eg a ion o bo h BUP and TDP (Cassidy e al., 2016, 2021b) has p o en e ec i e in signi ican ly imp o ing sequence co e age and iden i ica ion con idence o SEP. Inco po a ing TDP no only s eng hens he e idence o he exis ence o SEP bu also enables he de ec ion o N- and C- e minal neo- e mini and PTMs, achie emen s o en challenging wi h BUP alone (Tholey and Becke , 2017). Applica ion o TDP in his s udy iden i ied 26 canonical p o eo o ms (FIGURE V-4) and inc eased he a e age sequence co e age by 19% compa ed o BUP. Fu he mo e, he iden i ica ion o unca ed p o eo o ms, po en ially o igina ing om p o eoly ic p ocessing o al e na i e ini ia ion, may sugges he po en ial impac o s uc u al a ia ions on he biological unc ion o SEP (Melo e al., 2023). The iden i ica ion o se e al p o eo o ms wi h PTMs sugges s ha he iden i ied SEP can unde go pos ansla ional p o ein p ocessing. The esul s o his s udy, alongside hose o Pe uschke and colleagues (Pe uschke e al., 2021), sugges ha he p oduc ion o ce ain SEP is no solely dependen on in e species in e ac ions wi hin he SIHUMIx co-cul u e sys em. Ins ead, speci ic SEP can also be p oduced unde monocul u e condi ions and a e in luenced by a ious g ow h condi ions and ex acellula ac o s. Fac o s such as he p esence o endo oxins (LPS) o pH a ia ions, V | PROTEOGENOMIC ANALYSIS OF B. PRODUCTA P A G E | 118 known o play signi ican oles in gu in lamma o y diseases such as IBD (Bai e al., 2016; Candelli e al., 2021), appea o ac as s imuli o egula o s o he p oduc ion o speci ic SEP (FIGURE V-9). Mo eo e , a high numbe o iden i ica ions independen o cul u e condi ions indica e a uni e sal ole o SEP in he su i al and g ow h o B. p oduc a. O e all, he esul s om his s udy, alongside hose o Pe uschke and colleagues, sugges a complex in e play be ween SEP and en i onmen al ac o s (Pe uschke e al., 2021). None heless, i 's wo h conside ing why some o hese po en ially uni e sal SEP we e no iden i ied in Pe uschke and colleagues' s udy (Pe uschke e al., 2021). Va ious ac o s may ha e limi ed he dep h and de ec ion capabili ies o hei p o eomic analysis. One c i ical ac o impac ing p o eomic wo k lows is he da abase sea ch. In eg a ing a la ge p o eogenomic da abase like SIHUMIx, including eigh bac e ial s ains o in e es , poses challenges in p o eogenomic s udies and subsequen FDR analysis (Nes izhskii, 2014). This challenge a ises due o he inc eased numbe o candida es compe ing o ma ching o an expe imen al MS/MS spec um, inc easing he isk o inco ec ma ches and dis inguishing ue om alse iden i ica ions (Nes izhskii, 2010). Consequen ly, la ge p o eogenomic da abase sea ches may gene a e ewe non-canonical and o al pep ides compa ed o con en ional sea ches wi h a e e ence da abase (Agga wal e al., 2022). I 's also essen ial o no e ha he absence o ce ain SEP in speci ic cul u e condi ions does no necessa ily imply hei comple e absence om hose condi ions. Ins ead, hei p esence may be a concen a ions alling below he limi s o de ec ion o he me hods o ins umen s used o analysis. Fu he mo e, in e e ing ac o s such as co-elu ing compounds ha compe e o ioniza ion can lead o educed ioniza ion e iciency o he a ge pep ides (Kelle e al., 2008), esul ing in educed signal in ensi ies and sensi i i y o low-abundance pep ides, making iden i ica ion challenging (Cassidy e al., 2023). To add ess hese challenges, a ious echniques can be applied such as isola ing, en iching, o deple ing p o eins o in e es (Cassidy e al., 2019), applying a second ch oma og aphic sepa a ion (Cassidy e al., 2021a), o using gas-phase sepa a ion (Swea ingen and Mo i z, 2012). These echniques can ex end he limi s o de ec ion o low-abundan species in complex samples. To add ess some o he challenges Pe uschke and colleagues conduc ed a comp ehensi e in es iga ion o a ious p o eomic echniques and app oaches. They compa ed di e en en ichmen s a egies (C8-ca idge and GELF EE), global p o eomics me hods (SP3, FASP, in-gel, and in-solu ion), and mul iple p o ease clea age app oaches ( ypsin and Asp-N) o iden i y small p o eins in he SIHUMIX sys em (Pe uschke e al., 2020). Thei in es iga ion p o ided aluable insigh s in o he s eng hs and limi a ions o hese echniques. By applying hem o he SIHUMIX sys em, hey iden i ied se e al no el SEP, he eby con ibu ing o he ield o human gu mic obiome esea ch (Pe uschke e al., 2021). They also acknowledged ha di e ences in g ow h and cul i a ion condi ions could impac he de ec ion o speci ic SEP. PROTEOGENOMIC ANALYSIS OF B. PRODUCTA | V P A G E | 119 The esul s p esen ed in his s udy complemen he wo k o Pe uschke and colleagues, sugges ing ha he p oduc ion o ce ain SEP in B. p oduc a is no exclusi ely elian on in e species in e ac ions. En i onmen al ac o s can also impac he p oduc ion o ce ain SEP in B. p oduc a. Al hough SEP esea ch is complex, and he challenges in me hodology a e acknowledged, bo h s udies p o ide unique insigh s in o he in ica e na u e o SEP biology. The e o e, i is c ucial o ega d hese e o s as complemen a y, wi h each s udy con ibu ing signi ican ly o ou unde s anding o SEP p oduc ion. A comp ehensi e app oach conside ing a ious expe imen al ac o s, including g ow h condi ions, sample p epa a ion echniques, and p o eomic wo k lows, will be pi o al in ad ancing he ield and unco e ing he ull spec um o SEP. 4.2 Fu u e Di ec ions o SEP Resea ch Gi en he ocus o his s udy on soluble p o eins, i is no su p ising ha he biochemical p ope ies o he iden i ied SEP exhibi ed a dec eased ela i e equency o hyd ophobic amino acids and an inc eased equency o cha ged amino acids, pa icula ly lysine and a ginine esidues (FIGURE V-10C). These cha ged esidues, especially hose loca ed a he C- e mini o pep ides esul ing om yp ic diges ion, ypically enhance p o on a ini y, ioniza ion e iciency, and CID agmen a ion, leading o imp o ed MS sensi i i y (Dup ee e al., 2020). The small o e lap o AMP p edic ions o h ee SEP (BP16, BP34, BP40; FIGURE V-10A), can be likely a ibu ed o a ia ions in aining se s, physicochemical p ope ies, and amino acid equencies a each sequence posi ion, hus in luencing he p edic ion model o classi ying an ibac e ial pep ides (Gabe e and Noble, 2017). No ably, one SEP (BP27) was p edic ed o be a sho ansmemb ane p o ein (FIGURE V-10E). Sho memb ane-associa ed SEP a e p edic ed o cons i u e 35% o he SEP popula ion in he human mic obiome (Sbe o e al., 2019). Gi en hei po en ial oles in essen ial cellula p ocesses such as anspo , signaling pa hways, cell di ision, espi a ion, spo ula ion, and memb ane in eg i y (Yada alli and Yuan, 2022), hese ansmemb ane SEP should be a ge ed o u u e analysis. Consequen ly, specialized, MS-compa ible memb ane p o ein en ichmen p o ocols should be p io i ized in u u e s udies o analyze hese memb ane- associa ed SEP (Cap i and Whi elegge, 2017; Ah ens e al., 2022; Meie -C edo e al., 2022). Despi e hei po en ial signi icance, one o he majo challenges associa ed wi h SEP lies in hei lack o sequence homology wi h known p o eins. Thei small size and compac olding make adi ional anno a ion and s uc u e p edic ion ools less e ec i e, as hese ools ypically depend on homology sea ches (Ah ens e al., 2022). This limi s he unde s anding o he SEP unc ion and emphasizes he need o expe imen al alida ion. Func ional p o eomics eme ges as a p omising app oach o add ess hese challenges and e eal he unc ional p ope ies, biological oles, and mechanis ic con ibu ions o SEP. V | PROTEOGENOMIC ANALYSIS OF B. PRODUCTA P A G E | 120 Va ious p o eomic me hodologies, such as biochemical ac iona ion o soluble complexes coupled wi h mass spec ome y (Ha ugimana e al., 2022), a ini y pu i ica ion mass spec ome y (AP-MS) (Gnanaseka an and Pappu, 2023), o c oss-linking mass spec ome y (XL-MS) (Pie simoni e al., 2022), a e a ailable o de ec na i e p o ein complexes wi hin cellula ex ac s and gene a e ne wo ks o p o ein-p o ein in e ac ions (Low e al., 2021). These me hodologies can signi ican ly con ibu e o he iden i ica ion o SEP wi hin es ablished p o ein ne wo ks, he eby enhancing ou unde s anding o hei oles and con ibu ions o cellula unc ions (Ga cia-del Rio e al., 2023; Leblanc e al., 2023). Addi ionally, gene ic sys ems like he yeas wo-hyb id assay can complemen hese e o s by mapping p o ein- p o ein in e ac ions, p o iding insigh s in o he oles o SEP wi hin he cellula in e ac ome (Mehla e al., 2015). Fu he mo e, in eg a ing ansc ip omic da a c ea ed using RNA-Seq o ibosome p o iling wi h p o eomic analysis o he same biological samples can inc ease he numbe o expe imen ally alida ed SEP (Guilloy e al., 2023; Hadje as e al., 2023). This in eg a ed omics app oach enables a mo e comp ehensi e explo a ion o he SEP p o eome, leading o a be e unde s anding o hei biological unc ions. Fu u e esea ch in his ield will likely p o ide mo e insigh in o he signi icance o hese small p o eins. OUTER MEMBRANE VESICLES ANALYSIS | VI P A G E | 127 comp ised 10 mM DTT and 50 mM IAA wi hin a 100 mM TEAB bu e en i onmen . All samples we e diges ed wi h ypsin a a 1:40 enzyme- o-subs a e a io (20 h, 37°C, 800 pm), wi h he addi ion o 0.01% (w/ ) n-dodecyl-β-D-mal oside (DDM) o 0.5% (w/ ) SDC. Fo SDC diges s, samples we e addi ionally p ocessed using a modi ied phase ans e p o ocol (Masuda e al., 2008) (chap e II.3.3). TABLE VI-1 | O e iew o P o ocol-speci ic Sample P ocessing S eps. All lis ed alues ep esen end concen a ions o he in-solu ion diges ion (ISD), single-po , solid-phase-enhanced p o ein ex ac ion (SP3), o il e -aided sample p epa a ion (FASP) p o ocol. PROTOCOL STEP CONDITIONS ISD- CLASSIC ISD-I MPROVED SP3- SDS - TEAB SP3- SDS - SDC SP3- SDS - DDM SP3- SDC FASP- SDC FASP-SDS-SDC OMV LYSIS F eeze hawing ü 2% SDC ü ü ü 1% SDS ü ü ü ü RED. & ALK. 10 mM DTT & 50 mM IAA ü ü ü ü ü ü ü ü SDS REMOVAL 8 M u ea, 100 mM TEAB ü TRYPTIC DIGESTION 0.5% SDC, 100 mM TEAB ü ü ü ü ü 0.001% DDM, 100 mM TEAB ü 100 mM TEAB ü ü SDC REMOVAL 0.5% TFA & 100% e hyl ace a e ü ü ü ü ü 2.5 Caco-2 Wound-Healing Assay An impo an ea u e o OMVs is ha he p o eins associa ed wi h hem exhibi a ious biological ac i i ies. Pa icula ly, OMVs p oduced by E. coli can exhibi an inhibi o y e ec on cell p oli e a ion and induce p o-in lamma o y esponses in in es inal epi helial cells (Cañas e al., 2016; Pa en e al., 2017). To e alua e he po en ial biological ac i i y o isola ed E. coli OMVs, a wound-healing assay u ilizing he human in es inal epi helial cell line Caco-2 was conduc ed. Wound closu e was measu ed immedia ely a e emo ing he inse , as well as a 12 and 30 hou s a e incuba ion wi h inc easing OMV concen a ions (10, 50, and 100 µg/ml). Posi i e and nega i e con ols included incuba ion wi h 5 ng/ml TGFβ and 1 µg/ml LPS, espec i ely. Cells solely incuba ed wi h he medium (0.1% FCS) and he OMV elu ion bu e se ed as e e ences o compa ison wi h he ea ed g oups. These con ols acili a ed he calcula ion o ela i e wound closu e. Fu he de ails on he cul i a ion o Caco-2 cells and he wound healing assay can be ound in chap e II.2.3. VI | OUTER MEMBRANE VESICLES ANALYSIS P A G E | 128 3 Resul s 3.1 P o eomic Wo k low Compa ibili y Gi en ha he eagen s o he OMV ki a e undisclosed, conduc ing a ho ough compa ibili y check, speci ically ocusing on LC-MS/MS analysis and p o ein diges ion, was essen ial o i s in eg a ion in o a p o eomic wo k low. MALDI MS analysis o dilu ed elua e om a blank ki un e ealed no cha ac e is ic PEG ion se ies o o he polyme ic impu i ies. While he absence o cha ac e is ic impu i ies indica es compa ibili y o he OMV ki wi h mass spec ome y echniques (Kelle e al., 2008), i does no gua an ee comple e compa ibili y. Addi ional LC- MS analysis o wo dilu ions (1:10 and 1:100) using cy och ome C diges o e en ion ime and HeLa diges o pep ide iden i ica ion con ols showed no pep ide e en ion ime shi s and compa able pep ide iden i ica ion. This indica es ha he elu ion beha io o pep ides and pep ide de ec ion emained la gely una ec ed, a leas o subsequen LC-MS uns. Moni o ing signal s abili y and backg ound noise e ealed a singly cha ged peak (309.125 m/z) a 53 minu es, wi h in ensi ies o app oxima ely 1.8 x 108 and 6.4 x 108 in he 1:100 and 1:10 dilu ions, espec i ely. The obse ed minimal impac on ch oma og aphic uns and MS analysis con i ms he compa ibili y o he OMV ki wi h LC-MS analysis. Fu he mo e, he compa ibili y o he OMV elu ion bu e wi h yp ic diges ion was analyzed using p o ein mix u es consis ing o six p o eins (6P), and ou p o eins (4P), he la e excluding myoglobin and alcohol dehyd ogenase (FIGURE VI-2A). The mix u es we e diges ed using ei he TEAB-bu e ed OMV elu ion bu e (KIT) o 100 mM TEAB (TEAB) and we e e alua ed h ough SDS-PAGE analysis (FIGURE VI-2B). While he compa ison o he wo di e en diges ion condi ions showed sligh ly educed p o ein band in ensi ies wi h he TEAB- bu e ed OMV elu ion bu e (FIGURE VI-2B), he esul s s ill con i med he compa ibili y o he OMV ki wi h yp ic diges ion, allowing in eg a ion o he ki in o a p o eomic wo k low. FIGURE VI-2 | SDS-PAGE Analysis o P o ein Diges ion using he Exobac e ia Elu ion Bu e . (A) Molecula weigh dis ibu ion and composi ion o he six-p o ein mix u e. (B) E alua ion o he yp ic diges ion o he 6-p o ein (6P) o 4-p o ein (4P) mix u es (excluding myoglobin and alcohol dehyd ogenase) using TEAB-bu e ed OMV elu ion bu e (KIT) o 100 mM TEAB. kDa 212 118 66 A 20 BSA ADH CA & β-Cas CA & Myo Cy C 43 14 29 TEAB KIT B OUTER MEMBRANE VESICLES ANALYSIS | VI P A G E | 129 3.2 Biophysical Cha ac e is ics and Ki Loading Capaci y Biophysical Cha ac e is ics – The isola ed OMVs we e i s pla ed on LB aga o con i m he absence o bac e ial con amina ion. Subsequen ly, hey we e cha ac e ized using dynamic ligh sca e ing wi h NTA, which allowed di ec , eal- ime isualiza ion o he isola ed OMVs. FIGURE VI-3A illus a es a sc eensho om one o he eco ded ideos. Examina ion o he isola ed OMVs e ealed nanopa icles anging om 117.2 ± 2.1 nm (mode ± s anda d e o ) o OMVs isola ed om ace a e supe na an o 95.3 ± 4.1 nm o hose isola ed om glucose supe na an (FIGURE VI-3B and TABLE VI-2). The a e age pa icle concen a ion was highe o OMVs isola ed om supe na an ob ained om ace a e cul i a ion (9.4 x 1010 ± 0.9 pa icles/ml) compa ed o hose isola ed om supe na an ob ained om glucose cul i a ion (7.8 x 108 ± 0.8 pa icles/ml). The obse ed di e ences in pa icle concen a ion be ween he wo supe na an s, while p o iding a gene al e e ence, equi e addi ional eplica es o ho ough alida ion. Fu he mo e, NTA measu emen s indica e a ela i ely na ow size dis ibu ion o pa icles, sugges ing a mo e monodispe se sample a he han a polydispe se one. FIGURE VI-3 | Nanopa icle T acking Analysis o E. coli OMVs. (A) Rep esen a i e isualiza ion o OMV pa icles cap u ed om he eco ded ideo. (B) Pa icle size dis ibu ion o OMVs isola ed om E. coli supe na an cul u ed in ace a e o glucose M9 medium. TABLE VI-2 | Summa y o Nanopa icle T acking Analysis PARAMETER ACETATE GLUCOSE Mode pa icle size 117.2 ± 2.1 nm 95.3 ± 4.1 nm D10 82.9 ± 3.4 nm 79.2 ± 3.8 nm D50 124.3 ± 1.7 nm 103.4 ± 2.6 nm D90 204.3 ± 5.7 nm 207.4 ± 19.7 nm Pa icle concen a ion 9.4 x 1010 ± 0.9 pa icles/ml 7.8 x 108 ± 0.8 pa icles/ml A B 0 1 2 3 4 0 1 2 3 4 0200 400 600 800 1000 Size (nm) Pa icles x 10 7 Pa icles x 10 9 Ace a e Glucose VI | OUTER MEMBRANE VESICLES ANALYSIS P A G E | 130 Loading Capaci y – The assessmen o he ki 's loading capaci y e ealed ha an inc ease in he ini ial supe na an olume om 20 o 40 o 60 ml esul ed in a signi ican doubling o p o ein concen a ions (FIGURE VI-4A) and pa icle concen a ions (FIGURE VI-4B). No ably, he pa icle size and dis ibu ion o pa icles emained cons an a 106 ± 3.0 nm (FIGURE VI-4C and FIGURE A-24), indica ing ha OMVs main ained hei size and dis ibu ion e en wi h an inc eased pa icle concen a ion. Fu he mo e, he pu i y a io ( he a io o esicle coun s o p o ein concen a ion) o OMV p epa a ions, which conside s bo h p o ein con amina ion and loss o OMVs du ing isola ion (Webbe and Clay on, 2013), emained ela i ely cons an a 4.3 x 106 ± 0.1 pa icles/µg o p o ein (FIGURE VI-4D). Despi e a ia ions in he loaded supe na an olume, a compa able pa icle size and pu i y a io could be main ained, indica ing he ki 's e iciency in handling inc eased sample olumes. FIGURE VI-4 | Loading Capaci y E alua ion o he ExoBac e ia OMV Isola ion Ki . Di e en E. coli supe na an olumes (20, 40, and 60 ml) we e p ocessed wi h he OMV isola ion ki and analyzed using NTA and BCA. (A) P o ein concen a ion, (B) pa icle concen a ion, (C) pa icle mean size, and (D) no malized pu i y a io (pa icle/p o ein a io). Da a ep esen mean ± s anda d e o om 3 independen expe imen s. Signi ican di e ences we e calcula ed ia one-way ANOVA wi h Dunne ’s co ec ion. * (p < 0.05); ** (p < 0.01). Biological Ac i i ies – The abili y o isola ed OMVs o exhibi biological ac i i ies was e alua ed using an in i o wound closu e assay, which examined he mig a ion and p oli e a ion o human colonic Caco-2 cells. The e alua ion o wound size educ ion a e 12 and 30 hou s indica ed a no able educ ion o wound healing compa ed o un ea ed con ol cells (FIGURE VI-5A). The quan i a i e assessmen o wound healing, wi h he medium a bi a ily se a 100% as a e e ence, e ealed educed wound closu e wi h inc easing concen a ions o OMVs. A concen a ions o 10 µg/ml, 50 µg/ml, and 100 µg/ml, OMVs exhibi ed dec easing pe cen ages o wound closu e a e 12 hou s (45 ± 1.1%, 68 ± 3.0%, and 82 ± 2.8%, espec i ely FIGURE VI-5B). Despi e s a is ical analysis using one-way ANOVA wi h Dunne ’s co ec ion, no signi ican changes in wound healing (p < 0.05) we e obse ed. This lack o s a is ical signi icance may be a ibu ed o he limi ed sample size o only h ee biological expe imen s. Al hough he esul s we e no s a is ically signi ican , hey sugges a possible dose-dependen inhibi o y e ec o OMVs on wound closu e, wi h highe concen a ions ha ing a g ea e e ec . While he OMV elu ion bu e showed a modes 64 113 153 0 50 100 150 200 20 mL 40 mL 60 mL P o ein concen a ion (µg/ml) 4.2 4.2 4.4 0 2 4 6 8 20 mL 40 mL 60 mL Pu i y a io (10 6 pa icles/µg p o ein) 20 ml 40 ml 60 ml 109 106 102 0 50 100 150 200 20 mL 40 mL 60 mL Pa icle mean size (nm) 2.7 4.8 6.6 0 2 4 6 8 20 mL 40 mL 60 mL Pa icle concen a ion (10 8 /ml) A B CD * * ** OUTER MEMBRANE VESICLES ANALYSIS | VI P A G E | 131 s imula o y e ec on wound closu e a e 30 hou s (9 ± 2.0% FIGURE VI-5B), his e ec may be a ibu ed o common bu e componen s like calcium and phospho ic acid, which can in luence he wound healing p ocess (Na a o-Requena e al., 2018; Sim e al., 2022). The nega i e con ol (LPS) esul ed in a educ ion o 22 ± 2.5%, while he posi i e con ol (TGFβ), known o i s ole in p omo ing p oli e a ion (Penn e al., 2012), s imula ed wound closu e by 36 ± 6.2%. FIGURE VI-5 | Caco-2 Wound Healing Assay. (A) Time-lapse mic oscopy images o wound closu e o Caco-2 cells wi h medium, TGFβ (5 ng/ml), LPS (1 µg/ml), and OMVs (50 µg/ml). Images we e cap u ed a 0 h, 12 h, and 30 h (10- old magni ica ion). (B) Wound closu e a e 12 and 30 h incuba ion wi h a ying OMV concen a ions (10, 50, and 100 µg/µl), LPS (1 µg/ml), OMV elu ion bu e , medium alone, o TGFβ (5 ng/ml). Medium alone was a bi a ily assigned as 100%. Da a ep esen mean ± s anda d e o om h ee independen expe imen s. Ba s ep esen 200 μm. 3.3 P o eomic Analysis o E. coli OMVs The p o eomic changes in E. coli OMV p o ein con en unde di e en g ow h s a es and cul u e condi ions we e analyzed by a bo om-up p o eomic analysis. Changes in p o ein abundance and localiza ion we e es ima ed by anno a ing subcellula loca ions using STEPdb 2.0 (Loos e al., 2019), which ca ego izes p o eins in o 13 dis inc subcellula classes (FIGURE A-23A). Due o he dynamic na u e o p o ein localiza ion and hei abili y o mo e o a ious ex acy oplasmic compa men s, se e al p o eins may exhibi mul iple subcellula loca ions (FIGURE A-23B). To acili a e anno a ion, p o eins om di e en subcellula compa men s we e classi ied in o ou dis inc subcellula opological g oups: he cy oplasm (F1, A, R, and N), he inne memb ane (B), he pe iplasm (I, G, F2, F3, and E), and he ou e memb ane/ex acellula g oup (H, X, and F4) (FIGURE A-23C). These subcellula opological classi ica ions conside ed mos o he dynamic p o ein mo emen , p o iding obus insigh s in o hei cellula localiza ion. Fo mid-loga i hmic g ow h phases, a o al o 334 and 298 p o eins we e iden i ied in OMVs isola ed om supe na an ob ained om glucose and ace a e cul i a ion, espec i ely (FIGURE VI-6A). Among hese p o eins, 258 (69%) we e iden i ied in bo h isola ions, while 76 p o eins we e iden i ied exclusi ely unde glucose and 40 p o eins exclusi ely unde ace a e condi ions A B 0% 20% 40% 60% 80% 100% 120% 140% 160% 12 h 30 h % Wound healing OMV 100 (µg/ml) OMV 50 (µg/ml) OMV 10 (µg/ml) LPS Medium OMV elu ion bu e TGFβ LPSOMV MediumTGFβ 0 h 12 h 30 h VI | OUTER MEMBRANE VESICLES ANALYSIS P A G E | 132 (FIGURE VI-6A). The ele ance o he exclusi ely iden i ied OMV p o eins emains unce ain due o he absence o clea unc ional o pa hway en ichmen s. Compa ison o he subcellula loca ions o iden i ied OMV p o eins showed ha OMVs isola ed om supe na an ob ained om ace a e o glucose cul i a ion we e en iched in pe iplasmic and cy oplasmic p o eins, whe eas only 9% o he o al p o eins we e classi ied as ou e memb ane o ex acellula p o eins (FIGURE VI-6B-C). The majo i y o iden i ied cy oplasmic p o eins we e associa ed wi h ibosomal unc ions o in ol ed in glycolysis, such as enolase (P0A6P9), glyce aldehyde-3-phospha e dehyd ogenase (P0A9B2), and phosphoglyce a e kinase (P0A799). Howe e , mos o he abundan and essen ial cy oplasmic p o eins equi ed o bac e ial su i al we e no de ec ed (Goodall e al., 2018). FIGURE VI-6 | Subcellula Topological Dis ibu ion o OMV P o eins based on To al P o ein Iden i ica ions. (A) O e lap o p o eins iden i ied in OMV isola ed a mid-loga i hmic g ow h om glucose and ace a e cul i a ion. (B) Subcellula opological dis ibu ion o OMVs isola ed a mid- loga i hmic g ow h om glucose and (C) ace a e cul i a ion De e mina ion o median abundance alues om he wo independen OMV isola es, each measu ed in h ee echnical eplica es, allowed e alua ion o p o ein abundance wi hin subcellula opological ca ego ies. Compa ing he dis ibu ion based on he median abundance wi h he dis ibu ion based on he o al numbe o iden i ied p o eins o each subcellula opological ca ego y e ealed a dis inc pa e n (FIGURE VI-7). Fo OMVs isola ed om supe na an ob ained du ing he mid-loga i hmic g ow h phase o glucose cul i a ion, 68.4% o he median abundance o igina ed om he ou e memb ane/ex acellula , 20.5% om pe iplasmic, 4.3% om he inne memb ane, and 6.8% om cy oplasmic p o eins (FIGURE VI- 7A). Simila ly, OMVs isola ed om supe na an ob ained du ing ace a e cul i a ion exhibi ed a dis ibu ion whe e 73.5% o he median abundance o igina ed om ou e memb ane/ex acellula , 18.8% om pe iplasmic, 2.8% om inne memb ane, and 4.9% om cy oplasmic p o eins (FIGURE VI-7B). This obse a ion sugges s ha , while a subs an ial numbe o cy oplasmic p o eins we e p esen wi hin he OMVs isola ed om supe na an ob ained om glucose (143 p o eins) o ace a e cul i a ion (119 p o eins), hei median abundance is no ably lowe compa ed o ha o he 29 glucose o 27 ace a e ou e memb ane/ex acellula p o eins. Howe e , i is essen ial o no e ha solely elying on median 39.3% 3% 48.8% 8.9% C A Cy oplasm Inne memb ane Pe iplasm Ou e memb ane 8.6% 46.3% 2.9% 42.2% B Ace a e 298 Glucose 334 40 (11%) 258 (69%) 76 (20%) OUTER MEMBRANE VESICLES ANALYSIS | VI P A G E | 133 abundance may unde es ima e he signi icance o less abundan p o eins which could also play impo an unc ional oles o con ibu e o biological p ocesses. The e o e, bo h dis ibu ions o he subcellula opological ca ego ies should be conside ed complemen a y, p o iding dis inc insigh s in o p o ein abundance and di e si y. Despi e an inc ease in he o e all p o ein con en o he OMV isola es ( om 0.23 o 0.83 µg/µl), he subcellula opological dis ibu ion o OMVs isola ed om supe na an ob ained om glucose cul i a ion emained s able h oughou he mid-loga i hmic o s a iona y phase (FIGURE VI-7A). As cul i a ion ime inc eased, only a 3.2% inc ease in he median abundance o cy oplasmic p o eins and a 4.8% dec ease in ou e memb ane/ex acellula p o eins we e obse ed o OMVs isola ed om supe na an ob ained om glucose cul i a ion (FIGURE VI- 7A). In con as , OMVs isola ed om supe na an ob ained om ace a e cul i a ion exhibi ed a no able 13.5% dec ease in he median abundance o ou e memb ane/ex acellula p o eins, accompanied by a 6.4% inc ease in cy oplasmic p o eins and a 9.4% inc ease in inne memb ane p o eins spanning he mid-loga i hmic o dea h phase (FIGURE VI-7B). The dec ease in ou e memb ane/ex acellula p o eins may be a ibu ed o he di ec g ow h inhibi ion ha occu ed upon en e ing he s a iona y phase (FIGURE A-22). Addi ionally, he ac i a ion o p og ammed cell dea h mechanisms may ha e con ibu ed o he inc ease in cy oplasmic and inne memb ane p o eins (Juodeikis and Ca ding, 2022). FIGURE VI-7 | Subcellula Topological Dis ibu ion o OMV P o eins based on Median Abundance. (A) OMVs isola ed om supe na an ob ained du ing he mid-loga i hmic, p e-s a iona y, and s a iona y g ow h phases o glucose cul i a ion. (B) OMVs isola ed om supe na an ob ained du ing he mid- loga i hmic, la e-s a iona y, and dea h g ow h phases o ace a e cul i a ion. Cy oplasm Inne memb ane Pe iplasm Ou e memb ane 68.4% 20.5% 4.3% 6.8% A 8.3% 2.2% 15.6% 73.9% P e-s a iona yMid-loga i hmic 10%2.5% 23.5% 64% S a iona y 4.9% 2.8% 18.8% 73.5% BMid-loga i hmic 9.5% 2% 20.4% 68.1% La e-s a iona y 11.3% 12.2% 16.4% 60% Dea h phase VI | OUTER MEMBRANE VESICLES ANALYSIS P A G E | 134 Examina ion o majo componen s o ou e memb anes (TABLE A-10), sugges ed as ubiqui ous ma ke s o OMV alida ion (Daleke-Sche me ho n e al., 2014; Hong e al., 2019), e ealed a consis en inc ease in iBAQ alues in OMVs isola ed om supe na an ob ained om glucose cul i a ion (FIGURE VI-8A). Ce ain ma ke p o eins, such as OmpT and OmpF, exhibi ed inc eased iBAQ alues in isola ed OMV samples (FIGURE VI-8A) compa ed o ull p o eome p epa a ions o E. coli (FIGURE VI-8B), which may sugges he selec i e so ing and encapsula ion o ce ain p o eins in o OMVs. In e es ingly, o he ma ke p o eins, such as OmpA, OmpX, and OmpC, consis en ly showed high iBAQ alues o bo h p o eomes. FIGURE VI-8 | iBAQ Dis ibu ion o Po en ial E. coli OMV P o ein Ma ke s. (A) The OMV p o eome isola ed om he supe na an ob ained om glucose cul i a ion and (B) he comple e E. coli p o eome. P o ein names a e lis ed in TABLE A-10. 3.4 Imp o ing OMV Lysis and T ypsin-Based Diges ion The impac o he non-ionic de e gen SDC on OMV in eg i y and he suscep ibili y o OMV p o eins o enzyma ic deg ada ion we e e alua ed (FIGURE VI-1). OMV samples subjec ed o yp ic diges ion exhibi ed p o ein bands wi hin he 43 o 66 kDa ange (FIGURE VI-9A). The applica ion o 2% (w/ ) SDC be o e enzyma ic diges ion esul ed in he educ ion o hese bands. A con ol expe imen in ol ing only 2% SDC con i med ha he obse ed bands co esponded o p o eins encapsula ed wi hin he OMVs. P o ein bands wi h a molecula mass o app oxima ely 23.5 kDa co espond o ypsin i sel . O e all, he esul s indica e he capabili y o SDC o induce OMV lysis and he subsequen suscep ibili y o p o eins ini ially p o ec ed wi hin OMVs o enzyma ic deg ada ion. E alua ion o SDC and DDM impac on yp ic diges ion o he six-p o ein mix u e, compa ed agains a TEAB con ol, indica ed imp o ed yp ic diges ion using 0.5% (w/ ) SDC (FIGURE VI- 9B). Con e sely, 0.01% (w/ ) DDM exhibi ed p o ein bands wi h simila in ensi ies o hose o he TEAB con ol, sugges ing mino di e ences in p o ein abundance be ween he wo diges ion condi ions (FIGURE VI-9B). Especially o TEAB-bu e ed OMV elu ion bu e (KIT), 0100 200 300 400 500 2 4 6 8 10 0500 1000 1500 2 4 6 8 10 OmpA OmpC Lpp OmpT OmpF SlyBSlp OmpW LpoA Tsx RlpA BamD BamB RcsF BamE BamC BamA LolB OmpX OmpA OmpC OmpX Slp Lpp SlyB OmpT BamC BamB BamA BamE RcsF Tsx BamD OmpF LolB LpoA RlpA OMV log10 iBAQ Ranked p o eins A Po ines Lipop o eins Assembly p o eins Full p o eome log10 iBAQ Ranked p o eins B OUTER MEMBRANE VESICLES ANALYSIS | VI P A G E | 135 he esul s indica e an imp o ed ypsin diges ion e iciency in he p esence o SDC compa ed o DDM o TEAB. FIGURE VI-9 | E alua ion o Non-Ionic De e gen s on OMV Lysis and T yp ic Diges ion. (A) SDC-Media ed OMV Lysis. F om le o igh : molecula weigh ladde (in kDa), un ea ed OMV sample, yp ic- ea ed OMV sample, OMV sample ea ed wi h 2% (w/ ) SDC ollowed by yp ic ea men , and OMV sample ea ed wi h 2% (w/ ) SDC. (B) Non-ionic de e gen s e ec on yp ic diges ion using TEAB-bu e ed OMV elu ion bu e (KIT) o 100 mM TEAB (TEAB). To imp o e ypsin-based diges ion o OMV samples, a ious sample p epa a ion p o ocols we e es ed, including on-bead SP3, on-memb ane FASP, and in-solu ion me hods. These p o ocols u ilized bo h SDC and DDM o e ec i e lysis and yp ic diges ion o OMVs. To al iden i ied p o ein g oups anged om 347 o 710 (FIGURE VI-10A), wi h pep ide iden i ica ions anging om 1140 o 5043 (FIGURE VI-10B). The ISD-imp o ed p o ocol, employing SDC o bo h OMV lysis and yp ic diges ion, exhibi ed he highes numbe o p o ein iden i ica ions (FIGURE VI-10A). The SP3 p o ocol, in eg a ing SDS o OMV lysis and SDC o yp ic diges ion (SP3-SDS-SDC), exhibi ed he mos iden i ied pep ides (FIGURE VI-10B). Compa ison o di e en SP3 p o ocols showed ha he in eg a ion o SDC o DDM o yp ic diges ion esul ed in highe p o ein and pep ide iden i ica ions compa ed o de e gen - ee diges ion using only TEAB. (FIGURE VI-10A-B). Despi e he po en ial in e e ence om SDS, which could ha e a ec ed ypsin's ac i i y and led o incomple e p o ein diges ion (Masuda e al., 2008), less han 3% o he iden i ied pep ides had 2 missed clea ages (FIGURE VI-10C). These esul s indica ed e icien emo al o SDS and success ul p o ein diges ion. An excep ion was he FASP-SDS-SDC me hod, which exhibi ed he lowes numbe o p o ein and pep ide iden i ica ions, as well as he lowes sequence co e age (FIGURE VI-10D), ac oss all h ee biological eplica es. Small le o e s o SDS, which may no ha e been su icien ly emo ed, could po en ially ha e in e e ed wi h yp ic diges ion o led o signal supp ession du ing LC-MS measu emen s (Rundle and A ms ong, 1996; Masuda e al., 2008). A BSA ADH CA & β-Cas CA & Myo Cy C 6P KIT TEAB B 118 29 43 20 66 kDa kDa 212 118 20 43 14 29 66 212 VI | OUTER MEMBRANE VESICLES ANALYSIS P A G E | 136 Fu he mo e, sample loss, due o small le o e s emaining in he il e ese oi o he il e i sel , migh ha e occu ed du ing sample p ocessing. FIGURE VI-10 | Pep ide and P o ein Iden i ica ion Da a o Di e en OMV Sample P epa a ions. (A) P o ein iden i ica ions, (B) pep ide iden i ica ions, (C) missed clea age e en s, and (D) p o ein sequence co e age. Box-and-whiske plo s cap u e lowe qua ile and uppe qua ile wi h he median displayed as a ho izon al line and he mean depic ed as a c oss; whiske s ep esen minimum and maximum alues ha all wi hin 1.5 imes he in e qua ile ange. O e all, all me hods achie ed simila sequence co e ages o 23% (FIGURE VI-10D), wi h each me hod co e ing la gely simila ac ions o he E. coli p o eome (FIGURE A-25). Compa ed o he de e gen - ee ISD-classic p o ocol which employed eeze- hawing o OMV lysis, he in eg a ion o SDS o SDC o OMV lysis and SDC o DDM o diges ion no ably imp o ed pep ide and p o ein iden i ica ions, esul ing in highe sequence co e age. Fo his eason, subsequen analysis ocused on e alua ing hese imp o ed me hods. No able me hodological a ia ions we e obse ed in he subcellula opological dis ibu ion o OMV p o eins, wi h cy oplasmic p o eins anging om 2.2% o 4.2%, inne memb ane p o eins om 0.2% o 27.8%, pe iplasmic p o eins om 2.4% o 24%, and ou e memb ane p o eins om 64% o 87% (FIGURE VI-11A). The ISD-imp o ed me hod exhibi ed he highes abundance o ou e memb ane p o eins (87%) and he lowes abundance o inne memb ane p o eins (0.2%) (FIGURE VI-11A). In e es ingly, FASP-SDC and SP3-SDC, which also employed SDC o diges ion and lysis, showed he highes abundance o inne memb ane p o eins a 26% and 457 710 471 597 597 531 657 347 3162 3906 3885 5043 4720 4466 4445 1140 ISD-Classic ISD-Imp o ed SP3-SDS-TEAB SP3-SDS-SDC SP3-SDS-DDM SP3-SDC FASP-SDC FASP-SDS-SDC 0 200 400 600 800 1000 Σ P o eins A 0 1500 3000 4500 6000 Σ Pep ides B C D 0 20 40 60 80 100 Sequence co e age (%) 0 20 40 60 80 100 Missed clea age (%) 2 1 0