Application of Liquid Chromatography-Ion Mobility Spectrometry-Mass Spectrometry-Based Metabolomics to Investigate the Basal Chemical Profile of Olive Cultivars Differing in Verticillium dahliae Resistance

Applica ion o Liquid Ch oma og aphy-Ion Mobili y Spec ome y- Mass Spec ome y-Based Me abolomics o In es iga e he Basal Chemical P o ile o Oli e Cul i a s Di e ing in Ve icillium dahliae Resis ance I ene Se ano-Ga cía, Ioannis C. Ma akos, Lucía Olmo-Ga cía,*Lo enzo León, Raul de la Rosa, Ana M. Gómez-Ca a aca, Angjelina Belaj, Alicia Se ano, Ma ilena E. Dasenaki, Nikolaos S. Thomaidis, and Aleg ía Ca asco-Panco bo Ci e This: J. Ag ic. Food Chem. 2024, 72, 27561−27574 Read Online ACCESS Me ics & Mo e A icle Recommenda ions * sı Suppo ing In o ma ion ABSTRACT: The limi ed e ec i eness o cu en s a egies o con ol Ve icillium wil o oli e (VWO) p omp s he need o inno a i e app oaches. This s udy explo es he basal me abolome o 43 oli e cul i a s wi h a ying esis ance le els o Ve icillium dahliae, o e ing al e na i e insigh s o oli e c ossb eeding p og ammes. The use o an inno a i e UHPLC-ESI-TimsTOF MS/MS pla o m enabled he anno a ion o mo e han 70 compounds ac oss di e en oli e o gans ( oo , s em, and lea ) and he c ea ion o a p elimina y compila ion o TIMSCCSN2 expe imen al da a o mo e eliable me aboli e anno a ion. Mo eo e , i allowed he documen a ion o nume ous isome ic species in he s udied oli e o gans by esol ing hidden compounds. Mul i a ia e s a is ical analyses e ealed signi ican me abolome a iabili y be ween highly esis an and suscep ible cul i a s, which was u he in es iga ed h ough supe ised PLS-DA. Key ma ke s indica i e o VWO suscep ibili y we e anno a ed and cha ac e is ic composi ional pa e ns we e es ablished. S em issue exhibi ed he highes disc imina i e capabili y, while oo and lea issues also showed signi ican p edic i e po en ial. KEYWORDS: Olea eu opaea L., LC-MS p o iling, TIMS, oli e oo s, oli e s ems, oli e lea es, pa hogen esis ance, Ve icillium wil o oli e 1. INTRODUCTION Olea eu opaea L. has coexis ed wi h mankind since p ehis o ic imes, unde going a leng hy p ocess o in en ional o acciden al domes ica ion. 1 The ui is he mos alued pa o he ee and, due o i s p o i abili y, i s cul i a ion has been s eadily inc easing wo ldwide. Indeed, oli e-g owing a ea is cu en ly 19% highe compa ed o he beginning o he 21s cen u y. 2 Concu en ly, he mode niza ion o oli e managemen has wi nessed he eme gence o high-densi y g owing sys ems and he widesp ead adop ion o d ip i iga ion sys ems wo ldwide, wi h pa icula p ominence in Andalusia, Spain. 3,4 Howe e , a downside o hese signi ican changes has been he apid sp ead o some pes s and diseases, such as Ve icillium wil o oli e (VWO), ac oss oli e-g owing egions, esul ing in subs an ial economic losses o p oduce s. 5,6 This se e e pa hology is caused by he soil-bo ne ungus Ve icillium dahliae Kleb. and was i s diagnosed in 1946 in I aly and, la e , in he en i e Medi e anean basin. 7 Nume ous ac o s con ibu e o i s uncon olled expansion, bu pa icula ly no ewo hy is he ungus’s excep ional esis ance, acili a ed by mic oscle o ia, which a e igge ed o ge mina e by he oo exuda es o he plan . 8 V. dahliae pene a es he oli e ee ia oo s and dissemina es apidly h ough o he o gans ( unk, ba ks, lea es, e c.) o colonize he xylem essels causing he hos plan ’s wa e and nu ien s collapse. 8 The se e i y o plan symp oms will a y depending on se e al ac o s, including he ype o in ec ing isola es, he densi y o inoculum, he suscep ibili y o he cul i a , and en i onmen al condi ions. In se e e cases, his can lead o he comple e dea h o he ee. 9,10 Owing o he ex ended su i al o mic oscle o ia and he limi ed e icacy o ungicides, he managemen o V. dahliae p edominan ly ocuses on combining p e en i e s a egies wi h sus ainable ag icul u al p ac ices. 9,11 In his con ex , he use o oli e cul i a s possessing inhe en esis ance o V. dahliae as a componen o in eg a ed con ol s a egies has been widely ad oca ed o mi iga e disease incidence. 9,12 Many s udies ha e al eady ca ego ized in e ms o esis ance and/o suscep ibili y a subs an ial numbe o oli e cul i a s by using mul iple disease pa ame e s ela ed o physical symp oma ology and/o ungus in ec ion a e. 13−16 Howe e , he key ac o s de e mining VWO esis ance as a selec ion c i e ion in oli e b eeding p og ams emain unclea , and u he esea ch is equi ed. In plan biology, me abolomics has been pi o al in elucida ing he physiological and biochemical esponses o hos s o bio ic and abio ic s esses. 17 Me abolomics is Recei ed: Augus 6, 2024 Re ised: No embe 13, 2024 Accep ed: No embe 15, 2024 Published: No embe 22, 2024 A iclepubs.acs.o g/JAFC © 2024 The Au ho s. Published by Ame ican Chemical Socie y 27561 h ps://doi.o g/10.1021/acs.ja c.4c07155 J. Ag ic. Food Chem. 2024, 72, 27561−27574 This a icle is licensed unde CC-BY 4.0 Downloaded ia CSIC on Janua y 27, 2025 a 11:33:55 (UTC). See h ps://pubs.acs.o g/sha ingguidelines o op ions on how o legi ima ely sha e published a icles. ca ego ized in o a ge ed and non a ge ed app oaches, which di e mainly in he me hodologies and pu sued objec i es. Rega ding VWO, only a ge ed app oaches ha e been employed so a , co e ing a limi ed sec ion o me abolome. Thus, se e al seconda y me aboli es (mainly phenolic compounds) ha e been e alua ed in a ious in ec ed oli e o gans and issues ( oo s, s ems, co ex, xylem, e c.) o explo e hei ole in he plan ’s de ense mechanisms agains V. dahliae. 18−21 Indeed, some o hese me aboli es such as u in, oleu opein, lu eolin-7-glucoside o hyd oxy y osol ha e been p e iously desc ibed o exhibi in i o an i ungal ac i i y agains his ascula pa hogen. 19,22 Mo e ex ensi ely, Ca doni and coau ho s de e mined 31 seconda y me aboli es belonging o simple phenols and glycosides, secoi idoids and de i a i es, lignans, and i e penic acids o e alua e majo changes in me abolic p o iles o in ec ed-oli e oo ex ac s. 23 In ha wo k, a s ong ela ionship be ween he quan i a i e basal me abolic p o ile and oli e cul i a suscep ibili y was poin ed ou . Building on hese indings and p o iding addi ional e idence, Se ano-Ga cia and coau ho s, in a ecen s udy, depic ed he dis ibu ion o 56 basal me aboli es in h ee oli e o gans, emphasizing key quan i a i e di e ences obse ed in ela ion o VWO- esis ance le els. 24 These au ho s also demons a ed he abili y o he quan i ied me aboli es o di e en ia e oli e cul i a s based on ungal esis ance h ough he use o bo h supe ised and unsupe ised s a is ical analyses. Al hough non a ge ed me abolomics has no been applied in VWO-pa hology o da e, his holis ic app oach has p o ided aluable insigh s ying o elucida e he esis ance mechanisms o oli e ee agains Xylella as idiosa, 25 co on agains Aspe gillus ubingensis 26 o obacco agains Phy oph ho a pa asi ica a . nico ianae. 27 The p ima y objec i e o non- a ge ed me abolomics is o sc een he me abolome o samples exhibi ing speci ic ai s, such as esis ance, as well as o iden i y disc iminan bioma ke s wi hou p io knowledge o hei iden i y. The mos ime-consuming s ep in his p ocess is me aboli e/ma ke iden i ica ion, which demands ca e ul and de ailed da a in e p e a ion. Con en ional LC-High Resolu ion MS (LC-HRMS) pla o ms widely used in me abolomics p o ide many ion desc ip o s (e.g., e en ion ime, accu a e mass, molecula o mula, iso opic dis ibu ion, and MS/MS agmen a ion). These desc ip o s acili a e me aboli e iden i- ica ion by compa ison wi h comp ehensi e da abases and published li e a u e. O e he pas decade, he in eg a ion o ion mobili y spec ome y (IMS) wi h HRMS has in oduced an addi ional molecula desc ip o known as he collision c oss sec ion (CCS) alue. The CCS is a unique physicochemical pa ame e ela ed o he size, shape, and cha ge o he molecules, which is measu ed wi h a speci ic bu e gas, p essu e, and empe a u e. 28 The mobili y dimension enhances me aboli e iden i ica ion wi h highe con idence and imp o es sensi i i y by educing he signal- o-noise a io. Addi ionally, i inc eases he selec i i y o he me hod by boos ing peak capaci y. 29 Fu he mo e, o isome s ha canno be esol ed ch oma og aphically o di e en ia ed by MS, ion mobili y p o ides an addi ional sepa a ion dimension, enabling he de ec ion and po en ial dis inc ion o p e iously hidden isome s. Due o hese ad an ages, he use o IMS-MS in he analysis o na u al p oduc s has g own signi ican ly, becoming a aluable ool o esea che s wo king wi h complex ma i- ces. 30,31 Consequen ly, IMS s ands ou as a powe ul echnique o enhance he pe o mance o non a ge ed LC-MS me hods. Howe e , he e emains a no able lack o expe imen al CCS da abases in plan me abolomics, pa icula ly o compounds wi hou a ailable pu e s anda ds, such as hose de i ed om oli e ma ices. Consequen ly, he CCS desc ip o emains incomple ely in eg a ed in o he wo k low o me aboli e cha ac e iza ion, and u he esea ch is needed o achie e widesp ead accep ance. Being awa e o he exis ence o a signi ican in o ma ion gap ega ding VWO disease and he capabili ies o he analy ical pla o m used, his s udy pu sued h ee main objec i es: (i) o e alua e he po en ial o he inno a i e UHPLC-ESI- TimsTOF MS pla o m o maximize me abolome in o ma ion om oli e-de i ed ma ices, leading o he c ea ion o a p elimina y lis o compounds based on collision c oss-sec ion alues (TIMSCCSN2); (ii) cha ac e ize he p esence o absence o hese seconda y me aboli es in a ious oli e plan o gans; and (iii) apply an un a ge ed app oach o comp ehensi ely in es iga e and delinea e basal me abolic di e ences in oo s, s ems, and lea es o 43 oli e cul i a s as a unc ion o hei esis ance o Ve icillium dahliae Kleb. in ec ion. 2. MATERIALS & METHODS 2.1. Plan Ma e ial and Sample P e ea men . Heal hy one- yea -old plan s om 43 di e en oli e cul i a s ob ained by ege a i e p opaga ion o semiha dwood s em cu ings we e p o ided om he Wo ld Oli e Ge mplasm Bank (WOGBC) o Cen o IFAPA “Alameda del Obispo” in Co doba, Spain. 32 Table 1 includes he cul i a s selec ed in he p esen s udy classi ied acco ding o he VWO- esis ance ca ego y. 9,14 Plan o gans ( oo s, s ems and lea es) we e sampled om h ee di e en plan s o he cul i a s unde s udy, esul ing hus in a comp ehensi e collec ion o 129 samples pe plan o gan (387 samples in o al conside ing all he issues). As plan p e ea men , oli e o gans we e ca e ully de ached om he ee, ollowed by ho ough we cleaning. A e wa d, he de ached issues we e ai -d ied a oom empe a u e in a da k en i onmen un il a cons an weigh was achie ed. The d ied ma e ial was hen inely powde ed, homogenized o uni o m pa icle size using a 0.5 mm me al sie e, and s o ed a −23 °C un il u he use. Table 1. Oli e Cul i a s Included in he S udy, Thei Classi ica ion Acco ding o VWO-Resis ance, and he Code Used o Thei Iden i ica ion ca ego y oli e cul i a s and code used highly esis an (HR) “I117−120” (G1), “F an oio” (G2), “I111−2” (G3), “I117−117” (G4), “Manzanille a de Hue cal-O e a” (G5), “Empel e” (G6) esis an (R) “Uslu” (G7), “Maa i” (G8), “Ko oneiki” (G9), “Leccino” (G10), “Ma eya” (G11), “Dokka ” (G12) medium suscep ible (MS) “Fs17” (G13), “Klon 14−1812” (G14), “A bequina” (G15), “UCI 2−35” (G16), “Mawi” (G17), “UCI 10−30” (G18), “Fishomi” (G19), “Changlo Real” (G20), “Pinone a” (G21), “UCI 2−68” (G22), “Lianolia Ke ky as” (G23), “Picual” (G24), “Ba i” (G25), “Picudo” (G26), “My olia” (G27), “Co nicab a” (G28), “Ba nea” (G29), “Ve dial de Velez Malaga-51” (G30), “Siki i a” (G31), “Manzanilla de Se illa” (G32), “Mo u ” (G33) suscep ible (S) “Chemlal del Kabylie” (G34), “Abbadi Abou Gab a” (G35), “Hojiblanca” (G36), “Majhol-152” (G37), “Abou Sal Mohazam” (G38), “Menya” (G39), “Temp ano” (G40), “Llume a” (G41), “Jabali” (G42), “Mas oidis” (G43) Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c07155 J. Ag ic. Food Chem. 2024, 72, 27561−27574 27562 2.2. Chemicals and Reagen s. Double deionized wa e , wi h a esis i i y o 18.2 MΩ·cm, was ob ained using a Milli-Q sys em (Millipo e, Bed o d, USA). High-quali y e hanol (E OH) wi h a minimum pu i y o 99% and LC-MS g ade me hanol (MeOH) we e supplied by P olabo (Pa is, F ance). ESI-L low concen a ion uning mix was p o ided by Agilen Technologies (San a Cla a, CA, USA). The pu e s anda ds o quinic acid, hyd oxy y osol, u in, oleu opein, maslinic acid, ca echin, lu eolin, lu eolin-7-O-glucoside, apigenin, y osol, oleanolic acid, and e bascoside we e acqui ed om Sigma- Ald ich (S . Louis, MO, USA), as well as he ammonium ace a e sal . Mobile phases we e il e ed h ough a Nyla lo 0.45 μm nylon memb ane il e (Pall Co po a ion (Michigan, MI, USA)) while Cla ine 0.22 μm nylon sy inge il e s (Bonna-Agela Technologies (Wilming on, DE, USA)) we e used o ex ac s and pu e s anda d mix u es. The s anda d solu ion mix used o quali a i e pu poses was p epa ed by mixing he exac amoun o all pu e s anda ds men ioned abo e in E OH/H2O (80:20, / ) o ob ain a concen a ion o a ound 15 mg/L o each compound. 2.3. Ex ac P epa a ion. The sample p epa a ion ollowed he solid−liquid ex ac ion p o ocol p e iously ou lined by Se ano- Ga cia e al. 24 B ie ly, lea ex ac s we e p epa ed by mixing 100 mg o d ied and homogenized powde wi h 10 mL o E OH/H2O (60:40, / ) in a 15 mL alcon ube. A e 1.5 min o shaking, he alcon was in oduced in o an ul asonic ba h wo king wi hin he ange o 50−60 kHz o 30 min and cen i uga ed o 10 min a 9000 pm. Once he i s supe na an was emo ed in a da k lask, he emaining solid unde wen e-ex ac ion using 10 mL o E OH/H2O (80:20, / ) in he subsequen s ep, ollowed by 10 mL o pu e E OH in he las ex ac ion cycle. All supe na an s we e combined in he same da k lask ( o aling 30 mL in lea ex ac s). Be o e injec ion, an addi ional 10- old dilu ion was pe o med using E OH/H2O (80:20, / ). S em and oo ex ac s we e p epa ed ollowing he same p o ocol as desc ibed abo e, wi h he ex ac an agen olume educed o 5 mL a each s ep, esul ing in a inal olume o 15 mL. A 5- old dilu ion was ca ied ou o bo h oo and s em ex ac s. All ex ac s we e s o ed a −23 °C un il analysis. A quali y con ol (QC) sample was p epa ed o each plan o gan ( oo , s em, and lea ) by combining aliquo s om he ex ac s o he cul i a s included in his s udy. These samples we e u ilized as ins umen al con ols. 2.4. Analy ical LC-IMS-MS/MS Pla o m Condi ions. The en i e sample se was analyzed using an ul ahigh pe o mance liquid ch oma og aphy (UHPLC) equipped wi h an elec osp ay ioniza ion sou ce (ESI) and coupled o apped ion mobili y spec ome y- ime- o - ligh sys em (TimsTOF P o) powe ed by he la es pa allel accumula ion se ial agmen a ion (PASEF) echnology om B uke Dal onics (B emen, Ge many). Acco ding o he me hod p oposed by Ma akos e al., 33 analy es we e elu ed using an Acclaim RSLC 120 C18 column (2.1 ×100 mm, 2.2 μm) om The mo Fische Scien i ic Inc. (Wal ham, MA, USA), equipped wi h an Acqui y UPLC BEH C18 VanGua d P e-Column (2.1 ×5 mm, 1.7 μm), and main ained a a empe a u e o 30 °C. The injec ion olume was se a 2 μL and he au osample was kep a 4 °C h oughou he sequence. Mobile phases we e composed by H2O/MeOH (90:10, / ) (phase A) and pu e MeOH (phase B), bo h bu e ed wi h 5 mM ammonium ace a e. The ch oma og aphic elu ion condi ions, including ime, low a e, and mobile phase composi ion, we e p og ammed as ollows: 0 min, 99.0% A and 0.2 mL/min; 1.0 min, 99.0% A and 0.2 mL/min; 3.0 min, 61.0% A and 0.2 mL/min, 14 min, 0.1% A and 0.4 mL/min, 16 min, 0.1% A and 0.48 mL/min, 16.1 min, 99.0% A and 0.48 mL/min, 19 min, 99.0% A and 0.48 mL/min, 19.1 min, 99.0% A and 0.2 mL/ min; and 20 min, 99.0% A and 0.2 mL/min. Ion mobili y spec ome e ope a ed wi h ni ogen (N2) as d i gas and 100.0 ms o amp ime, moni o ing ea u es om 0.40 o 1.37 V· s/cm2. The ESI ope a ed in nega i e pola i y and Full Scan mode (m/ z20−1300), wi h speci ic se ings including +2500 V o capilla y, −500 V o end-pla e o se , 10 L/min and 220 °C o d y gas, and 2.0 ba o nebulize p essu e. Two di e en MS acquisi ion modes we e employed depending on he objec i e pu sued. B oadband collision- induced dissocia ion (bbCID) based on da a-independen acquisi ion (DIA) me hod was employed o analyze he en i e sample se , p o iding enhanced sensi i i y. Addi ionally, PASEF, which elies on da a-dependen acquisi ion, was exclusi ely u ilized in ce ain QC samples o gene a e he au o MS/MS agmen a ion pa e n. In his la e mode, he same p ecu so ion was selec ed and agmen ed se e al imes o gene a e mul iple MS2spec a. The so wa e used o sys em con ol included Compass Hys a and O o Con ol, supplied by B uke Co po a ion. Da a Analysis 5.3 so wa e was applied o examine he acqui ed ch oma og ams. 2.5. Sys em calib a ion, Sys em s abili y assu ance, and Da a p ocessing. Be o e s a ing any sequence, bo h TIMS and MS sys ems we e subjec ed o ex e nal calib a ion using sodium o ma e and comme cial ESI-L Low Concen a ion Tuning Mix solu ions. In addi ion, a eshly p epa ed mix u e (3:1, / ) o hese solu ions was cons an ly in used o se e as in e nal calib a ion o da a p ocessing. Fo success ul calib a ion, a leas h ee e e ence m/zand ion mobili y alues om he calib a ion solu ion had o co espond wi h hose measu ed in he sys em. The QC sample was analyzed e e y 10 samples o e alua e he s abili y o he ins umen esponse. Addi ionally, pu e sol en (MeOH) injec ions we e pe o med a he same in e als o clean he column and ensu e i emained ee o con amina ion. Da a p ocessing was conduc ed using he Me aboScape 2023 so wa e, which u ilized he T-Rex 4D (LC-TIMS-QTOF MS) algo i hm o au oma ically ecalib a e he acqui ed MS da a. This in ol ed conduc ing molecula ea u e selec ion, il e ing, and scaling. Key pa ame e s we e con igu ed du ing p ocessing, such as se ing he minimum ex ac ed ea u es by he numbe o occu ences o #3 o each g oup (in his case, o each cul i a ) o ensu e consis en ea u e p esence ac oss all cul i a eplica es. The in ensi y h eshold o peak de ec ion was es ablished a 1000 coun s and he minimum 4D peak size was se a 100 poin s, while ecu si e ea u es we e de ined a 75 poin s. An EIC co ela ion o 0.8 ela ed o ion decon olu ion was applied. The p ima y ion was [M-H]−, wi h [M+Cl]−as seed ion and [M−H-H2O]−and [M+CH3COO]−as common ions. Du ing da a p ocessing, he Wi hin-Ba ch Co ec ion ool was u ilized o add ess po en ial d i s ha may ha e occu ed du ing he sequences. Ex ac ed ea u es om sol en analyses we e au oma ically excluded i he analysis/sol en a io exceeded 3.0. Following his, he ex ac ed ea u es we e cha ac e ized using a numbe o ools ha a e in eg a ed in o Me aboScape. These ools include (i) Sma Fo mula, which de i es he molecula o mula o each anno a ed compound based on i s accu a e mass and iso opic pa e n, aking in o accoun any de ec ed adduc s; (ii) Compound C awle , which sea ches molecula s uc u es o speci ied molecula o mulas in local (Analy eDB) and online public da abases (ChEBI, ChemSpide and PubChem); and (iii) Me F ag, which pe o ms in silico agmen a ion o po en ial s uc u es and compa es hem wi h acqui ed MS/MS spec a. The so wa e also suppo s anno a ion by compa ing wi h p e iously es ablished analy e lis s and MS/MS spec al lib a ies (such as B uke Sumne Me aboBASE Plan Lib a y o public MS/MS da abases). Typical bioac i e compounds p ima ily consis o ca bon, hyd ogen, and oxygen. The e o e, ou ocus was on anno a ing compounds con aining hese elemen s, aiming o e o s below 5 ppm. Addi ionally, he so wa e p o ides a CCS p edic ion ool, c ucial o ensu ing high- eliabili y analy e cha ac e iza ion. 2.6. S a is ical Analysis. SIMCA 14.1 so wa e was used o pe o m bo h unsupe ised p incipal componen s (PCA) and supe ised pa ial leas -squa es-disc iminan analysis (PLS-DA). The da a ma ix included 129 samples (obse a ions) and con ained all he de ec ed ea u es ( a iables) exp essed as peak in ensi y o each oli e o gan ype. S anda d da a no maliza ion and uni a iance (UV) scaling we e implemen ed as p ep ocessing me hods. PCA was conduc ed o in es iga e da a quali y, biological di e si y, and na u al clus e ing o samples based on VWO- esis ance. Ho elling’s T2 (95%) and DModX (DC i 0.05) plo s we e examined o de ec any po en ial ou lie s wi hin he mul idimensional space o PCA. Following a ho ough examina ion o he LC-MS da a, a supe ised PLS-DA s a is ical analysis was employed o u he explo e he cha ac e is ic me abolic pa e ns associa ed wi h he mos VWO- Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c07155 J. Ag ic. Food Chem. 2024, 72, 27561−27574 27563 esis an /suscep ible oli e cul i a s. The quali y o PLS-DA models was e alua ed wi h a c oss- alida ion es h ough he R2X,R2Y, and Q2pa ame e s. These pa ame e s indica e he ac ion o explained a iance in he Xand Yma ices and he p edic i e capabili y o he model, espec i ely. Addi ionally, pe mu a ions plo wi h 100 i e a ions we e ca ied ou o assess he class disc imina ion pe o mance by compa ing he goodness o i (R2and Q2) o he o iginal model wi h andomly gene a ed models whe e he o de o Y- obse a ions was pe mu a ed while keeping X-ma ix in ac . 3. RESULTS & DISCUSSION 3.1. Sc eening o Oli e O gans P o iles o Build a P elimina y TIMSCCSN2 Da abase. The limi ed a ailabili y o expe imen al CCS-lib a ies emains an un esol ed obs acle o in eg a ing ion mobili y in o me abolomics s udies. The e o e, he ini ial s ep o his in es iga ion was o conduc a p elimina y sc eening o he LC-IMS-MS me abolic p o iles o oli e-de i ed ma ices, aiming o build an explo a o y TIMSCCSN2 da a compila ion. O e 70 me aboli es we e anno a ed in he oli e ee o gans, including o ganic acids, i idoids, couma ins, simple phenols, lignans, secoi idoids, la onoids, pen acyclic i e penes, and hei de i a i es. The anno a ed cons i uen s a e lis ed in Table 1 o Suppo ing In o ma ion (Table S1) including he p oposed compound name, chemical amily, calcula ed molecula o mula, e en ion ime (R ), expe imen al m/z, e o o he mass p edic ion (ppm), mSigma alue, TIMSCCSN2 alue, and he main MS/MS agmen s obse ed. All da a p esen ed in Table S1 a e exp essed as dep o ona ed o m [M-H]−, as his was he mos commonly de ec ed ion in nega i e pola i y. In some cases, o he ions such as [M+Cl]−, [M−H-H2O]−, and [M +CH3COO]−we e also moni o ed, al hough hey we e no included in he able in o ma ion o con ain he size o he able. The p oposed compounds we e c oss-checked wi h ele an comp ehensi e s udies ocused on he in-dep h cha ac e iza ion o oli e-de i ed ma ices o ensu e hei iden i y o con i med using B uke spec al lib a ies. 23,24,34−36 The ion mobili y desc ip o was used o suppo me aboli e iden i ica ion whene e a s anda d was a ailable, o i he compounds we e desc ibed in he plan me abolomics TIMSCCSN2 lib a y gene a ed by Sch oede and collabo a o s in a p e ious wo k, 37 o in o he wo ks applying TIMS mobili y. 38,39 Addi ionally, i was used o p opose a candida e i he p edic ed CCS alue was consis en wi h he pu a i e anno a ion. The e o e, he in eg a ion o IMS has p o en o be c ucial in he disc imina ion o nume ous isome ic me aboli es wi hin he ma ices unde s udy. No ably, se e al o hese me aboli es we e anno a ed o he i s ime in his s udy. This b eak h ough may be a ibu ed o he ac ha , un il now, LC-MS has p ima ily p o ided isome di e en ia ion based solely on e en ion ime and accu a e mass. In speci ic cases, hidden isome s we e dis inguishable wi hin a single ch oma o- g aphic peak solely h ough he IMS dimension. Fu he mo e, TIMS has shown i s capabili y o e ec i ely sepa a e widely o e lapping peaks ha canno be en i ely esol ed based only on e en ion ime and accu a e mass. This capabili y is especially c ucial o quan i a i e applica ions and ep esen s a signi ican enhancemen o a ge ed s udies. The de ailed wo k low u ilized in bo h scena ios is desc ibed in he ollowing sec ion, along wi h an examina ion o he dis ibu ion o he anno a ed me aboli es h oughou he oli e ee. 3.1.1. Exhaus i e Quali a i e Cha ac e iza ion o he Anno a ed Compounds wi hin he Me abolome o Oli e Roo , S em, and Lea Samples. In acco dance o p e ious s udies, he quali a i e me abolic p o ile is closely linked o he oli e o gan assessed. 24,34−36 Table S1 lis s he me aboli es ha we e consis en ly de ec ed in all es ed cul i a s o each ma ix. The able, as speci ied in he p e ious sec ion, includes ele an in o ma ion o each o he subs ances conside ed. As expec ed, mos o he compounds anno a ed a e o phenolic na u e, such as simple phenols, secoi idoids, la onoids, e c. In he case o o ganic acids, only wo me aboli es o his chemical class we e anno a ed: quinic acid (C7H12O6) wi h a CCS o 134.3 Å2, and ci ic acid (C6H8O7) wi h 126.5 Å2. These compounds we e consis en ly p esen in all o gans unde in es iga ion. Th ee ins ances o i idoids (compounds cha ac e ized by a six-membe ed ing con aining an oxygen bound o a cyclopen ane ing) we e anno a ed in he e hanolic ex ac s. Loganic acid (375.1296 m/z), wi h a CCS o 184.7 Å2, was ound in he h ee o gans examined. I is cha ac e ized by he calcula ed molecula o mula C16H24O10 and shows a agmen a ion pa e n wi h MS signals o ce ain in ensi y a 213.0764, 169.0876, 151.0752, 125.0606, 113.0244, and 107.0499 m/z. The me aboli es anno a ed as 11-hyd oxyi - idodial glucoside pen aace a e (555.2082 m/z; 222.3 Å2) and 7-deoxyloganic acid (359.1347 m/z; 182.4 Å2) we e exclusi ely de ec ed in oo s and s ems. The la e inding is no en i ely in line wi h he esul s epo ed by Michael and coau ho s, who obse ed he p esence o 7-deoxyloganic acid exclusi ely in oo ex ac s o “Ko oneiki” and “Che oui” cul i a s. 36 Se ano- Ga cia and co-wo ke s also ound 7-deoxyloganic acid only in oo s in a ecen pape wo king wi h 10 cul i a s. 24 These di e ences can be easily explained, aking in o accoun he cul i a s conside ed in each s udy and he analy ical me hod- ologies employed. Two me aboli es belonging o he couma ins g oup we e also ound in he oli e-de i ed issues. Aesculin (C15H16O9; 174.6 Å2), also known as escule in hexoside, was ound exclusi ely in oli e oo s and s ems. The agmen a ion pa e n o his compound e ealed he de ach- men o he suga moie y, eleasing i s aglycone a m/z 177.0192. In con as , aescule in (C9H6O4; 127.5 Å2), a dihyd oxycouma in, was de ec ed in oo s, s ems and lea es, and exhibi ed MS agmen a ion wi h signals a m/z149.0244, 133.0300, 105.0345, and 89.0401. Bo h me aboli es had been p e iously documen ed in a ious ma ices de i ed om oli e ees. 34,36 In gene al, simple phenols and de i a i es we e dis ibu ed h oughou he plan , wi h mos o hem being de ec ed in he h ee o gans unde s udy, al hough some excep ions we e obse ed. Fo example, hyd oxy y osol (153.0557 m/z; 128.8 Å2) was de ec ed in lea es and s ems bu i was no ound in oo s in he dilu ions o ex ac s analyzed. Con a y o ou esul s, Michel and colleagues epo ed he p esence o hyd oxy y osol in he oo s o “Ko oneiki” and “Che oui” cul i a s, albei a low concen a ions. 36 Se ano-Ga cia and coau ho s only quan i ied his simple phenol in he lea es o en oli e cul i a s. 24 Amma e al. obse ed he p esence o hyd oxy y osol in he wood o he oli e cul i a “Chemlali”, bu did no de ec i in ex ac s o “oli e lea es + s ems”. 34 In he same oli e cul i a , Toumi and collabo a o s desc ibe he p esence o hyd oxy y osol in oo s. 40 The subs ance anno a ed as iso e bascoside (C29H36O15; 6.17 min and 223.4 Å2) was ound only in oo issue, whe eas i s isome e bascoside ( 5.73 min and 223.2 Å2) was ound in all Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c07155 J. Ag ic. Food Chem. 2024, 72, 27561−27574 27564 o gans. The e we e o he 4 me aboli es de ec ed in he h ee o gans: wo isome s o hyd oxy y osol glycoside (C14H20O8), y osol glycoside (C14H20O7; 161.4 Å2) and phenyle hyl p ime e oside (C19H28O10: 202.2 Å2). The wo isome s o hyd oxy y osol glucoside we e anno a ed by obse a ion o a dual signal peak in he mobilog am (163.1 Å2and 171.8 Å2) accompanied by a agmen a ion pa e n wi h m/zsignals a 153.055, 135.045 and 123.045; he peak a 163.1 Å2p o ed o be he p edominan one. Acco ding o he li e a u e, one o hese isome s could coincide wi h he hyd oxy y osol 4-O- glucoside p e iously desc ibed in oli e lea es. 41 Lignans and de i a i es we e ound exclusi ely in oli e oo s and s ems. Howe e , al hough many epo s claim he absence o his amily o me aboli es in oli e lea es, o he au ho s ha e epo ed he p esence o ace amoun s o lignans in ha pa icula o gan. 35,42 In he p esen s udy, h ee po en ial isome s o cyclooli il glucoside (C26H34O12; 208.9 Å2, 214.5 Å2and 231.4 Å2), wo isome s o hyd oxypino esinol glucoside (C26H32O12; 229.5 Å2and 215.9 Å2), and wo isome s o ace oxypino esinol glucoside (C28H34O13; 210.7 Å2 and 227.9 Å2) ha e been desc ibed. In all cases, being glycosyla ed compounds, he HRMS/MS spec a consis en ly showed a loss o 162 m/z, con i ming he associa ion wi h a glucose uni a ached o he lignan aglycone. Se e al o hese isome ic s uc u es, al hough appea ing unde a single ch oma og aphic peak, could be elucida ed based on he molecula desc ip o s o he ions and he in ensi y o he peaks in he ion mobili y dimension, as illus a ed in Figu e 1. Fo ins ance, he highes signal o ace oxypino esinol glucoside wi h a CCS o 210.7 Å2was deno ed as (+)-1-ace oxypino - esinol-4′ß-D-glucoside in ag eemen wi h he p edominan s uc u e desc ibed in he li e a u e. 36 In con as , he signal o 227.9 Å2would be consis en wi h 8-ace oxypino esinol-4′- glucoside, based on he p edic ed CCS alue. The p esence o (+)-1-hyd oxypino esinol 4′-ß-D-glucoside and (+)-1-hyd oxypino esinol 1′-ß-D-glucoside has been documen ed o hese ma ices. 34,36 Howe e , al hough we ha e de ec ed 2 isome s, we ha e no been able o a ibu e hese iden i ies o he obse ed peaks, due o lack o consensus on he abundan species; u he s udies a e essen ial o cla i y his. Oli il (C20H24O7), wi h a CCS o 197.8 Å2, was anno a ed om he p ima y agmen s obse ed by HRMS/MS, namely he m/z360.1227, 345.1360, 327.1252, 195.0670, and 179.0713. In he case o cyclooli il (C20H24O7; 205.6 Å2) a agmen a ion pa e n wi h wo clea signals a 360.1228 and 345.1358 m/zwas ob ained. The lignan elu ing la e in he ch oma og aphic p o ile was 1-ace oxypino esinol, wi h a molecula o mula o C22H24O8. D akopoulou and co-wo ke s, in an in e es ing s udy, highligh ed he p esence o wo isome s o ace oxypino esinol a 203.5 Å2(1-ace oxypino esinol) and 285.5 Å2(8-ace oxypino esinol) in ex a i gin oli e oil. 38 Howe e , in ou case, only he signal linked o 1- ace oxypino esinol was de ec ed in he oo and s em ex ac s, wi h a CCS alue in line wi h ha desc ibed by he a o emen ioned au ho s. This p o ides a solid basis o anno a e wi h ce ain y his speci ic con o ma ion. The g oup wi h he highes numbe o me aboli es consis ed o secoi idoids and de i a i es, which a e undoub edly one o he mos ep esen a i e amilies o compounds in oli e ma ices. In Table S1, 27 compounds belonging o his chemical class ha e been desc ibed. P ac ically all o hem we e de ec ed in oli e oo , s em, and lea . Oleu opein (C25H32O13; 217.5 Å2) and some o i s de i a i es we e among he mos ele an subs ances o his g oup, including deme hyl oleu opein (C24H30O13; 213.5 Å2), wo po en ial isome s o hyd oxy oleu opein (C25H32O14; 218.9 Å2and 227.6 Å2), me hoxyoleu opein (C26H34O14; 223.2 Å2), oleu oside (C25H32O13; 217.1 Å2) and h ee isome s o oleu opein aglycone (C19H22O8; 186.0 Å2, 185.2 Å2and 184.8 Å2). Se e al signals de ec ed a 701.229 m/z, wi h a molecula o mula o C31H42O18, would be consis en wi h isome s o he glycosidic o m o oleu opein o neonuzhenide (245.5, 241.8, and 248.9 Å2). The i s wo isome s we e no de ec ed in lea es, and he la e , oge he wi h oleu oside, was absen in oo issue. Ano he no able subg oup o secoi idoids dis ibu ed in he h ee ma ices conside ed we e he Figu e 1. Example o he ex ac ed ion ch oma og am (EIC), mobilog am (EIM) and HRMS/MS spec a o ace oxypino esinol glucoside o p o e he po en ial o TIMS coupling o LC-MS/MS in he de ec ion o hidden isome ic species wi hou ch oma og aphic sepa a ion. Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c07155 J. Ag ic. Food Chem. 2024, 72, 27561−27574 27565 compounds ela ed o elenolic acid. Thei anno a ions we e achie ed by HRMS/MS analysis, e ealing wo isome s o aldehydic o m o deca boxyme hyl elenolic acid glucoside (C16H26O10; 188.8 Å2and 188.6 Å2), i e isome s o elenolic acid glucoside (C17H24O11; 192.5, 189.9, 192.1, 190.1, and 190.4 Å2), elenolic acid dihexose de i a i e (C25H38O18; 231.5 Å2), and elenolic acid dihexose (C23H34O15; 234.9 Å2). The signals de ec ed wi h m/z389.109 a 1.29 and 1.32 min, espec i ely, wi h a molecula o mula o C16H22O11, we e en a i ely anno a ed as oleoside/secologanoside (184.6 Å2 and 189.5 Å2) displaying a agmen a ion wi h m/zsignals o 345.116, 209.044, 183.066, 121.066, and 113.025. Finally, deme hyl ligs oside (C24H30O12: 208.8 Å2), nuzhenide (C31H42O17; 241.1 Å2), lucidumoside C (C27H36O14; 229.1 Å2), and ligs oside (C25H32O12; 214.7 Å2) we e also consis en ly anno a ed in all he oli e ma ices in es iga ed. Fla onoids p o ed o be ano he impo an g oup o phenolic compounds p esen mainly in oli e s ems and lea es. Dihyd okaemp e ol-O-glucoside (C21H22O11; 186.3 Å2), anno- a ed h ough he main MS/MS agmen s a 287.0550, 259.0633, 243.0664, 151.0034, and 125.0245 m/z, and wo isome s o dihyd oque ce in-O-glucoside (C21H22O12; 191.8 Å2and 192.5 Å2) we e ound exclusi ely in s ems o gan. Thei la anonol aglycones, axi olin (C15H12O7; 164.7 Å2) and dihyd okaem e ol (C15H12O6; 163.4 Å2), we e also de ec ed exclusi ely in he s em ex ac s. Bo h compounds we e con iden ly anno a ed as hey showed a ypical agmen a ion pa e n wi h MS signals a 285.0409, 177.0199, and 125.0263 m/z( o axi olin) and 259.0598, 243.0661, 177.0561, 151.0039, and 125.0244 m/z( o dihyd okaem e ol). 34−36 Among he la onoids ha we e sys ema ically ound in s em and lea ex ac s, i is possible o men ion he ollowing: na ingenin-O-glucoside (C21H22O10; 183.1 Å2), u in (C27H30O16; 232.4 Å2), h ee isome s o lu eolin-O-glucoside (C21H20O11; 210.2 Å2, 208.4 Å2, and 210.2 Å2), wo isome s o que ce in-O-glucoside (C21H20O12; 202.1 Å2and 210.9 Å2) and apigenin-7-O-glucoside (C21H20O10; 208.1 Å2). In all cases, he HRMS/MS spec a o hese glycosyla ed compounds e ealed a clea age o he suga (−162 m/z), eleasing he aglycone o m. I is wo h no ing ha he ollowing 4 compounds only appea ed in he oli e lea samples: wo isome s o apigenin-O- u inoside (C27H30O14; 232.7 Å2and 224.5 Å2), diosmin (C28H32O15; 231.8 Å2) and ch ysoe iol-7- O-glucoside (C22H22O11; 215.9 Å2). The wo isome s o apigenin-O- u inoside could no be ully dis inguished and anno a ed by LC-MS. Howe e , elying on he TIMS dimension and ollowing he s a egy illus a ed in Figu e 2, bo h peaks we e ully di e en ia ed. B ie ly, Figu e 2A shows he ex ac ed ion ch oma og am (EIC) o m/z577.1563 wi h a clea shoulde o he le o he main peak (min 6.55 and 6.65). Due o he absence o comple e ch oma og aphic sepa a ion o his glycosyla ed la onoid, an isome ic p o ile scan was pe o med in he mobili y dimension (Figu e 2B). As expec ed, wo dis inc peaks eme ged a 577.1563 m/zin EIM, indica ing he possible p esence o an isome , as hin ed abo e. Subsequen ly, speci ic mobili y alues o each segmen o he coelu ed peak we e e idenced by locking he elu ion ime (m/z577.1563; min 6.4−6.6 and 6.6−7.0) in EIM. HRMS/ MS spec a gene a ed by PASEF e ealed agmen a ion a 269.0458 m/z, p o iding use ul ex a in o ma ion o he anno a ion o he me aboli es. Finally, by me iculous e- ex ac ion o he ea u es by imposing mobili y cons ain s on he EIC, he ini ial o e lap o he apigenin-O- u inoside isome s was un a eled (Figu e 2C). The p edominan peak (224.5 Å2) was assigned as apigenin-7-O- u inoside acco ding o desc ibed in he li e a u e. 43−45 To conclude he o e iew o he desc ibed subs ances belonging o he la onoid amily, h ee me aboli es we e de ec ed in all o gans o all a ie ies: he la anone na ingenin (C15H12O5; 163.0 Å2), and wo la ones, lu eolin (C15H10O6; 160.6 Å2) and apigenin (C15H10O5; 157.6 Å2). Rega ding pen acyclic i e penes, maslinic acid (C30H48O4; 13.13 min and 223.4 Å2), be ulinic acid (C30H48O3; 14.00 min and 220.1 Å2) and oleanolic acid (C30H48O3; 14.14 min and 220.7 Å2) we e also egis e ed in all analyzed pa s o oli e ee. 3.2. Non a ge ed Me abolomics o he Anno a ion o Po en ial Ma ke s Rela ed o VWO-Resis ance Le el on he Basal Me abolic P o iles o Oli e Roo , S em, and Lea Tissues. To in es iga e he po en ial associa ion Figu e 2. Th ee-s ep-s a egy used o he comple e esolu ion o he o e lapping peaks o apigenin-O- u inoside in oli e lea ex ac s by inco po a ing he ion mobili y dimension in o LC-HRMS/MS me hodology. Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c07155 J. Ag ic. Food Chem. 2024, 72, 27561−27574 27566 be ween VWO esis ance le els and he basal me abolic p o iles o oli e o gans, all LC-TIMS-HRMS/MS da a, ex ac ed as de ailed in Sec ion 2.5, we e ho oughly analyzed using mul i a ia e s a is ical app oaches. Ini ially, unsupe ised PCA was employed o assess da a quali y, biodi e si y, and na u al clus e ing o samples by ma ix; howe e , his analysis did no e eal a clea na u al clus e ing be ween g oups. Despi e his, Ho elling’s T2 (95%) and DModX (DC i 0.05) plo s we e ca e ully e alua ed o de ec po en ial ou lie s ac oss he mul idimensional PCA space. Subsequen es s de e mined ha he dis an posi ioning o he suspec ed ou lie s was a ibu ed o he inhe en he e ogenei y o biological speci- mens. In e e y ins ance, hese samples emained in p oximi y o hei biological eplica es and hei exclusion did no enhance he model. The PCA model o oo s was ep esen ed by PC2 and PC3, explaining 9.28 and 7.74% o he a iance, Figu e 3. Two-class PLS-DA models and pe mu a ion es s o oli e oo , s em and lea issues o he disc imina ion o highly esis an (HR) and suscep ible (S) cul i a s o V. dahliae. Do s in PLS-DA plo s ep esen di e en cul i a s: highly esis an (HR: blue), esis an (R: o ange), medium suscep ible (MS: yellow), suscep ible (S: g een). Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c07155 J. Ag ic. Food Chem. 2024, 72, 27561−27574 27567 Table 2. Me aboli es om Oli e Roo , S em, and Lea O gans om VWO-Highly Resis an (HR) and Suscep ible (S) Oli e Cul i a s ha Exhibi ed he Highes Rele ance in PLS-DA Models (VIP ≥1.50) pu a i e compound iden i y chemical class a RT (min) m/zexp molecula o mula e o (ppm) mSigma CCS (Å2) main agmen s ia MS/MS R.C. VIP e . Oli e Roo s o HR-Cul i a s unknown 1 12.75 277.1661 C13H26O61.62 48.4 180.3 233.1531 −0.065 2.03 sinapyl alcohol(8- 5)coni e yl aldehyde de i a e lignans 6.95 337.1080 C20H18O5−0.02 8.4 189.7 322.0871; 307.0617; 291.0652 −0.056 1.95 45 unknown 2 (po en ial isome ) 1.31 267.0722 C9H16O90.91 14.2 200.8 113.0222; 75.0088 −0.023 1.93 anilloyl glucoside/ anillic acid hexoside phenolic acids 1.28 329.0876 C14H18O9−0.50 9.1 183.3 167.0357; 152.0107; 123.0450; 108.0218 −0.043 1.86 43,47 unknown 3 4.86 313.0929 C14H18O80.15 17.4 177.6 151.0406; 150.0333 −0.051 1.82 guaiaconic acid lignans 6.51 339.1238 C20H20O50.13 41.4 193.8 324.1001; 310.0779; 309.0770; 281.0840 −0.034 1.75 MS/MS Lib. me hyl galla e glucoside phenolic acids 1.35 345.0829 C14H18O10 0.41 43.6 172.7 - −0.034 1.70 48 unknown 4 1.33 557.2084 C22H38O16 −0.46 13.1 223.3 389.1088; 375.1288; 213.0769 −0.043 1.67 unknown 2 (po en ial isome ) 1.25 267.0721 C9H16O9−0.08 15.7 151.2 113.0230; 75.0080 −0.043 1.67 D-manni ol suga s 1.40 181.0719 C6H14O60.67 8.9 131.1 101.0257; 85.0307; 71.0145 −0.014 1.55 34 unknown 5 1.39 523.1878 C18H36O17 −0.24 11.0 207.2 341.1091 −0.005 1.53 lac one (es e wi h hyd oxy y osol) simple phenols 6.47 321.1342 C17H22O6−0.24 19.1 168.2 185.0820; 111.0823; 59.0142 0.028 1.68 49 unknown 6 7.37 465.2132 C24H34O90.39 28.3 208.9 - 0.051 1.63 elenolic acid dihexose de i a e secoi idoids 4.87 625.1986 C25H38O18 0.05 6.9 231.5 223.0601; 179.0564; 119.0353 0.051 1.61 23 hyd oxy y osol glucoside de i a i e simple phenols 6.81 481.2078 C24H34O10 −0.35 23.5 209.5 315.1078; 135.0441; 101.0231 0.049 1.56 36 unknown 7 1.30 237.0618 C8H14O80.66 16.3 144.8 87.0090 0.006 1.55 unknown 8 1.31 279.0512 C13H12O70.55 24.4 151.3 207.0705; 189.0584; 115.0180 0.050 1.54 unknown 9 6.97 569.2237 C27H38O13 0.13 11.0 214.6 537.1977; 403.1259; 223.0604; 121.0292 0.030 1.52 Oli e Roo s o S-Cul i a s cyclooli il glucoside (is. 3) lignans 5.16 537.1975 C26H34O12 −0.27 21.0 214.5 375.1449; 195.0665; 179.0700 −0.124 2.33 23 D-sedohep ulose suga s 1.27 209.0667 C7H14O70.47 10.1 139.0 85.0294; 84.0211; 78.9592; 59.1249 −0.111 1.93 44 unknown 10 (po en ial isome ) 1.30 207.0664 C11H12O4−0.12 7.8 146.6 −0.061 1.63 unknown 10 (po en ial isome ) 1.30 207.0661 C11H12O4−0.81 13.0 178.1 −0.062 1.63 unknown 11 6.70 199.1340 C11H20O30.09 11.3 148.1 0.078 1.64 maslinic acid monohyd oxyla ed de i a i e pen acyclic i e penes 11.22 487.3426 C30H48O5−0.35 11.3 225.4 0.077 1.57 35 phenyle hyl p ime e oside simple phenols 5.78 415.1609 C19H28O10 −0.12 4.6 202.2 149.0444 0.072 1.54 35 anilloyl glucoside/ anillic acid hexoside phenolic acids 1.28 329.0876 C14H18O9−0.50 9.1 183.3 167.0357; 152.0107; 123.0450; 108.0218 0.063 1.51 43,47 unknown 2 (po en ial isome ) 1.31 267.0722 C9H16O90.91 14.2 200.8 113.0222; 75.0088 0.032 1.50 Oli e S ems o HR-Cul i a s unknown 12 7.38 283.1187 C14H20O6−0.01 13.9 166.3 199.0959; 181.0492; 139.0378; 123.0447; 99.0450; 83.0167 0.111 3.36 elenolic acid-me hyl es e secoi idoids 5.59 255.0875 C12H16O60.28 13.3 156.4 153.0572; 101.0242; 83.0139 0.107 3.28 35 unknown 13 9.87 277.1804 C17H26O3−0.72 26.5 171.6 233.1531; 205.1627; 59.0144 0.088 2.94 hyd oxydeca boxyme hyl elenolic acid secoi idoids 1.40 199.0609 C9H12O5−0.70 20.7 138.5 155.0710 0.073 2.23 35 unknown 14 1.47 363.1659 C16H28O9−0.46 31.5 177.2 181.0717 0.054 1.90 unknown 15 1.33 353.0878 C16H18O90.09 19.3 185.8 191.0536; 111.0798 0.050 1.60 dihyd oque ce in-O-glucoside (is. 1) la onoids 4.67 465.1036 C21H22O12 −0.54 12.9 191.8 303.0505; 285.0399; 177.0191; 125.0261 −0.037 1.58 50 dihyd okaemp e ol la onoids 6.30 287.0560 C15H12O6−0.30 2.9 163.4 259.0598; 243.0661; 177.0561; 151.0039; 125.0244 −0.020 1.51 35 Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c07155 J. Ag ic. Food Chem. 2024, 72, 27561−27574 27568 Table 2. con inued pu a i e compound iden i y chemical class a RT (min) m/zexp molecula o mula e o (ppm) mSigma CCS (Å2) main agmen s ia MS/MS R.C. VIP e . Oli e S ems o S-Cul i a s sinapyl alcohol(8- 5)coni e yl aldehyde de i a e lignans 6.95 337.1080 C20H18O5−0.02 8.4 189.7 322.0871; 307.0617; 291.0652 0.126 2.67 45 unknown 16 1.21 333.0623 C16H14O82.58 28.7 165.1 241.0129; 217.0518; 78.9594 0.096 1.58 unknown 17 (po en ial isome ) 4.86 333.1555 C15H26O8−0.78 13.1 175.9 0.084 1.51 hyd oxypino esinol glucoside (is. 2) lignans 6.33 535.1822 C26H32O12 0.25 8.9 229.5 373.1290 −0.060 1.82 23 hyd oxypino esinol glucoside (is. 1) 535.1820 −0.16 2.8 215.9 373.1289; 355.1189; 295.0998 −0.049 1.81 unknown 18 7.39 315.1813 C16H28O6−0.03 2.9 176.8 297.1695; 187.1334; 145.05076; 101.0605; 83.0506 −0.102 1.66 Oli e Lea es o HR-Cul i a s unknown 19 1.39 395.1558 C16H28O11 −0.26 10.6 188.9 213.0771; 151.0765 0.109 2.19 loganic acid i idoids 1.30 375.1296 C16H24O10 0.07 22.7 184.7 213.0764; 169.0876; 151.0752; 125.0606; 113.0244; 107.0499 0.131 2.13 34 unknown 17 (po en ial isome ) 5.73 333.1553 C15H26O8−0.45 20.4 175.1 0.123 1.65 unknown 20 (po en ial isome ) 1.33 349.1502 C15H26O9−0.69 38.7 217.7 0.104 1.60 ihyd oxyoc adecadienoic acid a y acids 7.76 327.2178 C18H32O50.13 18.7 182.3 291.1971; 229.1439; 211.1338; 171.1006 0.078 1.54 35 unknown 15 1.33 353.0878 C16H18O90.09 19.3 185.8 191.0536; 111.0798 0.060 1.50 dihyd oxyhexadecanoic acid a y acids 8.69 287.2228 C16H32O4−0.06 17.0 170.3 −0.072 1.50 35 Oli e Lea es o S-Cul i a s 1-sinapoyl-2- e uloylgen iobiose phenolic acids 6.36 361.1041 [M−H−H]2−C33H40O18 1.94 4.9 315.0 0.126 2.22 51 unknown 21 7.98 481.1361 [M−H−H]2−C44H52O24 1.78 50.0 348.4 0.141 1.80 na ingenin la onoids 7.77 271.0613 C15H12O50.35 1.6 163.0 151.0028; 119.0500 0.057 1.78 52 dihyd oxyhexadecanoic acid a y acids 8.69 287.2228 C16H32O4−0.06 17.0 170.3 0.047 1.69 35 lu eolin-O-glucoside (is. 2) la onoids 6.85 447.0931 C21H20O11 −0.45 8.3 208.4 285.0405; 133.0297 0.030 1.67 34 unknown 11 6.70 199.1340 C11H20O30.09 11.3 148.1 0.101 1.60 unknown 20 (po en ial isome ) 1.30 349.1502 C15H26O9−0.57 18.7 178.7 0.108 1.55 couma oyl hexoside phenolic acids 4.95 325.0928 C15H18O8−0.24 14.0 180.7 145.0292; 119.0485; 117.0340 0.084 1.54 MS/MS Lib. apigenin 7-O-glucoside la onoids 6.73 431.0984 C21H20O10 −0.03 10.5 208.1 269.0459 0.032 1.53 52 aldehydic o m o deca boxyme hyl elenolic acid glucoside (is. 1) secoi idoids 1.27 377.1452 C16H26O10 −0.15 8.6 188.8 197.0821; 153.0921 0.069 1.53 45 apigenin la onoids 8.80 269.0456 C15H10O50.04 6.5 157.6 225.0554; 201.0547; 149.0238 0.075 1.51 52 hyd oxy y osol glucoside (is. 1) simple phenols 4.33 315.1084 C14H20O8−0.31 6.4 163.1 153.0559; 135.0451; 123.0459 0.003 1.50 45 ihyd oxyoc adecanoic acid a y acids 8.41 331.2488 C18H36O5−0.37 17.0 180.3 157.1272 −0.010 1.65 35 oc adecanedioic acid a y acids 9.86 313.2383 C18H34O4−0.32 12.2 180.4 295.2291; 277.2165 −0.015 1.62 53 maslinic acid pen acyclic i e penes 13.13 471.3479 C30H48O4−0.30 12.7 223.4 −0.088 1.57 35 ihyd oxyoc adecadienoic acid a y acids 7.76 327.2178 C18H32O50.13 18.7 182.3 291.1971; 229.1439; 211.1338; 171.1006 −0.117 1.56 35 a Including glycosyla ed o ms and de i a i es wi hin hese chemical classes; R.C: eg ession coe icien ; MS/MS Spec al Lib a y accessed: MSMS_Public_EXP_Neg_VS17; unde lined compounds indica e hei p e ious anno a ion in he ini ial sc eening (Sec ion 3.1). Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.4c07155 J. Ag ic. Food Chem. 2024, 72, 27561−27574 27569