Protein Biomarkers of Bovine Defective Meats at a Glance: Gel-Free Hybrid Quadrupole-Orbitrap Analysis for Rapid Screening

P o ein Bioma ke s o Bo ine De ec i e Mea s a a Glance: Gel-F ee
Hyb id Quad upole-O bi ap Analysis o Rapid Sc eening
En ique Sen and eu, Claudia Fuen e-Ga cía, Olga Pa do, Mamen Oli án, Nu ia León, Noelia Aldai,
Vicen Yusa
, and Miguel A. Sen and eu*
Ci e This: J. Ag ic. Food Chem. 2021, 69, 7478−7487
Read Online
ACCESS Me ics & Mo e A icle Recommenda ions *
sıSuppo ing In o ma ion
ABSTRACT: An unde s anding o biological mechanisms ha could be in ol ed in he s ess esponse o animal ca le p io o
slaugh e is c i ical o c ea e effec i e s a egies aiming a he p oduc ion o high-quali y mea . The sa coplasmic p o eome o di ec ly
ex ac ed samples om no mal and high ul ima e pH (pHu) mea g oups was s udied h ough a s aigh o wa d gel- ee s a egy
suppo ed by liquid ch oma og aphy hyb id quad upole-O bi ap high- esolu ion mass spec ome y (LC-HRMS) analysis. A
s epped p o eomic pipeline combining apid bioma ke hun ing suppo ed by quali a i e p o ein Masco sco es ollowed by a ge ed
label- ee pep ide quan ifica ion e ealed 26 desc ip o s ha cha ac e ized mea g oups assayed. The unc ional s udy o he
p oposed bioma ke s sugges ed hei ele an ole in me abolic, chape one/s ess- ela ed, muscle con ac ili y/fibe o ganiza ion,
and anspo ac i i ies. The efficiency, flexibili y, apidi y, and easiness o he me hodology p oposed can posi i ely con ibu e o he
c ea ion o inno a i e p o eomic al e na i es add essing mea quali y assessmen .
KEYWORDS: high- esolu ion p o eomics, mea bioma ke s, mea quali y assessmen , apid p o eomic sc eening, p e-slaugh e s ess
■INTRODUCTION
Consume s a e cu en ly wo ied abou e hics o ood
p oduc ion conce ning he implemen a ion o animal wel a e
policies, g ea ly influencing hei final decision on p oduc
selec ion.
1
In his ega d, he Eu opean Union is leading he
p omo ion o animal well-being ac ions as a way o achie e
sus ainable de elopmen in he p oduc ion o human ood-
s uffs.
2
P e-slaugh e s ess (PSS) is one o he mos ele an
issues among diffe en condi ioning ac o s ela ed o animal
ca e ha can g ea ly affec he quali y o mea , causing he
occu ence o de ec i e da k, fi m, and d y (DFD) mea ha is
no mally cha ac e ized by an ul ima e pH (pHu) ≥6.0. Food
au ho i ies conside ha pHu alues highe han 6.0 a 24 h
pos mo em a e in ima ely associa ed wi h PSS animals
3
and
DFD mea s.
4
The e o e, ea ly de ec ion o high pHu mea s in
he ood chain is c i ical o he indus y
5
since de ec i e aw
ma e ial causes significan economic losses.
6
Un o una ely, he
alue o pHu assessmen is comp omised since high alues do
no necessa ily gua an ee he p esence o ue DFD mea s,
6
equi ing new diagnos ic s a egies. The efficiency o
p o eomic esea ch o he hun ing o p edic i e high pHu
p o einbioma ke ss andsupasano elapp oach o
disc imina e mea om no mal and PSS animals.
7−11
Howe e ,
esul s achie ed o da e s ongly elied on gel-based me hod-
ologies ha p o ided an excellen esol ing powe while s ill
ha ing impo an cons ain s such as he limi ed numbe o
iden ified p o ein bioma ke s and inaccu a e quan i a i e
esul s. Fu he mo e, he applica ion o such app oaches can
be hinde ed by cu en ends in g een analy ical chemis y
conce ning he use o haza dous and nonsus ainable chemicals
(i.e., ac ylamide) and excessi e ene gy consump ion by
me ging wo p o ein pu ifica ion s eps.
12
Nowadays, he de elopmen o as , efficien , sus ainable,
and s aigh o wa d p o eomic s a egies is highly demanded
o mea quali y assessmen . As an affo dable gel- ee app oach
o he disco e y o pep ide bioma ke s, Sen and eu e al.
13
p oposed he use o OFFGEL ac iona ion ollowed by liquid
ch oma og aphy- andem mass spec ome y (LC-MS/MS)
analysis ea u ed by a con en ional explo a o y de ec o
( h ee-dimensional ion ap, 3D-IT) o simul aneous qual-
i a i e/quan i a i e analysis o he bo ine p o eome. Al hough
his accessible solu ion can be eadily inco po a ed by indus y,
he use o low- esolu ion mass spec ome y (LC-LRMS)
de ec ion es ic ed he esea ch o only no iceably abundan
pep ides, ha ing addi ional limi a ions such as he inclusion o
he OFFGEL ac iona ion s ep ha ex ended he sampling
p ocedu e. Such d awbacks can be o e aken by liquid
ch oma og aphy high- esolu ion mass spec ome y (LC-
HRMS) analysis conside ing he sensi i i y and high-ou pu
capaci y o cu en de ices. Mo eo e , minimal unce ain ies
achie ed by HRMS de ec ion a o simplifica ion o sample
p epa a ion h ough app op ia e backg ound disc imina ion.
Rega ding inno a i e analy ical al e na i es aiming a he
de elopmen o efficien holis ic p o eomic app oaches
eques ed by sys em biology s udies, LC-HRMS analysis
pe ec ly fi s ou ine and basic esea ch expec a ions by
minimizing he unce ain y o de e mina ions.
14
Howe e ,
Recei ed: Ap il 7, 2021
Re ised: June 9, 2021
Accep ed: June 10, 2021
Published: June 25, 2021
A iclepubs.acs.o g/JAFC
© 2021 The Au ho s. Published by
Ame ican Chemical Socie y 7478
h ps://doi.o g/10.1021/acs.ja c.1c02016
J. Ag ic. Food Chem. 2021, 69, 7478−7487
Downloaded ia UNIV DEL PAIS VASCO on Sep embe 21, 2021 a 10:44:06 (UTC).
See h ps://pubs.acs.o g/sha ingguidelines o op ions on how o legi ima ely sha e published a icles.
implemen a ion o LC-HRMS echnology in mea quali y
assessmen occu ed in e y ecen yea s conside ing mainly
bo om-up p o eomic s udies o ood au hen ica ion and he
de ec ion o adul e a ions in p ocessed p o ein-based ood-
s uffs.
15
In con as o sensi i e p o ein analysis commonly
suppo ed by a ge ed selec ed/mul iple eac ion moni o ing
(SRM/MRM) app oaches,
16
he e a e a limi ed numbe o
p o eomic al e na i es ca ying ou he simul aneous qual-
i a i e and quan i a i e esea ch o PSS bioma ke s by LC-
HRMS analysis. Recen s udies suppo ed by s a e-o - he-a
O bi apRand ion mobili y ime-o -fligh (TOF) echnologies
success ully de e mined p o eins linked o pHu in bo ine
mea .
5,17,18
Ne e heless, implemen a ion o hese efficien
s a egies can be discou aging by he complex sampling
p ocedu es p oposed, such as double ypsin diges ion,
p elimina y desal ing, molecula weigh cu offul afil a ion,
sample educ ion, p o ein sepa a ion by bidimensional
ch oma og aphy, and/o pe o ming a subp o eome assay
es ic ed o a pa icula cell o ganelle (mainly mi ochond-
ion). Thus, inno a i e app oaches a e needed o de elop
apid, easy, and high-ou pu LC-MS me hodologies o he
hun ing o PSS p o eomic p edic o s ha can be sui able o
bo h gene al o e iew and mo e a ge ed ou ine analyses in
mea quali y esea ch.
This wo k aims o demons a e he use ulness o a
s aigh o wa d quali a i e and quan i a i e LC-HRMS me h-
odology o he apid sc eening o p o ein bioma ke s linked o
mea quali y. Di ec analysis o sa coplasmic p o ein ex ac s
om no mal and high pHu mea g oups was s udied by hyb id
quad upole-O bi ap analysis. P elimina y p o ein cha ac e -
iza ion ollowed by a ge ed pep ide quan i a i e analysis led o
he en a i e iden ifica ion o po en ial mea bioma ke s.
Func ional analysis and s udy o he in e ac ion ne wo k o
he p oposed p o ein desc ip o s acili a ed he unde s anding
o diffe en biochemical pa hways ha could be in ol ed in he
PSS esponse. The simplici y and high efficiency o his
me hodology can acili a e i s easy implemen a ion in mul i-
pu pose ac i i ies add essing apid mea quali y assessmen .
■MATERIALS AND METHODS
Reagen s and Sol en s. LC-MS g ade ace oni ile (ACN),
o mic acid (FA), e hylenediamine e aace ic acid (EDTA, 99%
pu i y), T is buffe (99% pu i y), and 0.45 μm PVDF fil e s we e om
Scha lab (Scha lab S. L., Ba celona, Spain). Ul apu e g ade wa e was
om Millipo e (EMD Millipo e Co., Bille ica, MA). Suc ose, p o ease
inhibi o cock ail (P8340), and ammonium bica bona e we e om
Sigma-Ald ich (Sigma-Ald ich Co., S . Louis, MO). Modified ypsin
was om P omega (P omega, Madison, WI).
Sample P epa a ion. Mea samples om 12 c ossb ed animals
belonging o As u iana de los Valles xF iesian b eed we e collec ed
om a comme cial aba oi in no heas e n Spain. Muscle samples
we e om Longissimus ho acis e lumbo um (LTL) o yea ling bulls
slaugh e ed a 14−15 mon hs o age acco ding o EU egula ions
(Council Regula ion (EC) No. 853/2004 and No. 1099/2009). A 24
h pos mo em, 10 g o he LTL muscle was excised om he 13 h ib,
and he epimysium was dissec ed. Mea samples we e immedia ely
acuum-packed and s o ed a −80 °C un il p ocessed o p o ein
ex ac ion. Muscle samples we e classified in o wo diffe en g oups
acco ding o hei pHu alues: no mal pHu samples (NORMAL, n=
6) wi h pHu alues below 6.0 (in ou case: 5.53 ±0.14) and high
pHu samples (HIGH, n= 6) wi h pHu alues highe han 6.0 (in ou
case: 6.56 ±0.25). De e mina ion o pH was pe o med a he six h
ib o he LTL muscle a 24 h pos mo em.
Sa coplasmic p o eins we e ex ac ed acco ding o Fuen e-Ga ci

ae
al.
9
B iefly, hal a g am o he muscle sample was homogenized in 4
mL o ex ac ion buffe (10 mM T is pH 7.6 con aining 0.25 M
suc ose, 1 mM EDTA, and 25 μL o p o ease inhibi o cock ail),
cen i uged a 20 000g o 20 min a 4 °C, and he supe na an was
fil e ed h ough a 0.45 μm PVDF fil e . One hund ed mic oli e s o
each sample was mixed wi h 300 μL o chilled E OH (con aining
0.15% FA), o exed (20 s), s o ed a −20 °C o 20 min, and
cen i uged a 3600g o 30 min a 4 °C. The supe na an was
disca ded and he esul ing pelle was comple ely desicca ed in an
SPD121P SpeedVac acuum concen a o (The mo Scien ific, San
Jose, CA). Diges ion o d ied samples was ca ied ou by adding 15
μL o a sequencing g ade modified ypsin solu ion a a 12.5 μg/mL
concen a ion and mixed wi h 20 μL o 50 mM ammonium
bica bona e (pH 8.5), being he mix u e incuba ed o e nigh a 37
°C wi h con inuous gen ly shaking. T yp ic diges s we e acuum-d ied
as p e iously desc ibed. The samples we e esuspended in 80 μLo an
aqueous 0.1% FA solu ion. Fo y mic oli e s o each biological
eplica e (n= 6) was pooled acco ding o assayed g oups (no mal s
high), gi ing ise o a final alue o 240 μL each. Pooled sample
g oups we e spiked wi h 60 μL o he in e nal s anda d (IS) solu ion
composed by a yp ic hyd olysa e o an almond (P unus dulcis)
p o ein ex ac as desc ibed by Sen and eu e al.,
13
cen i uged a
20 000g o 5 min, sepa a ely pou ed in o LC ials and kep a −80 °C
un il LC-HRMS analysis.
The s udy o pooled sample g oups aimed a he de elopmen o a
s aigh o wa d me hodology o he apid sc eening o mea quali y
bioma ke s. Al hough his p ocedu e educes biological diffe ences o
indi iduals ( eplica es), i inc eases he powe o de ec changes
be ween he a e aged samples (mea g oups) o med. Despi e he
undesi able dilu ion effec ha pooling can cause insensi i i y o
pep ides om some low-abundan p o eins, he ad an ages o his
s a egy in e ms o obus ness and ime efficiency alo ize i s use o
apid bioma ke hun ing.
LC-HRMS Analysis. Ch oma og aphic analysis was pe o med on
a The mo Vanquish Flex UHPLC sys em equipped wi h a qua e na y
pump, a acuum degasse , and an open au osample wi h a
empe a u e con olle (The mo Fishe Scien ific, San Jose
, CA).
Ch oma og aphic sepa a ion o yp ic pep ides was pe o med on a
150 mm ×2.1 mm, 3 μm pa icle-size Luna Omega PS C18 column
(Phenomenex Inc, To ance, CA) wi h he ollowing sepa a ion
condi ions: sol en A, wa e /FA (99.9:0.1); sol en B, ACN/FA
(99.9:0.1); sepa a ion g adien , ini ially 0% B, held o 15 min, linea
0−20% B in 2 min, held o 4 min, 40% B in 0.1 min, held o 9.9 min,
100% B in 0.1 min, washing wi h 100% B o 9.9 min, 0% B in 0.1
min, and column equilib a ion o 54.9 min; o al un ime, 95 min;
flow a e, 50 μL/min; and injec ion olume, 5 μL. Column flow was
conduc ed in o he MS sys em du ing he 1.2−90 min ime ange
di e ing he es o he un ime o was e. Au osample and column
empe a u es we e se a 10 and 25 °C, espec i ely.
Mass spec ome y analysis was ca ied ou on a hyb id quad uple-
O bi ap The mo Q Exac i e de ec o equipped wi h a hea ed
elec osp ay (H-ESI) sou ce ope a ing in posi i e ion mode (The mo
Fishe Scien ific, B emen, Ge many). The samples we e s udied by
me ging ull MS1and da a-dependen MS/MS (dd-MS2) analyses. A
ull desc ip ion o HRMS de ec ion condi ions is de ailed in he
Supplemen a y File.
The LC-MS pla o m o analysis was con olled by a PC ope a ing
he Xcalibu . 2.2 SP1.48 so wa e package (The mo Scien ific, San
Jose, CA).
Quali a i e Analysis o MS/MS Da a (P o ein Mapping).
P o eins we e iden ified h ough in e oga ion o dd-MS2da a by a
licensed Masco .2.7 sea ch engine (www.ma ixscience.com)
loading UP9136_B-Tau us and NCBIp o da abases wi h he
ollowing se ings: enzyme, ypsin; no fixed o a iable modifica ions
bu enabling he “E o ole an ”op ion; pep ide ole ance
(monoiso opic) was 6 ppm and 0.02 Da o ull MS1and MS/MS
analyses, espec i ely; pep ide cha ge, +1 o +4; and axonomy
es ic ion pa ame e , Mammalia. The samples we e u he
in e oga ed by loading he NCBIp o da abase, indica ing “ i idi-
plan ae, g een plan s”as a axonomy o he elucida ion o almond
pep ides used as IS. The decoy op ion was used o es ima e alse
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.1c02016
J. Ag ic. Food Chem. 2021, 69, 7478−7487
7479
posi i e a es by means o a alse disco e y a e (FDR) h eshold o
1%. Only hose iden ifica ions ha ha e a p o ein sco e de i ed om
indi idual pep ide ion sco es indica ing iden i y o ex ensi e
homology (p< 0.05) we e conside ed as ue p o ein iden ifica ions.
Quan i a i e Analysis o MS1Da a (Label-F ee Pep ide
Quan ifica ion). Iden ified p o eins om mea g oups assayed we e
apidly sc eened acco ding o hei indi idual Masco p o ein sco e
achie ed by loading he UP9136_B-Tau us da abase. Assignmen s
exclusi ely ound in no mal o high samples we e immedia ely
conside ed (p ima y bioma ke candida es) o u he quan i a i e
MS1analysis. Then, p o ein sco e a ios o he iden ified p o eins
sha ed by bo h mea g oups we e calcula ed as sugges ed by
Sen and eu e al.
13
ega ding he u ili y o p o ein sco es as a coa se
indica o o hei abundance. Only hose p o ein pai s (seconda y
bioma ke candida es) ha ha e a minimum 2- old change a ia ion
in hei p o ein sco e a ios we e conside ed. Bo h p ima y and
seconda y candida es popula ed he p elimina y lis o po en ial
p o ein bioma ke s (Table S3, discussed below). Label- ee MS1
quan ifica ion o p o eo ypic pep ides (wi h he maximum ion
sco e) om sugges ed candida es helped o efine p e ious ough
esul s jus conside ing p o ein sco es om Masco analysis. F eely
a ailable MZmine 2 .2.53 (h p://mzmine.gi hub.io/download.h ml)
loading an in-house lib a y (Table S1), lis ing he a o emen ioned
cha ac e is ic pep ides o p elimina y p o ein candida es, p ocessed
MS1da a as indica ed by Sen and eu e al.
13
wi h some modifica ions.
Fo be e esul s, me ged MS1−dd-MS2 aw da a files needed
demul iplexing (h p://p o eowiza d.sou ce o ge.ne /)andonly
isola ed MS1in o ma ion was handled o accu a e quan ifica ion
using he ollowing op imized se ings: mass ole ance, 5 ppm;
minimum scans-ac oss-peak (scan a e) o eliable quan ifica ion, 8;
19
e en ion ime ole ance o lib a y in e oga ion, 0.5 min; and
e en ion ime ole ance o ch oma og ams alignmen , 1 min.
Ch oma og aphic esul s we e app op ia ely no malized h ough
spiked IS and pep ide a ios om mea g oups assayed (no mal/
high o high/no mal) we e finally con o med o elabo a e he
defini i e lis o p o ein bioma ke s discussed in p o ein unc ional
analysis.
P o ein Func ional Analysis. P oposed p o ein bioma ke s we e
classified conside ing hei biological p ocess (BP) and cellula
componen (CC) om Gene On ology (GO) e ms powe ed by
AmiGO websi e (h ps://amigo.geneon ology.o g/amigo/), KEGG
pa hway, anno a ed keywo ds (UniP o da abase), and local ne wo k
clus e ound in unc ional en ichmen analysis pe o med by
STRING .1.11.1 eewa e (ELIXIR, Wellcome Genome Campus,
Hinx on, Camb idgeshi e, U.K., h ps://s ing-db.o g). P o ein−
p o ein in e ac ion s eng h among bioma ke s s udied was assessed
by STRING analysis, selec ing “Bos Tau us”as he o ganism o
pe o m in e oga ions. The esul s we e u he p ocessed by
Cy oscape .3.8.2 (h ps://cy oscape.o g)
20
o elucida e p o ein
ne wo ks.
■RESULTS AND DISCUSSION
O iginal LC-HRMS (bo h me ged MS1−dd-MS2and isola ed
MS1expe imen al esul s in he mzXML o ma ) and Masco
gene ic o ma (mg ) da a files gene a ed in his s udy a e
eely a ailable a h p://hdl.handle.ne /10261/228237.
Accu acy o High-Resolu ion MS/MS Da a In e -
oga ion o P o ein Mapping. FDR and ex ensi e
homology iden ifica ion cons ain s conside ed in his s udy
(see he Ma e ials and Me hods) made he Masco s udy
p opose hose eliable en a i e p o ein iden ifica ions ha
ha e a minimum indi idual ion sco e o 19 (Figu es S1A and
S1B o no mal and high, espec i ely). Table S2 lis s iden ified
p o eins om no mal (cu si e ed) and high pHu (bold black)
pooled samples acco ding o iden ifica ion cons ain s
conside ed. P o ein amilies will be men ioned h oughou
he ex acco ding o ac onyms de ailed in Table S2.
Since he sea ch o bioma ke s can be limi ed by
unce ain ies de i ed om LC-MS de ec ion, his s udy
ocused on he acquisi ion o unambiguous esul s based on
accu a e HRMS analysis. As an example, Figu e S2 shows he
p o ein sequence co e age o p o ein amilies iden ified in
mea g oups assayed wi h ei he high o low p o ein sco es
achie ed (GAPDH wi h a sco e o 523 and BIN1 wi h a sco e
o 22 in he no mal sample, Figu e S2A,B, espec i ely, and
HSPA8 wi h a sco e o 315 and DBI wi h a sco e o 43 in he
high pHu sample, Figu e S2C,D, espec i ely). Obse ed
pep ide mass de ia ions we e below 5 ppm in mos cases (see
he ppm column and dispe sion o mass accu acy e o s
molecula weigh in Figu e S2). High mass accu acy allowed
ha in gene al, Masco analysis could g ea ly educe
unce ain ies h ough he p oposal o a unique amino acid
sequence pe in e oga ed que y (MS/MS agmen a ion
pa e n om yp ic pep ides), as shown in Figu e S3 (S3a
and S3b o pep ides om GAPDH and BIN1 in he no mal
sample, espec i ely, and S3C and S3D o HSPA8 and DBI in
he high sample, espec i ely). In addi ion o uni ocal amino
acid sequence ma ches, some p o ein amilies de ailed in Table
S2 lis ed que ies wi h diffe en easible possibili ies. Thei
unambiguous amino acid sequence assignmen was eased
h ough he e y no iceable sco e diffe ence mos ly ound
be ween he fi s and he es o he p oposed sequence
al e na i es (Figu e S4), co esponding o a pep ide om
PYGM in he no mal sample.
P elimina y Elucida ion o P o ein Bioma ke s om
Mea G oups Assayed. A apid elucida ion o po en ial
bioma ke s was achie ed h ough he elimina ion o hose
p o eins om Table S2 ha we e sha ed by bo h mea g oups
assayed (Table S3A, 30 p o eins). Ve y clea ly, he numbe o
specific p o ein bioma ke s (p e iously men ioned as p ima y
bioma ke candida es) was significan ly highe (24 s 6) in he
high pHu sample compa ed o i s no mal coun e pa (bold
black s i alic ed assignmen s in Table S3A). The esul s can
be unde s ood conside ing p e ious s udies in sa coplasmic
p o ein ex ac s de ailing how high pHu mea om s essed
animals has an al e ed p o eome exhibi ing cha ac e is ic
p o eins such as hea shock p o eins and α-c ys allin B.
9
Fu he mo e, low p o ein sco es shown by assignmen s lis ed
in Table S3A (22−115 ange) sugges ed hei low abundance
in he sa coplasmic p o eome o mea g oups assayed. In
con as , mos p o eins ound in bo h sample g oups had
simila Masco sco es and yielded sco e a ios up o a 1.5- old
diffe ence (Table S2), sugges ing hei sca ce ele ance as
disc iminan s. In e es ingly, ew sha ed species (AHNAK,
HSPA8, HSPB1, LDHA, PGM1, and TF, p e iously
men ioned as seconda y bioma ke candida es; Table S3B)
showed ema kable diffe ences in hei abundance as sugges ed
by hei old change a ia ion (minimum Masco sco e a io o
2).
MS1Label-F ee Quan i a i e Analysis o Tes he
Reliabili y o P elimina y Bioma ke Elucida ion. The
use ulness o he p o ein sco e (isola ed and a ioned) as a
coa se bu apid semiquan i a i e indica o was assessed by
label- ee MS1quan ifica ion o p o eo ypic pep ides om
candida es lis ed in Tables S3A and S3B (a o al o 36 pep ides
me ged in Table S1). Table S4 shows he ela i e MS1
quan i a i e esul s o he a ge ed LC-MS analysis ca ied
ou in his esea ch. F om he ini ial 36 candida es, only DBI
and ACAA2 we e exclusi e o he high pHu sample g oup
(Table S4). The emaining 34 po en ial candida es we e
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.1c02016
J. Ag ic. Food Chem. 2021, 69, 7478−7487
7480
de ec ed in bo h mea g oups bu wi h significan diffe ences.
As men ioned in he Ma e ials and Me hods sec ion, a
minimum a e o 8 scans/peak was conside ed o eliable
quan i a i e pu poses. Lowe alues can lead o posi i e
p o ein iden ifica ions (unambiguous high- esolu ion MS/MS
analysis) bu wi h no eliable pep ide quan ifica ions (below
limi o quan ifica ion, BLQ, in Table S4). In ou case, mos
BLQ assignmen s we e defined by only one scan, finding all o
hem in he 1−3 scanning a e ange. Since scans-ac oss-peak is
co ela ed wi h pep ide abundance, a pseudo-quan i a i e alue
o a 100- old change was g an ed o no mal/high o high/
no mal no malized a ios o pep ides ound a he BLQ le el in
one o he mea g oups s udied bu no in he o he (i.e.,
HSPA8 and PRDX6; Table S4). Despi e i s inaccu acy, his
s a egy eased he apid confi ma ion o many p o ein
candida es as eliable bioma ke s. Finally, pep ides obus ly
quan ified in bo h mea g oups (i.e., PGM1 and CRYAB;
Table S4) enabled he accu a e de e mina ion o hei old
change a ia ion. As a esul , om he 36 po en ial bioma ke s
ini ially p oposed by he coa se p o ein sco e app oach (Table
S3), label- ee quan i a i e analysis ce ified 26 o hem (Table
1) as obus desc ip o s ha exhibi ed a minimum o 2- old
change a ia ion (no malized peak a ea a io o 2; Table S4)in
mea g oups s udied.
Biological Func ions o P oposed Bioma ke s. To
acili a e he unde s anding o esul s, a discussion abou he
unc ional analysis o p o ein bioma ke s is fi s o e iewed
and hen discussed in de ail in diffe en subsec ions.
The unc ional analysis o he 26 p o ein bioma ke s lis ed in
Table 1 e idenced hei pa icipa ion in diffe en biological
p ocesses, as depic ed in Figu e 1. P o eins belong o diffe en
loca ions such as ex acellula space, cell memb ane, and inside
cells as a cons i uen componen o he cy oplasm, cy osol, o
mi ochond ion (Figu e 1). The in e ac ion ne wo k o p o eins
(Figu e 2 om Cy oscape analysis wi h yellow and blue colo s
o cha ac e is ic no mal and high pHu p o eins, espec i ely)
and in e ac ion s eng h among hem (Figu e S5 om
STRING analysis) e ealed se e al ema kable ac s. All
p o eins we e clus e ed in o a single ne wo k wi h he only
excep ion o NEFH and QDPR. Fu he mo e, p o eins we e
mainly g ouped acco ding o biological unc ions, as shown in
Figu e 2: me abolic p o eins (ATPIF1, CS, DBI, EEF2, GOT2,
MDH2, LDHA, PCMT1, PGM1, PRDX6, QDPR, and
UGP2), p o eins belonging o he chape one amily
(CRYAB, HSPA8, HSPB1, and HSPB6), s uc u al-con ac ile
p o eins (FHL1, FLNC, PDLIM3, TNNT3, and TPM2), and
anspo unc ions (HBB and TF). Only NEFH, NPEPPS,
and PLEC we e ou side s ega ding hese ou p incipal
unc ionali ies and we e classified as “o he s”. Me abolic
p o eins had he la ges numbe o in e ac ing pa ne s and
e idenced hei key ole in he esponse mechanism induced
by PSS. Mo eo e , he clus e con o med by chape ones
connec ed me abolic enzymes wi h s uc u al-con ac ile
p o eins. This finding can be explained conside ing he
pa icipa ion o chape ones in diffe en p o ein configu a ion
p ocesses (assembly/disassembly, olding/un olding, ans-
loca ion, and ac in o ganiza ion) and hei in e ac ion wi h
damaged p o eins unde s essed condi ions.
21
Me abolic P o eins. This g oup is composed o 12
me abolic enzymes ha in gene al a e di ec ly o indi ec ly
Table 1. P o ein Bioma ke s Cha ac e izing No mal (Cu si e Red) and High (Bold Black) pHu Pooled Mea Sample G oups
Assayed
a
Biological unc ions a e de ailed in he main ex (see he Biological Func ion o P oposed Bioma ke s sec ion).
b
Iden ifica ion de ails a e
o iginally om Table S2. All desc ip o s ound in bo h mea g oups we e assayed a diffe en significance le els wi h he excep ion o DBI ha was
exclusi e o high pHu mea (Table S4).
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.1c02016
J. Ag ic. Food Chem. 2021, 69, 7478−7487
7481
Figu e 1. Classifica ion o he p oposed p o ein bioma ke s (Table 1) om no mal (■) and high (□) pHu mea samples conside ing Gene
On ology (GO) e ms, KEGG pa hways, anno a ed keywo ds (UniP o ), and local ne wo k clus e (STRING so wa e). GO e ms: BP, biological
p ocess; CC, cellula componen .
Figu e 2. Cy oscape p o ein−p o ein in e ac ion ne wo k o he p oposed p o ein bioma ke s (Table 1) om no mal and high pHu mea g oups
s udied. Ne wo k nodes (ci cles) ep esen p o eins and lines deno e p o ein−p o ein unc ional associa ions ( h eshold: >0.4 medium confidence
le el). Yellow and blue colo s ep esen up egula ed p o eins ound in no mal and high pHu samples, espec i ely. Dashed lines delimi p o ein
clus e ing acco ding o he unc ional ole.
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.1c02016
J. Ag ic. Food Chem. 2021, 69, 7478−7487
7482

ela ed o ca bohyd a e me abolism (CS, LDHA, MDH2,
PGM1, and UGP2), ica boxylic acid (TCA) cycle (CS and
MDH2), lipids (DBI and PRDX6), amino acids (GOT2 and
QDPR), ca abolism/deg ada ion, and o he me abolic p o-
cesses (ATPIF1, EEF2, and PCMT1). Among hem, only
GOT2, LDHA, and PGM1 we e o e abundan in he no mal
pHu g oup (Table 1).
I has been widely desc ibed ha he PSS esponse is
associa ed wi h he deple ion o muscle glycogen s o es p io
o slaugh e , causing a educ ion in he subs a e a ailabili y o
anae obic glycolysis pos mo em.
22
This pe u bs glycoly ic
po en ial, which is a measu emen o he emaining amoun o
glycogen and lac a e in he muscle, affec ing he me abolism o
key enzymes in ol ed in his pa hway.
22−24
Al hough he
associa ion be ween p e-slaugh e s ess and muscle glycogen
deple ion has been ex ensi ely s udied in uminan s, he
linkage be ween PSS and he pos mo em glycoly ic a e
emains unclea .
25
Rega ding CS, which ca alyzes he ini ial
eac ion o he TCA cycle pe o ming he i e e sible
condensa ion o ace yl (CoA) wi h oxalace a e o o m ci a e,
he e a e no s udies epo ing any ela ionship wi h high pHu
mea s. Howe e , some s udies demons a ed he o e -
abundance o some TCA me aboli es such as ci ic acid in
high pHu mea s,
26
explaining he up egula ion o his enzyme
in ou s udy.
MDH2, which is in ol ed in glucose p oduc ion
27
when
glycogen is no a ailable, was cha ac e is ic o he high pHu
sample (Table 1) as p e iously epo ed.
18
In line wi h his,
some au ho s also obse ed highe le els o UGP2 in high pHu
mea s. This could be due o he enhanced gluconeogenesis
equi ed o eplenishing he low glycogen le els by p omo ing
he flux o glucose owa d glycogen syn hase.
17
UGP2
pa icipa es in he biosyn hesis o glycogen by ans e ing a
glucose moie y om glucose 1-phospha e o MgUTP, gi ing
ise o he o ma ion o UDP-glucose and MgPPi. The e a e,
howe e , o he au ho s ha epo ed a lowe abundance o
UGP2 in high pHu mea samples.
5
PGM1 and LDHA we e ound as o e abundan p o eins in
he no mal g oup. P e ious s udies e idenced he down-
egula ion o PGM1 in high pHu mea s,
9,17
sugges ing ha his
could be ela ed o glycogen deple ion be o e slaugh e since
PSS no ably educes he me abolism o his enzyme. In his
line, Fuen e-Ga ci

a e al. claimed ha his ac migh depend
on i s phospho yla ed o m.
9
P e ious esul s s a ed ha
PGM1 unde wen phospho yla ion changes be ween high and
no mal pHu mea s,
10
sugges ing ha he phospho yla ion s a e
may al e he a e o con e sion o glucose 1-phospha e o
glucose 6-phospha e, inducing diffe ences in he a e o pH
decline.
28
Conce ning LDHA, which ca alyzes he e e sible
con e sion o py u a e o lac a e, he e we e epo s on i s
p eponde ance in no mal pH mea s,
18
confi ming he esul s
achie ed in cu en esea ch. Howe e , o he au ho s ound
dec eased le els o LDHA in no mal pHu muscle ex ac s,
sugges ing ha his migh be due o muscle physiology and no
pHu a ia ions.
8
The li e a u e also epo ed ha inc eased
LDHB concen a ions could be associa ed wi h an accele a ed
pos mo em pH decline.
29
Taking in o accoun ha PSS
animals ha e limi ed glucose ese es, inc eased ene gy
demands caused by he PSS esponse may al e ca bohyd a e
me abolism, hus enhancing he ac i i y o hose enzymes ha
a e di ec ly ela ed o ATP p oduc ion. I was demons a ed
how ca casses yielding abno mal da k cu ing mea (pHu ≤
5.8) migh ha e educed glycolysis a es a ea ly pos mo em
imes, gi ing ise o low concen a ions o ene gy- ela ed
p o eins.
8
The e o e, u he esea ch is needed o be e
unde s and he ole ha each enzyme plays in pos mo em
muscle me abolism.
Ou side hei key ole in ca bohyd a e me abolism, CS and
MDH2 (o e abundan in he high mea g oup) a e also
in ol ed in he TCA cycle by ca alyzing he ini ial eac ion o
he cycle and he oxida ion o mala e o oxaloace a e,
espec i ely. The TCA cycle is he final common pa hway
o he oxida ion o uel molecules (i.e., amino acids, a y
acids, and ca bohyd a es) associa ed wi h he p oduc ion o
ene gy and educ ion equi alen s (NADH and FADH2)
pa icipa ing in mi ochond ial elec on ans e . Since PSS
animals ha e less glycogen s o es p io o slaugh e , muscle
cells would need al e na i e ene gy sou ces such as he TCA
cycle o es o ing ATP le els, explaining he o e abundance o
CS and MDH2 in he high pHu sample g oup. Al hough
al e na i e ene gy pa hways would be p e e ably ac i a ed
be o e slaugh e , some au ho s epo ed ha mi ochond ia can
s ill consume oxygen in pos mo em muscle e en a e 60 days
o s o age unde acuum packaging.
30
This sugges s ha
oxida i e me abolism (i.e., TCA cycle and/o oxida i e
phospho yla ion) migh be also ac i a ed in pos mo em
muscle o main ain cell homeos asis.
17,18
In da k cu ing
bee , highe NADH le els lead o highe oxygen consump ion
and influence he myoglobin edox s a e,
17,30
affec ing he mea
colo . NADH and o he subs a es such as mala e can also limi
a ailable oxygen o myoglobin, p omo ing he o ma ion o
deoxymyoglobin,
26
gi ing ise o a da ke colo , and limi ing
b own o me myoglobin (Me Mb) o ma ion. In his line,
se e al s udies s a ed ha a highe muscle pHu can inc ease
he ac i i y o se e al enzymes in ol ed in Me Mb, educing
he ac i i y and oxygen consump ion.
30,31
Fa y acid me abolism, especially a y acid β-oxida ion, is an
impo an pa hway o ene gy me abolism. In his s udy, DBI
and PRDX6 we e cha ac e is ic o he high pHu mea g oup
(Table 1). Al hough hese p o eins ha e no been p e iously
ela ed o high pHu mea s, i was obse ed ha o he enzymes
in ol ed in a y acid me abolism we e up egula ed in da k
cu ing mea s.
18
Again, his clea ly sugges s ha high pHu
mea s p obably exploi al e na i e me abolism pa hways such
as lipid oxida ion o ob ain ene gy om oxida i e phospho -
yla ion, imp o ing bo h mi ochond ial oxygen consump ion
and mi ochond ial espi a ion.
17,18,32
Apa om i s ole in
a y acid me abolism, PRDX6 is an an ioxidan enzyme ha
con ibu es o he de oxifica ion o eac i e oxygen species and
i was p oposed as a po en ial indica o o oxida i e s ess.
33
Rega ding amino acid deg ada ion as ano he al e na i e
sou ce o ene gy when o he subs a es a e limi ed, QDPR,
which is an essen ial enzyme o phenylalanine and y osine
deg ada ion, was cha ac e is ic o he high pHu mea g oup.
On he con a y, GOT2, which pa icipa es in aspa a e
me abolism h ough he e e sible ansamina ion o aspa a e
and 2-oxoglu a a e o o m oxaloace a e and glu ama e, was
ep esen a i e o he no mal pHu sample g oup. Al hough
some au ho s di ec ly linked GOT2 o high pHu mea s,
18
i
also akes pa in diffe en biological p ocesses, dese ing
u he esea ch o unde s and i s egula ion in mea depending
on pHu.
The emaining me abolic p o eins ATPIF1, EEF2, and
PCMT1 we e up egula ed in he high pHu mea g oup. EEF2
ca alyzes he GTP-dependen ibosomal ansloca ion s ep
du ing ansla ion elonga ion, playing an impo an ole in
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.1c02016
J. Ag ic. Food Chem. 2021, 69, 7478−7487
7483
p o ein syn hesis. Some s udies epo ed ha inhibi ion o
EEF2 and euka yo ic ansla ion ini ia ion ac o 2 (EIF2)
ac i i y by phospho yla ion migh occu as a esponse o
cellula s ess, con ibu ing o he supp ession o p o ein
syn hesis du ing exe cise/con ac ile ac i i y.
34−36
Simila ly,
o he esul s sugges ed ha EIF2, also in ol ed in p o ein
syn hesis h ough he ini ia ion s ep o RNA ansla ion,
showed highe abundance in high pHu mea s.
18
Since he
occu ence o high pHu mea s is in ima ely linked o he
animal s ess condi ion, his can explain inc eased EEF2
phospho yla ion in he high pHu sample ha inhibi ed skele al
muscle p o ein syn hesis. O he p o eins no p e iously
desc ibed as bioma ke s o high pHu mea s we e ATPIF1
and PCMT1. The o me is an enzyme ha nega i ely
egula es ATPase ac i i y, educing he a e o ATP hyd olysis
when he po en ial o he mi ochond ial memb ane alls,
explaining i s o e abundance in he high pH sample g oup.
Second, PCMT1 ca alyzes he me hyl es e ifica ion o L-
isoaspa yl and D-aspa yl esidues in pep ides and p o eins
esul ing om he spon aneous decomposi ion o no mal L-
aspa yl and L-aspa aginyl esidues. I plays an essen ial ole in
he epai and/o deg ada ion o damaged p o eins, especially
in me hionine deg ada ion, hen explaining i s up egula ion in
he high pHu sample.
Chape one and S ess-Rela ed P o eins. P o eins
CRYAB, HSPA, HSPB6, and HSPB1 belonging o he
chape one amily we e displayed in he co e o he ne wo k
(Figu e 2) and cha ac e ized he high pHu mea g oup in
acco dance wi h p e ious esul s.
9,18
These p o eins we e
commonly s udied by hei ole in he s ess esponse, ac in
s abili y, and apop o ic signaling pa hways. Unde s ess ul
condi ions, hey play a majo pa as essen ial molecula
chape ones in e ac ing wi h damaged p o eins o p ese e hei
unc ion.
37,38
Conside ing he p o ec i e ac i i y o chape -
ones, i is logical o assume ha PSS animals would ha e
highe le els o HSPs o main ain cell in eg i y and o p e en
he ac i a ion o apop osis signaling pa hways. T igge ing o
his p ocess depends on he na u e o he ini ial s imulus,
finding animal s ess as one o he mos ele an ac o s whe e
he in ol emen o HSPs has been desc ibed as an iapop o ic
playe s coun e ac ing he caspase ac i i y.
37,38
Pa icula
unc ionali ies o HSPs seem o be linked o hei
phospho yla ion s a us as demons a ed by Ma o e al.,
10
finding no iceable diffe ences in HSPB1 and HSPB6 om
no mal and high pHu mea s. The e o e, addi ional esea ch is
needed o comple ely unde s and he specific ole o HSPs
acco ding o pos - ansla ional modifica ions such as phos-
pho yla ion.
S uc u al-Con ac ile P o eins. Du ing he con e sion
o muscle in o mea , he e a e complex in e ac ions be ween
biochemical p ocesses ha influence he final mea ex u e
cha ac e is ics such as agmen a ion o myofib ils. P o eoly ic
deg ada ion o se e al s uc u al p o eins (i.e., i in, nebulin,
oponin T, desmin, filamin, and inculin) plays a majo ask in
he de elopmen o mea ende ness.
39−41
I is no ewo hy ha
p o eins in ol ed in muscle con ac ion a e insoluble and
should be ep esen ed in he myofib illa subp o eome.
Howe e , high pHu mea s a o hei solubiliza ion
24
and
acili a e hei ex ac ion wi hin he sa coplasmic ac ion.
9
This can explain he main occu ence o FHL1, FLNC,
PDLIM3, TNNT3, and TPM2 in he high mea g oup assayed
(Table 1). Howe e , o he s udies poin ed ou ha FLNC,
which is a p o ein ha c oss-links ac in cy oskele on filamen s
in o a dynamic s uc u e,
42
was o e egula ed in he no mal
pHu sample g oup.
5
TPM2 binds o ac in filamen s in muscle
cells and plays a cen al ole, in associa ion wi h TNNT3, in
he calcium-dependen egula ion o e eb a e s ia ed muscle
con ac ion. Pa icipa ion o TPM2 and TNNT3 in PSS
muscle is qui e con o e sial since bo h p o eins we e ound
up- and down egula ed in bo h no mal and high pHu mea s.
While Ma o e al.
10
ound highe TNNT3 phospho yla ion
le els in high pHu samples, o he au ho s epo ed ha
opomyosin α-1 chain le els we e lowe in high pHu mea s.
8
In his line, some au ho s s udied he influence o pHu in
muscle con ac ion, finding ha high pHu mea s p e en ed
p o ein dena u a ion, educing muscle ans e se sh inkage and
inc easing WHC, hus con ibu ing o da k colo .
43
These
findings could explain he up egula ion o TPM2 and TNNT3
in he high pHu sample assayed (Table 1).
This s udy desc ibes, o he fi s ime, he p esence o
p o eins FHL1 and PDLIM3 in ela ion o high pHu mea
cha ac e is ics. While PDLIM3 may be ele an in he
o ganiza ion o ac in filamen a ays wi hin muscle cells,
FHL1 is in ol ed in he egula ion o muscle de elopmen .
Despi e hese findings, u he esea ch is needed o efine he
unde s anding o he influence o hese p o eins in he
occu ence o high pHu mea s.
T anspo P o eins. HBB is a he e o e ame ic oxygen
anspo p o ein ound in ed blood cells and in ol ed in
oxygen anspo om he lungs o a ious pe iphe al issues. I
was p e iously epo ed ha espi a ion machine y has an
enhanced unc ionali y in high pHu muscles.
17,18,24,26
As a
esul , inc eased oxygen consump ion would a o i s ans-
po a ion by specific p o eins such as HBB, explaining i s
o e abundance in he high pHu sample (Table 1). In con as ,
he i on anspo a ion TF p o ein was less abundan in his
assayed mea g oup. Al hough TF is necessa y o hemo-based
p o ein biosyn hesis, p e ious s udies epo ed ha TF le els
dec eased in he case o inflamma ion,
44
as occu s unde s ess
si ua ions. In any case, u he esea ch is needed o cla i y he
ole o anspo p o eins on he appa i ion o de ec i e mea .
O he s. O he p o eins no assigned o any o he
a o emen ioned biological unc ions ha we e ound as
desc ip o s o he high pHu mea g oup we e NEFH, NPEPPS,
and PLEC (Table 1). A highe abundance o NPEPPS, an
essen ial aminopep idase o pep ide ca abolism, migh be a
sign o a g ea e amino acid deg ada ion as an al e na i e
ene gy sou ce due o he lack o ca bohyd a e ene gy supply.
P e ious s udies desc ibed he o e abundance o some amino
acid me abolic enzymes
18
and educed amino acid concen-
a ions in high pHu mea s.
32
An inc eased pHu in mea a o s
he solubiliza ion o myofib illa con ac ile p o eins such as
PLEC,
9,24
explaining i s o e abundance in he high pHu
sample. Howe e , up egula ion o NEFH, in ol ed in DNA
binding, axon de elopmen , and nucleosome assembly, is s ill
unclea and dese es u he in es iga ion.
O e all, he esul s ob ained in his s udy sugges ha
diffe en al e na i e ene gy sou ces could be ac i a ed in PSS
animals as a consequence o he educed glycoly ic
me abolism. This would indica e ha cellula ene gy a ises
no only om muscle glycogen bu also o he compounds
could con ibu e o ATP p oduc ion in animals, yielding high
pHu mea s by he ac i a ion o o he biochemical pa hways
such as lipid and amino acid me abolism/deg ada ion, TCA
cycle, and oxida i e phospho yla ion. This also sugges s ha
he g ea e oxida i e s ess and ROS p oduc ion in PSS
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.1c02016
J. Ag ic. Food Chem. 2021, 69, 7478−7487
7484
animals could lead o an ea ly onse o apop osis, inc easing
he up egula ion o some an iapop o ic p o eins such HSPs in
high pHu mea s. Howe e , u he esea ch is needed o
unde s and whe he hese ene gy pa hways (i.e., TCA cycle
and/o oxida i e phospho yla ion) would s ill emain ac i e in
pos mo em muscle. Addi ionally, s uc u al-con ac ile p o-
eins seemed o be diffe en ially egula ed be ween high and
no mal pHu mea s, being he de ailed explana ion o his a
ma e o u he in es iga ions.
The efficiency, apidi y, and simplici y o he p o eomic
app oach p oposed in his wo k ga e ise o clea esul s
dealing wi h main biochemical pa hways unde lying he
occu ence o high pHu mea s. Rigo ous selec ion o p o ein
candida es yielded 26 mea bioma ke s ha clea ly cha ac-
e ized mea g oups assayed. Label- ee ela i e MS1pep ide
quan ifica ion analysis demons a ed he use ulness o he
coa se p o ein sco e a io indica o as an in e es ing s a egy
o he apid sc eening o po en ial p o ein bioma ke s. The
unc ional analysis o he p oposed disc iminan p o eins
allowed hei clus e ing acco ding o ou main biological
unc ions, namely, me abolic p o eins, chape one and s ess-
ela ed p o eins, s uc u al-con ac ile p o eins, and anspo
p o eins. In e es ingly, his s aigh o wa d p o eomic s a egy
fi s desc ibed some bioma ke s associa ed wi h high pHu
mea s.
The esul s achie ed can p omo e he implemen a ion o he
p oposed me hodology o c ea e new insigh s add essing he
apid assessmen o mea quali y.
■ASSOCIATED CONTENT
*
sıSuppo ing In o ma ion
The Suppo ing In o ma ion is a ailable ee o cha ge a
h ps://pubs.acs.o g/doi/10.1021/acs.ja c.1c02016.
Highligh s (PDF)
In-house lib a y used o label- ee pep ide quan ifica ion
(Table S1) (XLS)
Iden ified p o eins om no mal (cu si e ed) and high
(bold black) pHu pooled mea sample g oups assayed
(Table S2) (XLS)
Specific p o ein desc ip o s ound in no mal (cu si e
ed) and high (bold black) pHu pooled mea sample
g oups app oached by he p elimina y p o ein sco e
s a egy. P o eins sha ed by NORMAL and HIGH pHu
pooled mea sample g oups assayed wi h a minimum
indi idual p o ein sco e a io ( old-change) o 2 (Table
S3B) (Table S3A) (XLSX)
Label- ee MS1 ela i e quan ifica ion o p o eo ypic
pep ides om po en ial p o ein bioma ke s p oposed by
he p elimina y p o ein sco e app oach (Table S4)
(XLSX)
Pep ide sco e dis ibu ion achie ed by no mal (Figu e
S1A) and high (Figu e S1B) pHu mea samples assayed
o de e mine he indi idual ion sco e h eshold
indica ing iden i y o ex ensi e homology (P< 0.05)
(PDF)
P o ein sequence co e age and pep ide mass dispe sion
epo s o iden ified p o eins om no mal (GAPDH,
Figu e S2A and BIN1, Figu e S2B) and high (HSPA8,
Figu e S2C and DBI, Figu e S2D) pHu mea samples
assayed. The nomencla u e o p o ein amilies shown is
om Table S2 (PDF)
MS/MS uni ocal amino acid sequencing o yp ic
pep ides om p o eins de ailed in Figu e S3 ound in
no mal (GAPDH, Figu e S3A and BIN1, Figu e S3B)
and high (HSPA8, Figu e S3C and DBI, Figu e S3D)
pHu mea samples assayed. The nomencla u e o p o ein
amilies shown is om Table S2 (PDF)
MS/MS in e oga ion o a yp ic pep ide om he
PYGM p o ein ound in he no mal pHu sample wi h
se e al easible amino acid sequences wi h one clea ly
dis inguished. The nomencla u e o he p o ein s udied
is om Table S2 (Figu e S4) (PDF)
STRING p o ein−p o ein in e ac ion ne wo k o he
p oposed p o ein bioma ke s (Table 1) om no mal and
high pHu mea samples. Ne wo k nodes (ci cles)
ep esen p o eins, lines deno e p o ein−p o ein unc-
ional associa ions, and line hickness indica es he
associa ion s eng h ( h eshold: >0.4 medium confi-
dence le el) (Figu e S5)(PDF)
Samples we e s udied by me ging ull MS1 and da a-
dependen MS/MS (dd-MS2) analyses (PDF)
■AUTHOR INFORMATION
Co esponding Au ho
Miguel A. Sen and eu −Ins i u o de Ag oquímica y
Tecnología de Alimen os (IATA-CSIC), 46980 Pa e na,
Valencia, Spain; o cid.o g/0000-0001-5792-0148;
Email: [email p o ec ed]
Au ho s
En ique Sen and eu −Ins i u o de Ag oquímica y Tecnología
de Alimen os (IATA-CSIC), 46980 Pa e na, Valencia, Spain
Claudia Fuen e-Ga cía−Ins i u o de Ag oquímica y
Tecnología de Alimen os (IATA-CSIC), 46980 Pa e na,
Valencia, Spain; Lac ike Resea ch G oup, Depa men o
Pha macy and Food Sciences, Uni e si y o he Basque
Coun y (UPV/EHU), Vi o ia-Gas eiz 01006, Spain
Olga Pa do −Founda ion o he P omo ion o Heal h and
Biomedical Resea ch o he Valencia Region, FISABIO-Public
Heal h, 46020 Valencia, Spain; Analy ical Chemis y
Depa men , Uni e si y o Valencia, 46100 Bu jasso , Spain
Mamen Oli án−Se icio Regional de In es igación y
Desa ollo Alimen a io (SERIDA), 33300 Villa iciosa,
As u ias, Spain
Nu ia León−Public Heal h Labo a o y o Valencia, 46020
Valencia, Spain
Noelia Aldai −Lac ike Resea ch G oup, Depa men o
Pha macy and Food Sciences, Uni e si y o he Basque
Coun y (UPV/EHU), Vi o ia-Gas eiz 01006, Spain
Vicen Yusa−Founda ion o he P omo ion o Heal h and
Biomedical Resea ch o he Valencia Region, FISABIO-Public
Heal h, 46020 Valencia, Spain; Analy ical Chemis y
Depa men , Uni e si y o Valencia, 46100 Bu jasso , Spain;
Public Heal h Labo a o y o Valencia, 46020 Valencia, Spain
Comple e con ac in o ma ion is a ailable a :
h ps://pubs.acs.o g/10.1021/acs.ja c.1c02016
No es
The au ho s decla e no compe ing financial in e es .
■ACKNOWLEDGMENTS
Depa men o Economic De elopmen and In as uc u es o
he Basque Go e nmen is acknowledged o he doc o al
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.1c02016
J. Ag ic. Food Chem. 2021, 69, 7478−7487
7485
ellowship o C.F.G. Funding om P ojec RTA2014-00034-
C04 (Spanish Ins i u o Nacional de In es igación y Tecnología
Ag a ia, INIA), unding and con ac o E. Sen and eu om
P ojec RTI2018-096162-R-C22 (Spanish Agencia Es a al de
In es igación), and FEDER unds a e also ully acknowledged.
■REFERENCES
(1) Le e , J.; E ans, A. Co po a e social esponsibili y and a m
animal wel a e: owa ds sus ainable de elopmen in he ood
indus y?. In S ages o co po a e social esponsibili y; Sp inge , 2017;
pp 205−222.
(2) Vogele , C. S. Ma ke -Based Go e nance in Fa m Animal
Wel a eA Compa a i e Analysis o Public and P i a e Policies in
Ge many and F ance. Animals 2019,9, 267.
(3) Chambe s, P. G.; G andin, T.; Heinz, G.; S isu an, T. Guidelines
o humane handling, anspo and slaugh e o li es ock; 2001.
(4) Loudon, K. M. W.; Lean, I. J.; Pe hick, D. W.; Ga dne , G. E.;
G ubb, L. J.; E ans, A. C.; McGilch is , P. On a m ac o s inc easing
da k cu ing in pas u e inished bee ca le. Mea Sci. 2018,144, 110−
117.
(5) Pole i, M. D.; Moncau, C. T.; Sil a-Vigna o, B.; Rosa, A. F.;
Lobo, A. R.; Ca aldi, T. R.; Neg ao, J. A.; Sil a, S. L.; Ele , J. P.; de
Ca alho Baliei o, J. C. Label- ee quan i a i e p o eomic analysis
e eals muscle con ac ion and me abolism p o eins linked o ul ima e
pH in bo ine skele al muscle. Mea Sci. 2018,145, 209−219.
(6) Ponnampalam, E. N.; Hopkins, D. L.; B uce, H.; Li, D.; Baldi,
G.; Bekhi , A. E. Causes and con ibu ing ac o s o “da k cu ing”
mea : Cu en ends and u u e di ec ions: A e iew. Comp ehensi e
Re iews in Food Science and Food Sa e y 2017,16, 400−430.
(7) F anco, D.; Ma o, A.; Salgado, F. J.; López-Ped ouso, M.;
Ca e a, M.; B a o, S.; Pa ado, M.; Galla do, J. M.; Zapa a, C.
Tackling p o eome changes in he longissimus ho acis bo ine muscle
in esponse o p e-slaugh e s ess. J. P o eomics 2015,122,73−85.
(8) Mahmood, S.; Tu chinsky, N.; Pa adis, F.; Dixon, W. T.; B uce,
H. L. P o eomics o da k cu ing longissimus ho acis muscle om
hei e and s ee ca casses. Mea Sci. 2018,137,47−57.
(9) Fuen e-Ga cia, C.; Aldai, N.; Sen and eu, E.; Oli án, M.; Ga cía-
To es, S.; F anco, D.; Zapa a, C.; Sen and eu, M. A. Sea ch o
p o eomic bioma ke s ela ed o bo ine p e-slaugh e s ess using
liquid isoelec ic ocusing (OFFGEL) and mass spec ome y. J.
P o eomics 2019,198,59−65.
(10) Ma o, A.; Rod íguez-Vázquez, R.; López-Ped ouso, M.; B a o,
S.; F anco, D.; Zapa a, C. The i s e idence o global mea
phosphop o eome changes in esponse o p e-slaugh e s ess. BMC
Genomics 2019,20, No. 590.
(11) Fuen e-Ga cia, C.; Sen and eu, E.; Aldai, N.; Oli án, M.;
Sen and eu, M. A

Cha ac e iza ion o he myo ib illa p o eome as a
way o be e unde s and di e ences in bo ine mea s ha ing di e en
ul ima e pH alues. P o eomics 2020,20, No. 2000012.
(12) Ku owska-Susdo , A.; Zwie zdzynski, M.; Be anda, A. M.;
Talic
, S.; I anko ic
, A.; Pło ka-Wasylka, J. G een analy ical chemis y:
Social dimension and eaching. T AC, T ends Anal. Chem. 2019,111,
185−196.
(13) Sen and eu, E.; Fuen e-Ga cía, C.; Na a o, J. L.; Sen and eu,
M. A. A s aigh o wa d gel- ee p o eomics pipeline assis ed by liquid
isoelec ic ocusing (OFFGEL) and mass spec ome y analysis o
s udy bo ine mea p o eome. Food Sci. Technol. In . 2021,27, 112−
122.
(14) Rocha , B. Quan i a i e and quali a i e LC-high- esolu ion MS:
The echnological and biological easons o a shi o pa adigm. In
Recen ad ances in analy ical chemis y; In echOpen, 2018.
(15) S achniuk, A.; Suma a, A.; Mon owska, M.; Fo nal, E. Liquid
ch oma og aphy−mass spec ome y bo om-up p o eomic me hods
in animal species analysis o p ocessed mea o ood au hen ica ion
and he de ec ion o adul e a ions. Mass Spec om. Re . 2021,40,3−
30.
(16) Wu, W.; Dai, R.; Bendixen, E. Compa ing SRM and SWATH
me hods o quan i a ion o bo ine muscle p o eomes. J. Ag ic. Food
Chem. 2019,67, 1608−1618.
(17) Kiyimba, F.; Ha son, S. D.; Roge s, J.; VanO e beke, D. L.;
Ma i, G. G.; Ramana han, R. Changes in glycoly ic and mi ochond ial
p o ein p o iles egula es pos mo em muscle acidi ica ion and oxygen
consump ion in da k-cu ing bee . J. P o eomics 2021,232,
No. 104016.
(18) Wu, S.; Luo, X.; Yang, X.; Hopkins, D. L.; Mao, Y.; Zhang, Y.
Unde s anding he de elopmen o colo and colo s abili y o da k
cu ing bee based on mi ochond ial p o eomics. Mea Sci. 2020,163,
No. 108046.
(19) Sen and eu, E.; Pe is-Díaz, M. D.; Sweeney, S. R.; Chiou, J.;
Munoz, N.; Tiziani, S. A su ey o o bi ap all ion agmen a ion
analysis assessed by an R Me aboLis package o s udy small-molecule
me aboli es. Ch oma og aphia 2018,81, 981−994.
(20) Shannon, P.; Ma kiel, A.; Ozie , O.; Baliga, N. S.; Wang, J. T.;
Ramage, D.; Amin, N.; Schwikowski, B.; Ideke , T. Cy oscape: a
so wa e en i onmen o in eg a ed models o biomolecula
in e ac ion ne wo ks. Genome Res. 2003,13, 2498−2504.
(21) Mym iko , E. V.; Sei -Nebi, A. S.; Guse , N. B. La ge po en ials
o small hea shock p o eins. Physiol. Re . 2011,91, 1123−1159.
(22) Ta an , P. V. Animal beha iou and en i onmen in he da k-
cu ing condi ion in bee -a e iew. I . J. Food Sci. Technol. 1989,1−21.
(23) Wul , D. M.; Emne , R. S.; Leheska, J. M.; Moelle , S. J.
Rela ionships among glycoly ic po en ial, da k cu ing (da k, i m, and
d y) bee , and cooked bee pala abili y. J. Anim. Sci. 2002,80, 1895−
1903.
(24) McKei h, R. O.; King, D. A.; G ayson, A. L.; Shackel o d, S. D.;
Geh ing, K. B.; Sa ell, J. W.; Wheele , T. L. Mi ochond ial abundance
and e iciency con ibu e o lean colo o da k cu ing bee . Mea Sci.
2016,116, 165−173.
(25) Fe guson, D. M.; Ge a d, D. E. Regula ion o pos -mo em
glycolysis in uminan muscle. Anim. P od. Sci. 2014,54, 464−481.
(26) Ramana han, R.; Kiyimba, F.; Gonzalez, J.; Ma i, G.; DeSil a,
U. Impac o up-and down egula ion o me aboli es and mi ochon-
d ial con en on pH and colo o he longissimus muscle om no mal-
pH and da k-cu ing bee . J. Ag ic. Food Chem. 2020,68, 7194−7203.
(27) Hung, G. C.; B own, C. R.; Wol e, A. B.; Liu, J.; Chiang, H. L.
Deg ada ion o he gluconeogenic enzymes uc ose-1,6-bisphospha-
ase and mala e dehyd ogenase is media ed by dis inc p o eoly ic
pa hways and signaling e en s. J. Biol. Chem. 2004,279, 49138−
49150.
(28) Ande son, M. J.; Lone gan, S. M.; Hu -Lone gan, E.
Di e ences in phospho yla ion o phosphoglucomu ase 1 in bee
s eaks om he longissimus do si wi h high o low s a p obe alues.
Mea Sci. 2014,96, 379−384.
(29) Gagaoua, M.; Te louw, E. C.; Micol, D.; Boudjellal, A.;
Hocque e, J.; Pica d, B. Unde s anding ea ly pos -mo em bio-
chemical p ocesses unde lying mea colo and pH decline in he
Longissimus ho acis muscle o young Blond d’Aqui aine bulls using
p o ein bioma ke s. J. Ag ic. Food Chem. 2015,63, 6799−6809.
(30) Tang, J.; Faus man, C.; Hoagland, T. A.; Mancini, R. A.;
Sey e , M.; Hun , M. C. Pos mo em oxygen consump ion by
mi ochond ia and i s e ec s on myoglobin o m and s abili y. J. Ag ic.
Food Chem. 2005,53, 1223−1230.
(31) English, A. R.; Wills, K. M.; Ha sh, B. N.; Ma i, G. G.;
VanO e beke, D. L.; Ramana han, R. E ec s o aging on he
undamen al colo chemis y o da k-cu ing bee . J. Anim. Sci. 2016,
94, 4040−4048.
(32) Consolo, N. R. B.; Rosa, A. F.; Ba bosa, L. C. G. S.; Maclean, P.
H.; Higue a-Padilla, A.; Colnago, L. A.; Ti o, E. A. L. P elimina y
s udy on he cha ac e iza ion o Longissimus lumbo um da k cu ing
mea in Angus×Nello e c ossb eed ca le using NMR-based
me abolomics. Mea Sci. 2020,172, No. 108350.
(33) Pa edi, G.; Raboni, S.; Bendixen, E.; de Almeida, A. M.;
Mozza elli, A. “Muscle o mea ”molecula e en s and echnological
ans o ma ions: The p o eomics insigh . J. P o eomics 2012,75,
4275−4289.
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.1c02016
J. Ag ic. Food Chem. 2021, 69, 7478−7487
7486