Intact Cell MALDI-TOF Mass Spectrometry on Single Bovine

Intact cell MALDI-TOF mass spectrometry on single bovine oocyte and follicular cells combined with top-down proteomics: A novel approach to characterise markers of oocyte maturation Valérie Labas, Ana-Paula Teixeira-Mechin, Laura Bouguereau, Audrey Gargaros-Ratajczak, Lucie Spina, Aurélie Marestaing, Svetlana Uzbekova

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Valérie Labas, Ana-Paula Teixeira-Mechin, Laura Bouguereau, Audrey Gargaros-Ratajczak, Lucie Spina, et al.. Intact cell MALDI-TOF mass spectrometry on single bovine oocyte and follicular cells combined with top-down proteomics: A novel approach to characterise markers of oocyte maturation. Journal of Proteomics, Elsevier, 2017, 175, pp.56-74. 10.1016/j.jprot.2017.03.027. hal-01605876

HAL Id: hal-01605876 https://hal.archives-ouvertes.fr/hal-01605876 Submitted on 26 May 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Version postprint Uzbekova, S.(2018). Intact cellMALDI-TOF massspectrometry on singlebovine oocyteandfollicular cells combined with top-down proteomics: Anovel approach to characterise markers ofoocyte Labas, V., Teixeira-Gomes, A.P.,Bouguereau, L., Gargaros, A.,SPINA, L.,Marestaing, A., maturation. Journal of Proteomics, 175, 56-74. , DOI : 10.1016/j.jprot.2017.03.027 journal pertain. be it manuscript a This authors. Pleasecheckifappropriate.Jprot(2017),doi: maturation. with MALDI-T Audrey Please Accepted date: Revised date: Received date: T Reference: DOI: PII: Uzbekova Audrey V A novelapproachtocharacterisemarkersofoocytematuration oocyte Intact Accepted Manuscript o appearin: alérie is service discovered published is top-down cell cite and a Labas, Gar Gar PDF to OF this will MALDI-T follicular The garos, garos, our mass in which file article Ana-Paula under address proteomics: customers its of Comment citer cedocument: Lucie Lucie spectrometry final could go cells an OF as: 31 March2017 23 February2017 27 October2016 Journal ofPr JPROT 2815 doi: S1874-3919(17)301 copyediting, unedited for form. V Spina, mass Spina, combined 10.1016/j.jprot.2017.03.027 T alérie af we the eixeira-Gomes, fect A

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de Masse, France 37380Nouzilly, INRA, Plateforme de d’AnalyseIntégrative Laboratoire Spectrométrie Biomolécules, des INSA/CNRS 5504 INSA/CNRS UMR de 37380Nouzilly, ISP, France Tours, INRA, Université UMR Un PRC, INRA 85,CNRS, Intact cell MALDI

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conjugatedgoat anti bands were detected inallthreewere detected where cell types,

Using this approach, 271, 182 and 248 m/z were m/z Usingand approach,248 this 271,182

Biotechnology, CA 133 , 296), which were296),found which tobesignificantly more [

54 w

peaks , France) as ICM ] . peaks

analy Primary p of follicular cells follicular of

< 0.05).

- (ANOVA, (ANOVA, MS spectraMS compared, were with107,33 , presented as aheatmap, presented z and were used ed (Fig.

- using using

) and anti) rabbit olyclonal in vitro

(Amersham Biosciences Orsay, p 3A Scion < 0.001, maximum FC >3)< 0.001,maximum antibody ( . ).

Membranes were visualized Membranes mature (n=50) oocytes

- anti ubiquitin antibodies ubiquitin

Image for Windows - human H2Bhuman Cell SignalingCell (Fig. (Fig. as 23.9,24.3 3B (Fig. ),

or - 3C 14 )

HR we to µLC peaks re - HR [ MS 50 µLC used to generate a peak list (using Flex AnalysisFlex a togenerate peak containingused (using software) list more ] - es

) - MS proteomic analyses without priorchemical were performed treatment of

). MS spectra. MS (with redundancy). In anbiomolecules(with overview redundancy). the to obtain order of -

ACCEPTEDMS/MS MANUSCRIPT

of the intactof orfragmented the range werefrom proteins inamass detected β HR These peptidoThese

ACCEPTED MANUSCRIPT 4, thymosin 4, thymosin proteomic analysis proteomic - 1500 Comment citer cedocument: MS/MS , usingIn thesame extract. protein orderaddition, in toenrich the 43, 54, 46 and 40 fractions collected from RP1, RP2, RP3 and GF collected RP3 43, 54,46and 40fractions fromRP2, RP1, - 20,000 Da massrange.

. After direct injectionof total proteinextract samples, we softwaregenerate ofthe angel four toview expanded - 6 - β

to 9.92×10 ( and proteoforms wereand proteoforms detected inthe2 - supplementary

10, thymosin rifugation enrichment was combined with RP enrichmentwaswith RP rifugation combined

E - ) wasperformedTD tothe prior values ranging×values 10 from 6.98 - 91 . Among previously been them,ninehad - α fractionationabundant ofsample themost

we combined - Table 1), thioredoxin, ubiquitin,1), hemoglobin, thioredoxin,

( esponding to the thymosin familyesponding tothe thymosin - supplementary TOF MS. DB h 17,15and 20molecular species 1 - A

four separation methods separation four T in Data inBriefin Data article he spectra MS Table Table , 000 - DB1 6 TD HR

to 1.45×10 HR - 9 , - 000 Da mass of these 183 of - B in inData MS - MS

- 59 15 - .

. infusion,

Comparison the of accession biomoleculesgene numbers identified, and names

peptido Conventional µLC , respectively, only thethree to twomasses with common approaches. A , respect [ 50 - was to20kDa increased

µLC 25 - and proteoforms in values ranging9.96×values 10 from ] - ). MS were compared, only 136 common masses were wereMS compared, 136common only observed ion numbers and 54gene 97,33,42and respectively names, ( - themass range 1500 ively, in of

ACCEPTED MANUSCRIPTnumber of molecularspecies fold more identified biomolecules by thedirect thanthose infusion

From theseFrom redundancy subsets,after had been - HR ACCEPTED MANUSCRIPT

HR peaks Comment citer cedocument: . From the439 11,3 masses identified, - MS/MS MS/MS - T - MS MS/M op a total of 386 different moleculara sp total of386different

in -

was obtained obtained by from merging lists theprotein D

1 [ own own 50 peaks - ,

in the massrange of1 S analysisS identified ofall 1424molecular species. fractions 500 redundant and ]

). HR -

Thereno common biomolecules fo were prefractionation 15

detected, 115, 44, 60 and 83 m/z were 44,60and 83m/z detected, 115, specific toRP1,

(Fig. (Fig. ,000 Da mass range,000 Da - ( MS supplementary liquid chromatographic liquid molecular species

4E approaches - - 6

direct

with those with

). and proteoformsand to 2.87×10

– 20,000 m/z 20,000 m/z

infusion, approaches , 000 Table

determinedICM by – 17,000 Da were17,000 Da identified - detected 65

were identified forwere identified theRP1, 9 and 316were tothe specific . These (Fig. µLC ecies werein amassecies identified DB

(Fig. identified inbovine identified

combined toµLCcombined suppressed

1 separations and439 separations - 4B - HR C using fractionationsusing 4A represented 190unique

). in Data inBrief article -

MS/MS ). Comparison of

For the RP1, RP2, For RP2, theRP1, , und between - a total of372 (Fig. MS, MS, Fig. fter (Fig.

with or - 1A in however 4C HR 4D ). peaks - )

was 16

] ) ) .

[ [ Regarding range molecu themass distribution, Among speciescorresponded towholeproteins, the386molecular identified, 15%

The peptido From the328 protein fragments (N 50 50 - MS spectra, the number spectra, ofproteforms the MS iden - ] ] terminal fragments of original proteins, 23% to C oforiginalterminal proteins, fragments ) ). .

Although theoverall rangethatobserved weight molecular asas wasbroad for , 055 and 16,790Daand ([M+H] 055

ACCEPTED- MANUSCRIPT - terminal methionine excision (101 m/z); (ii) (101 excision m/z); methionine terminal

and proteof ACCEPTED MANUSCRIPT a similar proportion a similar

- had previously beenhad at the protein described 82atthetranscript level, cies ofthecies topfive aminoare acids presented Comment citer cedocument:

to 2.87×10 - - ter or internal fragments, and thoseter above internal orinternal fragments, the orC tryptic sequences (cleavagestryptic sequences atthe C orms derivedthe386 identified from species correspondedto

- 65 ( supplementary of - ter, C ion (3 m/z), or disulfide bridgesm/z), (4 m/z). ion (3 - + isoelectric points for biomolecules theidentified ter or C ter or

theoretical E mass), average with - aromatic (F)aromatic amino acid Toobtainan residues. terminal Gln into pyroglutamate Glninto andterminal (4 m/z); - ter and internal and fragments),ter 105 tified inthe range of10 - ter of aminocl acids. Aseries lar species identified bylar identified species a TDshowed Table Table -

or C - terminal fragments oforiginal DB ( -

Fig. N terminal were also directions Fig. - - 1 terminal proteinsblocked methylation (3 m/z), methylation (3 m/z), - 2B 2B D - in Fig. Fig. in ter R or K), and R the ter 2A

in Data inBrief articlein Data in Data inBrief in Data in Data inBrief in – 5 17 kDa largely ,

with the most with the most - values eaved

ies - ter ter 17 -

were characterised from profiles specific to GC, CC, oocyteswere fromGC,CC, characterised profiles to global and specific the follicular

accession numbers corresponding numbers accession tomasses Thus, our ucleosome assembly (13

- related top - c activity oxidase (7proteins) likeandendopeptidases orkallikreinenzymes other specific targeting sites ). 136 qualitative ACCEPTED MANUSCRIPT proteins aret Also, 36 involved in

[ low weighten low 50 ACCEPTED MANUSCRIPT Table S1 Table - 247 DB

[ in gene The regulation. expression pathways w ackaging of telomere ends and DNA double strand breakackaging and oftelomere DNA ends double response strand Comment citer cedocument: ] 50 386 ) 2 . The representative functions most were protein (126 binding

out of439 ]

) in Data inBrief article

was co ICM proteins identified was - 1 ). TheMALDI observed - - MS/TD mitochondrial oxidative phosphorylationoxidative ( mitochondrial TOF MS TOF MS dogenous andpeptide protein with several species different mpar proteins), proteins),

peaks

S2 ed to ICM ed to

. HR

). observed in the ICMobserved inthe The biologicalidentifiedThe proteins the processesof peaks ) including mRNA3’ The peptido The chrom - MS

byTD . [

strategy for allowed thedirect 50

- MS spectralMS follicular profiles cells of ransport to <17From kDa.58,66and these, 60 57, osome dimerization activitydimerization proteins) and (17

in relation their to molecularfunction, ]

). HR T

7 - op

peaks

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and derived proteoforms from 91 MS organisation (13 proteins),organisation (13 - D own own

Goldgi andsubsequent ( ( were aligned totheoreticalwere aligned or - Supplementary t Supplementary table MS spectra wereMS identified - UTR binding (IGF2BP3, HR ith the most significant most the ith - MS

12 proteins, able .

Among the DB1 DB 5.8 2

- D in 18 -

respectively. Using TD respectively. UsingTD

- maturation.ICM Comparison the of I), and metaphase

Afterchanges oocyte the beenICM throughout had maturation determined, In were after foundIVM 9ofdecreased theoocytes, 21peaks - 10) andat4 that 10) , - (Fig. 718.77 m/z and theotherm/z as 718.77

ACCEPTED MANUSCRIPTand down 572.15 m/z and RM52_BOVIN m/z L52, (39S572.15 ribosomal protein S , Table Table 555.11 m/z. m/z. 555.11

ACCEPTED MANUSCRIPT 3 cell MALDI cell ) were allowed) for which of for used PCA, immatu thediscrimination 6A F1MPB2_BOVIN (insulin - Comment citer cedocument: II(Meta ) ter IVMter

HR S3 and oocytes - the , 764.44 m/z. Twelve 764.44 m/z. andIVMregulated oocytes inCC after in are shown ). - MS

Fordiffered 20peaksgroups CC, these( between , oocytes 18andIVM. 24hours These of at0,6,10, time - 964.87 m/z II),respectively.

proteomics, s -

TOF , 717.15 m/z wasas annotated 717.15 m/z TYB10_BOVIN (Fig. -

MS spectra from immature single spectra MS

beta MS analysis to identify potential markers of of markers potential identify to analysis MS

was TYB4_BOVIN beta (thymosin 6B G3X8G9_BOVIN (probable E3ubiquitin G3X8G9_BOVIN (probable - 10) at 4 10) 21 m/z species ( m/z 21 ), ix peaks wereix abundant significantlymore in

- peaks significantly were found tobe as shown by the PCA score plots. Examples as scoreExamples shownby plots. PCA the Thirty different ( peaks like growth factor2mRNA es five inimmature of CC, these were , 516.33 m/z, E1BK75_BOVIN516.33 m/z, , - 885.31 m/z, F6S1Q0_BOVIN885.31 m/z, points waspoints presente etween and mature immature - inducing gene 46protein)gene atinducing p

- < 0.01,FC > 2; I (Meta (Fig. (Fig. 7A p - I), anaphase

6B < 0.05,

). oocytes (n= 10)

d as aheat map, Hierarchical ). - binding protein

- Two of theseTwo 4). On the 4). Onthe p Fig.

- < 0.01,FC MS MS - 6A 6A - I - and - in 19 re

Table 2presents asummary ofthe p Among the30 In thedifferences tovalidate order inproteins (Fig.

< 0.01 n CC afterIVMn CC when compared toimmature cells ( p

< 0.01 8D , FC=1.86 )

ACCEPTED MANUSCRIPT peaks whereas oteins identified by TD , Fig. Fig. , ACCEPTED MANUSCRIPT .37 peaks luster 3, such as m/z 8 3,such asluster m/z - Comment citer cedocument: MS, this molecular this MS, species wasIVM significantly upregulated during , representing clusters,four, isshownin main the

(clusterIVM.decreased 2) of expression progressively The during 8B ) but not in ) butnotin

the abundance of ubiquitin wasthe abundance inimmature similar ofubiquitin and mature

). characterised by the IVM werecharacterised study, kinetic by the six

Another protein, ubiquitin, was identified protein,by TDatAnother ubiquitin, level ofthe oocyte maturation. maturation. oocyte - , - MS analysisMS showed thatthis 519 MS, we performed immunoblots tofollow immunoblots the change in weMS, performed -

4) at 4 the .76

oocytes( - (cluster 1)at was 6hours foundtodrop drastically 136 protein ligaseatHECTD4) 2 whole H2B(~14 whole kDa) protein , HR 965.66 m/z, m/z, 965.66 , 718

identified m/z correspondingm/z proteinsidentified towhole - MS 30 m/z m/z 30

16 m/z (represented m/z IDs)protein16 by 10 .57 Fig.

proteomics and inthe CC oocytes before H

wasat of increasedmaturation the end abundance 2B, characteriseda2B, fragment as at 8C (represented by 1 UBC_BOVIN(polyubiquitin ). Ubiquitin was detectedas was). Ubiquitin a (Fig. p

, peak < 0. 718.57 m/z. 718.57 m/z. between the immaturebetween and the .31 7B 0

was significantly less ) progressively increased 1, ).

, The relativeThe FC>2, FC>2, 764.44 m/z, 764.44 m/z, immature than CC 9 was Fig.

protein IDs)protein -

10) at 4 identified: identified: also decreased 7C Fig. .

8 The 8A , 565 , 519.76 - these C) atC) ). were

. When When 53 20

- [ MS and powerful statistical tests enables tests determination andheterogeneityMS powerfulthe of statistical and cellular 39

Indeed,ICM ] , humanblood

s analysis without ofbiomolecules biological intheir any context risk of - ACCEPTED onwholeMS intact cells hasproved MANUSCRIPT - [ MS allowed for allowed ofMS thedirect lowmolecular detection peptideand weight ACCEPTED MANUSCRIPT 55 Comment citer cedocument: ] [

33 and 2154proteins humanoocytes in ]

and male germinal cells - This was achieved byThis was achievedICM quantitative differentialanalysis of MS was employed to annotateemployedas proteins was potential identified MS cyte of andlayers, thegranulosa part only cumulus or - cell level has been limited b cell limited been level has

- MS and ICM andMS

molecular (1 weight - TOF MS profiling ofovarian profiling TOF MS follicular

[ - to bea high remarkable 42 MS quantitative MS , - 43 - up proteomic approaches.A TD cell level, using bovine oocytecell level, using ] . Moreover, the combination of y Recent size. its technological [ 24 - ] MS spectra. Moreover,MS the – . These studiesreported 17 kDa) proteins17 kDa) and their [ er, the possible detectionpossible the er, 44 ] proteomic . [

24 ] . Here,ICM - - throughput

and analyses in - MS MS 21 -

Version postprint Uzbekova, S.(2018). Intact cellMALDI-TOF massspectrometry on singlebovine oocyteandfollicular cells combined with top-down proteomics: Anovel approach to characterise markers ofoocyte Labas, V., Teixeira-Gomes, A.P.,Bouguereau, L., Gargaros, A.,SPINA, L.,Marestaing, A., kinetics of progress thematuration reflectedby was molecular allowing fingerprints, oocyte be clearly theoocyte from discriminated pro and CC process.this and Usinghierarchical clustering,couldand oocytes PCA theimmature mature during revealed which significant inseveral maturation, variation species molecular during ICMperformed onthe support of organelles, areall ofwhich oocyte quality importantembryo for interms of development molecular and consequent PTMs, includes maturation, protein which synthesis, produce maturation meiotic oocytes, beaccompanied must competent by cytoplasmic and nucleus, GV metaphase tomature stage) transforming oocytes meiotic division, first resume (pr oocytes their from immature In study,we find markers oocytematuration. aimed our During process, to of thematuration cells cumulus Differential ce intact of theenclosed oocyte. proliferation other addition to apoptosis, non in and relatedfurther different forCC follicles tolook markersinGCand to compared between to annotate shareddetermined peaks peptidome/proteome from GC/CC characterisation. specific analyses when which investigating anopportunity theoocyteproteome, also provides showed profile a oocytes. similar much more thanwith toGC Thisconfirms all follicular cells.are from layer, Furthermore, as themural they CC differentiated GC differed and oocytes, GC,CC ofthe and between 15% only development and oocytes.expected function due This was of tothethese specific during cells oocyte ICMfrom thatused studies reflectingand CC, biological morevariation. consistentwithresults Thisis than technical with the CV%group between ranging44% samplesoffor from thesame theoocytes the 35to degradationIn preparation. dueICM tosample our the study, transition from events,the characterisation markersmeiotic GVBD of main the such potential ofas the or maturation. Journal of Proteomics, 175, 56-74. , DOI : 10.1016/j.jprot.2017.03.027

Oursignificant studyICM demonstrated differences in In quantitative analyses of toidentifyoocyte order markers maturation, were Therefore, andfinge have oocytes GC,CC molecular specific [ 57 ] . [

Meta ] ACCEPTED MANUSCRIPT . In detected of439fact, 133out m/z inthemass range1 - ll MALDI ll ACCEPTED MANUSCRIPT I to - MS profilesMS oocyt obtained individual from

Comment citer cedocument: Meta - MS forMS epithelial sperm cells and - II. InII. bovineoocytes, fact, and in protein synthesis - TOF MS analysisTOF MS oocytes ofbovine single surrounding and

- II whichare To able tobe oocytes, fertilised. - invasive biomarkers related tothequality files. Moreover, that we the demonstrated peaks - [ MS showed repeatable spectra, showed MS - 35 MS profiles between GC, CC profilesGC,CC MS between ,

detected werecommon to detected 42 es and surroundinges and CC - rprints, which canrprints, which be 44 ] . , 500

the need cell for – modification 18,00 ophase ophase 0 m/z 0 m/z

22 -

Thus, ICMThus, and detectedICM peptidoforms by [ HR 8 .57 , HR 22 - MS

peak at the Ana ] - [ . Consistent among this, species with the248m/z detectedICM by MS 61

proteomic approachproteomic analysis isbased ofintact allowing onthe proteins, for ] -

ACCEPTED MANUSCRIPTInterestingly,. several different ,

MS appears to befor an appears efficientthep to MS method ta 519 approach peptidome and tocharacterise the proteome offollicularcells - ACCEPTED MANUSCRIPT

II whereas 11 stage, them/z .76 Comment citer cedocument:

and - up approaches using digested peptides. Inup approaches toid digested using peptides. order - Iand Meta -

11 and proteoforms structural Therefore, and characterisation. urethe maturationor oocytes, inthe kinetics) or in , 119

a top [ 58

.37 - Iand Meta The abundance speciesThe of71 were m/z found tovary ] , and differs during progression meiosis - -

II drastically decrease down - MS in the oocytes, TD HR we in MS performed stages, suggestingstages, could be thatthese peaks HR , - ial 235 II was stages by marked an inthe increase - MS

peaks bothcell types, were common to .31 [ 62

proteomic strategy,

] increased GV from toMeta the

and proteomic analyses ofoocytes d from immatureat oocytesd from the - , 358, 2 invasive predictive markers invasive predictive of henotyping offollicular - TOF MS signaturesTOF MS , 885, 4 - cumulus complex complex cumulus oocyte shareoocyte a , 516, 4 which also - derived factors entify the [ - 59 , MS inthe MS 964 ] - . MS MS For .

and - I 23

Given thatuse ofHR The attributedtothecorresponding GeneOntology the most terms proteins included In HR total, the TD phase or gel was or phase coupled filtration, toµLC - terminal (20%) and C (20%) terminal o develop approach cells. aTD follicular for of pools direct µLC - HR - es and functions surroundinges cells ofthefollicular and inthe oocyte infusion, ACCEPTED MANUSCRIPTHR - [ ACCEPTED MANUSCRIPT MS/MS MS/MS 63 - MS/MS Comment citer cedocument: ession numbers were orgeneession names shared. ] . In. fact, inpoly(A) involved more proteins bindingwere abundant,

were complementary highly -

direct injectionto MS strategyMS resulted of inthe386lowmolecular identification - GC MS oran MS and , respectively, - 11, 39 and 316 masses outof386TD were11, 39and identifications 316masses -

t UTR ( protein extractsdecrease to complexity thesample and erminal fragments (23%) andinternal oforiginal proteins, prefractionations combined toµLC ,

no common masses were found methods between thefour - fractionation methods fractionation IGF2BP3, PUM1, µLC nd GF only were fractionations, common 19masses - HR and onl and

µLC

noted, as detection only transcriptnoted, at the level - as MS/MS strategy aloneMS/MS sufficientfor isnot - GC

- and proteoforms - HR available.Byour TD applying HR cellapplied we proteome, four different y betweencommon twomasses the were - . Of the 464 m/z detectedOf the464m/z intheaverage. enriched Thesepools). approaches - - MS/MS and prefractionations MS/MS MS/MS, and approximatelyMS/MS, 30% SERB1, YBX3 CPEB3), and tegies ( tegies based onreverse approximately

[ 48 ctionation methodsbased on ctionation - MS spectra wereMS ] . Thus, an off identified by identified TD direct

- HR - infusion, MS/MS 10 - phase and gelandphase – 50 µg for - line multi HR ), which direct HR - MS MS

- MS - 24

Version postprint Uzbekova, S.(2018). Intact cellMALDI-TOF massspectrometry on singlebovine oocyteandfollicular cells combined with top-down proteomics: Anovel approach to characterise markers ofoocyte Labas, V., Teixeira-Gomes, A.P.,Bouguereau, L., Gargaros, A.,SPINA, L.,Marestaing, A., fragments,weorigin andwere indefining interested the observed cleavages.We ofthese a for thelife and ofproteins death pathway, highlighting thus N theimportance of potential roles as signal adegradation of protein andthe targeting prevention tothe secretory for numerousthefunctio proteins includeimplications regulation ofprotein ortargeting interactions tomembranes, however, N began aftermethionine excision. the N phosphorylationmethylation,oxidation, andamong disulfide bridges,Of methionine others. protein N acetylationmethionine excision, after About 30% identified of386 molecules by TD follicular in cellsproteins Top representativepathways of inoocyte involved thekey maturation. bee which arecrucial reflect COX5BCOX6A1, these ATP5I, energy ATP5H, and COX6C) UQCR10,phosphorylation COX7A2, COX8A, UQCRC1, COX7B, NDUFAB1, (NDUFA4, acidand APOA2,APOC3, UBCand PPARGC1B), (FABP5, metabolism oxidative (HECTD4, MAN2BA, HSPG2, MDH2, PPP2R5D, GAPDH P MGAT4C, and Thus, theidentification of proteins inglucose and involved carbohydrate metabolism [ c metabolism is the oocyteand complete meiotic divisions, energy. energy requires Oocyte/cumulus In(H1, other and H2B,H3 andSMARCA5). factors (NPM1toresume and H4) meiosis order proteins, several of rearrangement frommetaphase prophase ofthechromatin to bovine oocytes, whichoccurs during meiotic maturation corroboratingimportancecritical anddegradationmaternal of the ofpolyadenylation RNAin P and ManyP S. are residues ofthese hydroph high cleavages frequency ofprotein aminoL, acidK,D, involvingF, A,G, nine residues: R, 66 maturation. Journal of Proteomics, 175, 56-74. , DOI : 10.1016/j.jprot.2017.03.027 ] n reported study inarecent onhumanoocytes - , which are bothenergy mitochondrialto make oxidation ATP. that require substrates down proteomics demonstrated post proteomics demonstrated down - terminus PTMs identifiedterminus PTMs in78proteins,

Importantly, the(328) masses 85%of identif Therefore, theTD using losely relatedtoglucose

ACCEPTED MANUSCRIPTfor maturation oocyte which werewhich inthe current including identified study, histones different ACCEPTED MANUSCRIPT Comment citer cedocument:

HR [ n remains unknown n remainsunknown 69 - - terminal amajor is and acetylation functional PTM, its

, MS 70 ] [

[ . approach, dataset were we able toobtaina protein 54 65 -

translational modif , ] obic or polar,obic targets and specific for represent 67 , transformed HR ] - 55 (70%) which acetylation, represented - . Among several detected, theproteins had terminal acetylationterminal terminal pyroglutamate, acetylation, [ - 24 MS ied by TD ied by ] [ . 68

carriedas N such PTMs [ ] 59 . Recent reports have suggested to pyruvate and fattyandacidsto pyruvate CC by , - II involves inthemany oocyte 64 ications and of degradation HR ] . Furthermore, meiotic - MS

as determinant amajor - providing mechanisms, mechanisms, providing

were protein were protein GAM2), fatty - terminal 25

Kallikreins A large ofhave proteins number alrea observedWe inthecleavage that theD was residue frequently involved sitesof13% [ . The list ofannotated substratestoincrease, continues and candidates lack most 7 ith the CASBAH database, theCASBAH comprehensiveith containing a substrates ofcaspases list 5 ] . Moreover, caspase - ic substrates (as caspases, metalloproteinases,cathepsin, orelastase plasmin ic substrates mediated deg

ACCEPTED MANUSCRIPT are tobe expressedrange ina tissues, known the ovary, wideincluding of ACCEPTED MANUSCRIPT [ 76 [ Comment citer cedocument: 72 ] - . like serine proteinases. radation. radation. ] -

. S cumulus active complexes, caspasesreported have been inCC, sed ome kallikreins are knowntopresent specificity, withastrong - oocytes 3 activity granulosacells tobecorrelated was3 inbovine found - like or chymotrypsinlike or

and with themolecular concentrationand of low weight [ 74 dy reported been tobe ] . The enzymaticare determined properties by [ Other endopeptidases targeting sites specific 71 [ 73 ] , as well as beingin involved ] . These cysteine - like specificity such as kallikreins, such like specificity a

dicator ofbovinedicator oocyte in vivo e putative caspasee putative - dependent aspartate dependent - related malignancies

caspase substratescaspase [ 71 rane ] . In our ions of ions of HR - MS - like) like)

- 26

Finally, TD our Therefore,according identified, datasetovarian cell toour ofbiomolecules follicular Althoughin the oocyte, peaks half only and were of70% detectable more of these than potential m/z markerspotential m/z ICM identified by - MS/ , -

718 were initiallyofsimilar identifications considered withtwo peak, as one

- obtained byand HR proteoforms TD TD MS allowed us to identify usto allowed inoocyteMS several markersinvolved valuable nuclear

eight ACCEPTED MANUSCRIPT - - ACCEPTED MANUSCRIPT MS II(mature ooc HR Comment citer cedocument: ribonucle proteins and fragments which showed andfragments abundance a which proteins difference inthe in

workflow,be which overcome may by using - MS peaks

approachformalallowed the annotation for of o roteins A1, C, F, Mandroteins F, U A1, C,

detected by ICM detected by

yte) or other ofoocytemeiotic stages progression specific [ 79 ] . Among them, . Among .

MS wereMS TD using annotated HR yICM - MS wereMS annotatedan considering error - MS inallthreeMS types offollicular cells. - IGF2BP3, ubi MS differentialMS analysis. Thus, (thymosin beta(thymosin - in like), or with histone binding,with histone like), or or to peptide fragmentsor topeptide derived CC. Thisis of CC. one limitation ogical changes, and provides - expressed moreexpressed than1000 ns could be attributedns could to - peptidomic profilingpeptidomic of quitin in situ in [ 80 - 10). However, ] - . Thisreinforces protein ligase ICM

MALDI TD 136 -

- MS spectraMS molecular MS, MS, - fold - 27 s

84 ] emoglobin beta and hydrolaseCMBL cysteine all specific tooocytes, were . tofish,Similar ACCEPTED MANUSCRIPT

showed thesame showed patterns – ACCEPTED MANUSCRIPT 43) ofa was43) predictedprotein lessabunda (F1MPB2_BOVIN), which mature cumulus [ 81 Comment citer cedocument: ] . Described as anti - proteasome and cumulus expansion oocyte pathway for meiosis de novo - the difference in relative abundancefragmentthe difference ofthepeptide inrelative like, thymosin beta like, thymosin [ - 85 expression ofexpression genes their corresponding during IVM, [

cewhich differedsurrounding of immature inCC or .

82 This list was completedThis list by CC

] synthesis of cyclin of the initiation synthesis of B1 isessential for , which requires activation of maturation compared tocompact ] , therefore, theycould potentially involved in be [ 83 d by ICM d by ] s of the translation and ofB1 s of other cyclin . - , and therefore,greater abundance the of apoptotic and antiapoptotic as by ICMas by -

UTR during meiosis. Pumilio 1 is also 1is UTR during awell Pumilio meiosis. - 4

HR - and MS, mayMS, withsimilar be associated - - MS thymosin beta thymosin MS, MS, - element (CPE) binding (CPE)(CPEB) element

immature - were H2B histoneH2Band - MS analysisMS was by validated in both CC and oocytes. CC in both The UTR of - inflammatoryfactors, these - specific markers, CCBN1 cumulus appearscumulus tobe - 10 differed10 in

- mRNA promoting wn to -

- like nt 28 -

and oocytes, suggesting thath ngIVM,IGF2BP3 tothe similar decreasesubstrate could inCPEB.caspasedue bea to , 556) differed cells, between abundance follicular 4.5 asthe was Interestingly,ICM using Therefore, ofdetecting thepossibility and quantif in vivo - oocy ,

including pre te level of h of te level

than in less competent oocytesIVM,than inless after s

ACCEPTED MANUSCRIPT environmentlived theoocyte hypoxic to before ovulation ACCEPTED MANUSCRIPT Comment citer cedocument: - fractionations followed byHR ,

emoglobin be highercompetent was inmore shownto mature these of form associated proteins part orcomplexes intheoocytes. in vitro - - MS, weMS, d specific protein and peptidespecific signatures. and protein analyses Differential [ emoglobin may actemoglobin may asa tha molecule 86

maturation. useof complementary The

- ] IIIndeed, of expression stages. the . Previously, we described a spatio

single

etectedabundance that ofh the inclu [ TD TD in vitro in 87 - oocyte level can help to improve IVM leveloocyte help improve can to - ] terminal position ofterminal theTD position ded molecularweight bothlow and proteins HR . Here, fragments internal -

- MS embryo production. - invasive methodpredicting for oocyte MS/MS, MS/MS, ying

workflow, suggestingthat upporting thehypothesis that markers using ICMmarkers using allowed theidentification for

endogenous molecules that endogenous - t sequestersO fold higherfold inthe - HBA HBA temporal expression expression temporal emoglobin HBB emoglobin TD

[ of CPEB3 and - 90 [ identified

and 88 analytical ] . Moreover, , - MS in MS 89 HBB

2 ] oocytes

. or NO

genes - 29

- Cumulus cells complexe; CC :Cumulu cells CC complexe; Cumulus

ACCEPTED MANUSCRIPT- TOF Mass Spectrometry;Top TOF TD: ACCEPTED MANUSCRIPT Committee - hermore, the identification of hermore, theidentification MS) projects.MS) onmarker supported Work was by identification the Comment citer cedocument:

, the French National Institute, theFrench National Agricultural Research for ean Development(ERDF), Regional FundVal the

high resolutionhigh mass spectrometeracquisition

- hange Consortium. hange Consortium. LiquidIVM: Chromatography;InVitro maturation. We acknowledgeTeamWe thePRIDE for that had not previously beenthat had notpreviously described in peaks - s Cells; GC: Granulosa GC: ICMs Cells; Cells; Down; HR

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Version postprint Uzbekova, S.(2018). Intact cellMALDI-TOF massspectrometry on singlebovine oocyteandfollicular cells combined with top-down proteomics: Anovel approach to characterise markers ofoocyte Labas, V., Teixeira-Gomes, A.P.,Bouguereau, L., Gargaros, A.,SPINA, L.,Marestaing, A., Development 1206 (2010) 22(8) upregulated during cells incumulus Reproduction meiotic maturation, Fertilityand Uzbekova, Thymosins beta M. Salhab, P.Papillier,[81] C.Perreau,Guyarder C. cancer, one 5(10) PloS (2010) e13133. Moore Y. Liu,[80] Shen, N.Tolic, T.Zhao, M.A. Gritsenko,Camp, R.J. B.O. D.G. R. Petritis, apoptotic properties, 53(2)92 (2006) Neoplasma K.P.Letsas, [79] Frangou M. death1 anddifferentiation A.U. Luthi, Martin,[78] The CASBAH: S.J. substrates, ofcaspase asearchable database Cell protease, ofbiological Journal The chemistry (2007) 282(20) 15011 a blueprint for CED Taylor,R.C. [77] Ito, S. Brumatti, G. Hengartner, Martin, Establishing W.B. M.O. Derry, S.J. quality, Biologyreproduction 72(4) of (2005) 796 molecular B. Nicholas,Fouladi R.Alberio,[76] A.A. developmental competence, (2005) Theriogenology 63(8) 2147 Peelman, oocytes, cells, incumulus not Apoptosis but embryonic may influence bovine Y.Q. Yuan, Leroy,Soom, A.Van[75] J.L.Zeveren, de Dewulf,A.Van Kruif, A. J. ofbiologicalJournal chemistry(2009) 284(33)21777 G.S. Pop, C. [74] Salvesen, Human specificity,The caspases: activation, and regulation, Spring Harborinb perspectives D.R. McIlwain,[73] T.Berger,functions cell in anddisease, death Mak, T.W. Caspase Cold and applications Molecular cancer, 2(5) in cancer :MCR (2004) research 257 Borgono,C.A. [72] I.P.Michael, D E.P. preferences, subsite individual The ofbiological Journal chemistry (2006) 281(35) 25678 Bode,Choe, W. Goettig, P. seven kallikreins Specificityhumantissue profiling reveals of M.Debela, V.[71] Magdolen, N. Schechter, M.Valachov biological chemistry (2008) 283(50) 34469 A.Varshavsky,[70] Discovery ofcellular by regulation degradation, The protein of Journal create Hwang,C.S. [69] A. Varshavsky, A.Shemorry, N biology(2011) 9(5)e1001074. T.Arnesen,[68] Towards afunctional N ofprotein understanding diversity/energy http://www.intechopen.com/books/meiosis 953 - N. [67] Songsasen, mammalianoocyte Meiosis Energy metabolism regulating in: maturation, roleB offatty and acids K.Dunning,Lipids Robker,[66] D.L. R. and oocytecompetence: developmental Russell, the 685 determiningmetabolism in developmental oocyte competence,139(4) Reproduction (20 M.L.[65] Sutton AnimReprodMPF Sci and kinase MAP activation, 73(3 polyadenylation of condensatio mRNAscodingchromatin for proteins Krischek,C. [64] B. ofbovineIn oocytes Meinecke, requires maturation vitro Dynamics Remodeling, and and Cytoskeleton Vitamins hormones 25 102(2016) Thym L.[82] Intrinsic, Functional, Renault, andStructural Properties of beta

Molecular Mechanisms andMolecular CytogeneticISBN: Diversity, Mechanisms edited by AndrewSwan. 978 maturation. Journal of Proteomics, 175, 56-74. , DOI : 10.1016/j.jprot.2017.03.027 - - 51 95. osin/WH2 Domains theRegulation in ofosin/WH2 Actin Self andCoordination signals, s specificdegradation (2010) 327(5968) 973 Science , S.O. Purvine, F.J. Esteva, R.D. Smith, Blood Esteva, Purvine,, S.O. F.J. peptidome R.D.Smith, -

0118 - weigh - 5, InTech,DOI: from:5, Available 10.5772/33752. - t insulin - metabolism ACCEPTED MANUSCRIPT Thompson, The R.B.McDowall, pivotal role Gilchrist, J.G. ofglucose - 3 ACCEPTED MANUSCRIPT - depende Comment citer cedocument: - oxidation, Reproductionoxidation, (2014). - like growth factor 4(4) (2007) 641 - 4 and beta - - regulating Lazaridis, Surfingprothymosin alpha on anti proliferation and

nt killing through identification of multiple substratesnt killingof throughfor multiple identification this iology a008656.(2013) 5(4) - 1221.

10 are ex - iamandis, Human kallikreins: tissue physiologiciamandis, roles mamma - - - 50. - Nashta, R. Webb, Relationship R.Webb, Nashta, between low molecular - 89. binding proteins, caspase proteins,binding

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- - oocyte Joly, J. Dupont, P. Mermillod, S. Mermillod, S. Dupont,P. J. Joly, - - mechanisms 81.

- 4) (2002) 129.4) (2002)

a, F. Lottspeich, C.S. Craik,Lottspeich,F. Y. C.S. a, -

maturation - - 63. degradome profile ofbreast - terminal acetylation,terminal PLoS - -

and 7. - 21.

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(2012) 173 (2012) cytogenetic - Assembly - 80. -

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binding inimmature repressesBiochemical oocytes, thetranslation protein that and - activity of the 26S proteasome germinal 26S activity ofthe isrequired for themeiotic resumption, 33.

ACCEPTED MANUSCRIPT , C. Perreau, C. Prigent,, C.Mermillod, P. Uzbekov, Spatio R. communications 448(1) 22 (2014) ACCEPTED MANUSCRIPT - - Comment citer cedocument: 76. binding proteinbinding zebrafishoocyte 3in aB1 novel as cyclin maturation

- Bonnemains, J. Dupont, R. DalbiesBonnemains, Dupont,R. J.

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ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Comment citer cedocument:

Version postprint Uzbekova, S.(2018). Intact cellMALDI-TOF massspectrometry on singlebovine oocyteandfollicular cells combined with top-down proteomics: Anovel approach to characterise markers ofoocyte resolution MS fragment I fragment C fragment N protein Whole Labas, V., Teixeira-Gomes, A.P.,Bouguereau, L., Gargaros, A.,SPINA, L.,Marestaing, A., nternal - - ter ter maturation. Journal of Proteomics, 175, 56-74. , DOI : 10.1016/j.jprot.2017.03.027 Table 1. Numberendogenous of pepti

61 9 8 8 Ds

in bovine follicular cells 1 8 7 5 I 3 5 60 45 PTM with Ds

Comment citer cedocument: I excision hionine ACCEPTED MANUSCRIPT 0 0 56 45 Met

acetylation do forms and proteoformsforms and ACCEPTED MANUSCRIPT 0 0 55 35 N

PyroGlu => 2 0 2 0 Q

acetylation ernal

with or without post with orwithout 1 0 1 3 Int

methylation 0 3 0 0 Tri

- - translational modifications identified by top

Oxidation hionine 0 0 0 3 Met

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bridge sulfide

0 2 0 2 Di -

down high - 41

Version postprint Uzbekova, S.(2018). Intact cellMALDI-TOF massspectrometry on singlebovine oocyteandfollicular F1MV04_BOVIN CO3_BOVIN F1N3H1_BOVIN MRP_BOVIN E1B7N2_BOVIN FIBB_BOVIN F1MP41_BOVIN RL39_BOVIN F1N2I5_BOVIN RM52_BOVIN HBB_BOVIN TYB10_BOVIN UBC_BOVIN F6S1Q0_BOVIN TYB4_BOVIN Q2KII5_BOVIN G3N053_BOVIN E1BK75_BOVIN G3X8G9_BOVIN F1MPB2_BOVIN name Entry cells combined with top-down proteomics: Anovel approach to characterise markers ofoocyte Labas, V., Teixeira-Gomes, A.P.,Bouguereau, L., Gargaros, A.,SPINA, L.,Marestaing, A., MS/MS maturation. Journal of Proteomics, 175, 56-74. , DOI : 10.1016/j.jprot.2017.03.027

Table 2. Table

proteomics

E3 ubiquitin E3 C3 Complement Calumenin MARCKS HistoneH4 chain beta Fibrinogen Metalloendopeptidase L39 ribosomalprotein 60S homolog Carboxymethylenebutenolidase mitochondrial L52, ribosomalprotein 39S beta subunit Hemoglobin beta Thymosin Polyubiquitin 18 cytoskeletal I type Keratin, beta Thymosin HistoneH2B HistoneH2B E3ubiquitin Probable Insulin

ACCEPTED MANUSCRIPT List ofpeptides/p in bovine ovarian cells. follicular - ACCEPTED MANUSCRIPT like growth factor 2 mRNA 2 factor growth like Protein description Protein Comment citer cedocument: -

related protein related

protein ligase ligase protein - C - - 10 4

- roteins

protein ligase HECTD4 ligaseHECTD4 protein

detected by ICMdetected by - binding protein 3 3 protein binding

- MS

and 2,098.45 8,295.55 8,182.39 8,565.84 8,451.74 3,680.93 5,044.46 4,922.46 4,495.00 4,161.67 4,964.46 8.96 2,886.36 6,026.87 4,572.26 2,766.04 4,718.37 [M+H] av Theoretical 4,281.55 5,134.96 5,088.54 5,183.29 6,107.23 3,790.37 11,309.24 2,209.33 2,192.33 1,951.04 5,460.2 5,199.71 7,036.74 6,620.22 5,547.1 5,330.9 5,286.76 5,043.5 3,626.05 6,276.49 1,604.89 11,560.21 3,485.97 5,193.06 3,557.05 8,718.97 4,004.44 4,517.99 4,717.24 6,418.29 id

entified byTD

3,39

UBC KRT18 TMSB4X HIST1H2BI HIST1H2BA HIST1H2BA HECTD4 IGF2BP3 Gene ZFP91 C3 CALU MARCKSL1 HIST1H4A FGB ASTL RPL39 CMBL MRPL52 HBB TMSB10

42 F F F - F - F - - F - - F F F F F F F F F CC - - F F Oo, - CC Oo, CC, F Oo, - - F F CC,F Oo CC - - - - CC Oo, - CC Oo, - CC Oo Oo Marker

Version postprint Uzbekova, S.(2018). Intact cellMALDI-TOF massspectrometry on singlebovine oocyteandfollicular NDUA4_BOVIN COX8A_BOVIN COX6C_BOVIN CX6A1_BOVIN QCR9_BOVIN A5D7Q4_BOVIN E1BIW2_BOVIN ATP5H_BOVIN APOA2_BOVIN F1MUZ9_BOVIN 68MP_BOVIN DSS1_BOVIN F1N312_BOVIN HBA_BOVIN IF4H_BOVIN CALM_BOVIN ALDR_BOVIN HSPB1_BOVIN VIME_BOVIN PTMA_BOVIN G3N1C9_BOVIN G3N0F3_BOVIN E1BGW2_BOVIN A6QQ28_BOVIN H13_BOVIN G3N135_BOVIN E1BB06_BOVIN E1BEL7_BOVIN CLPT1_BOVIN TMA7_BOVIN F1MMM4_BOVIN F1N6B7_BOVIN E1BAU3_BOVIN G5E6I9_BOVIN HP1B3_BOVIN cells combined with top-down proteomics: Anovel approach to characterise markers ofoocyte Labas, V., Teixeira-Gomes, A.P.,Bouguereau, L., Gargaros, A.,SPINA, L.,Marestaing, A., maturation. Journal of Proteomics, 175, 56-74. , DOI : 10.1016/j.jprot.2017.03.027

Synaptotagmin Serine/threonine member10 superfamily Immunoglobulin like 1 type HistoneH2B 1 protein Heterochromatin Cytochrome c oxidase subunit NDUFA4 subunit oxidase c Cytochrome mitochondrial 8A, subunit oxidase c Cytochrome 6C subunit oxidase c Cytochrome mitochondrial 6A1, subunit oxidase c Cytochrome b Cytochrome CSDAprotein domain BEN mitochondrial d, subunit synthase ATP A2 Apolipoprotein ACCEPTEDmitochondrial protein, kDaheat shock 60 mitochondrialkDaproteolipid 6.8 DSS1 subunit complex proteasome 26S 608 protein Zincfinger alpha subunit Hemoglobin 4H factor initiation translation Eukaryotic Calmodulin (20 Aldosereductase protein shock Heat MANUSCRIPTVimentin alpha Prothymosin HistoneH2B HistoneH2B HistoneH2B HistoneH2B HistoneH1.3 Formin beta protein shock Heat homolog 1 protein transmembrane palate and Cleft lip machinery Translation ACCEPTED MANUSCRIPT - like like

Comment citer cedocument:

protein 2 2 protein -

containing protein 4 protein containing -

c1 complex subunit 9 9 subunit complex c1 - like protein 5 likeprotein - protein phosphatase 2B 2B phosphatase protein

beta -

alpha - associated protein 7 protein associated - -

1 (HspB1) 1 1

binding protein 3 protein binding - HSD) HSD)

3,109.28 3,805.01 6,025.92 1,985.24 2,714.17 2,374.69 5,897.75 5,741.56 4,173.79 1,975.20 1,947.15 1,785.90 2,422.91 2,080.26 1,767.12 3,441.90 2,185.43 4,914.21 6,978.11 1,854.02 1,722.95 3,401.79 13,773.97 6,025.92 4,677.33 9,325.72 5,046.94 8,521.95 2,285.53 7,327.43 5,928.39 2,255.44 4,527.18 3,888.26 3,487.11 6,835.08 8,189.46 1,829.13 15,054.18 3,474.93 3,955.46 3,460.82 16749.39 6,612.23 3,807.29 2,994.50 3,978.44 2,997.33 2,244.97 5,041.67 4,323.66 4,066.37 11,983.68

PTMA (H2B) N/A (H2B) N/A HIST1H2BJ H2B HIST1H1D FMNL2 FMNL2 HSPB1 CLPTM1 TMA7 SYTL5 PPP3CC IGSF10 LOC107133263 HP1BP3 NDUFA4 COX8A COX6C COX6A1 UQCR10 YBX3 BEND4 ATP5H APOA2 HSPD1 MP68 SHFM1 ZNF608 HBA EIF4H CALM AKR1B2 HSPB1 VIM

43 ------F F F F F F F F

Version postprint Uzbekova, S.(2018). Intact cellMALDI-TOF massspectrometry on singlebovine oocyteandfollicular F1MC90_BOVIN F1MFG7_BOVIN UBXN1_BOVIN TR112_BOVIN TTHY_BOVIN G3X7U0_BOVIN THIO_BOVIN Q05B82_BOVIN RUXF_BOVIN A1L5B7_BOVIN SPA31_BOVIN E1BE29_BOVIN F1N0Q3_BOVIN F6RBQ9_BOVIN RT63_BOVIN THRB_BOVIN F1N5H8_BOVIN E1BG53_BOVIN PTMS_BOVIN F1N1Y3_BOVIN E1BPA2_BOVIN E1BIX5_BOVIN ATOX1_BOVIN F1N052_BOVIN F1MCA8_BOVIN KRT81_BOVIN E1BMT1_BOVIN HINT2_BOVIN HMGN3_BOVIN F1MZX2_BOVIN FIBA_BOVIN FABP5_BOVIN EPCR_BOVIN F1MWR3_BOVIN G5E5G8_BOVIN F1ME65_BOVIN cells combined with top-down proteomics: Anovel approach to characterise markers ofoocyte Labas, V., Teixeira-Gomes, A.P.,Bouguereau, L., Gargaros, A.,SPINA, L.,Marestaing, A., maturation. Journal of Proteomics, 175, 56-74. , DOI : 10.1016/j.jprot.2017.03.027

Fattyacid CD201) antigen(CD Creceptor protein Endothelial mitochondrial subunit alpha, flavoprotein transfer Electron Diacylglycerol Cytoskeleton Zinc finger protein 106 (Zfp 106 protein Zincfinger hydrolase (VACVase) Valacyclovir domain UBX homolog 112 methyltransferase tRNA (Prealbumin) Transthyretin 28 protein Transmembrane Thioredoxin protein TEAD2 F ribonucleoprotein nuclear Small 1 protein mRNA binding SERPINE1 A3 Serpin 2 receptor Ryanodine RNA RNA mitochondrial 63, protein Ribosomal II) factor (Coagulation Prothrombin ATP Probable 1 coactivator gamma PPAR Parathymosin Olfactoryreceptor Olfactoryreceptor (nucleoporin) Nup153 complex protein pore Nuclear ATX1 protein transport Metal chromatin matrix Lamina Hb1 cuticular II type Keratin, protein Homeobox mitochondrial nucleotide triad Histidine 3 protein containing nucleosome mobilitygroup High Glia chain alpha Fibrinogen

ACCEPTED MANUSCRIPT - derived nexin (Peptidase inhibitor 7) inhibitor nexin(Peptidase derived - - binding Raly binding 3 protein binding - ACCEPTED MANUSCRIPT associated actin associated - associated polypeptide 2 polypeptide associated - Comment citer cedocument: - binding protein 5, 5, protein binding

1 (Endopin 1

- -

associated protein 4 protein associated

containing protein 1 1 protein containing -

dependent RNA helicase RNA dependent kinase (DAG kinase) kinase) kinase(DAG

likeprotein

- 1A) -

dependent regulator of regulator dependent

- binding protein 2, 2, protein binding

- - beta beta 106) epidermal

binding domain binding

4,195.85 3,488.87 6,000.13 1,561.06 2,966.43 7,235.30 2,959.19 6,405.29 2,829.24 3,297.75 3,155.49 10,578.77 3,831.51 1,934.92 4,595.35 2,114.40 3,491.16 2,944.45 6,193.75 2,119.43 4,320.93 6,458.21 2,605.01 2,782.16 2,344.81 11,680.42 1,915.23 2,406.93 2,431.61 4,049.91 1,886.24 2,226.40 3,176.33 11,867.73 5,729.26 2,007.30

PPARGC1B PTMS OR5W2 OR2A7 NUP153 ATOX1 SMARCA5 TMPO KRT81 SIX3 HINT2 HMGN3 SERPINE2 FGA FABP5 PROCR ETFA DGKZ CKAP4 ZNF106 BPHL UBXN1 TRMT112 TTR FAM155B TXN TEAD2 SNRPF SERBP1 SERPINA3 RYR2 RALYL RBM3 MRPL57 F2 DDX31

44 ------

ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Comment citer cedocument: