Article Title: Statistical Strategies for Relating Metabolomics and Proteomics Data: A
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Article title: Statistical strategies for relating metabolomics and proteomics data: a real case study in nutrition research area.
Journal name: Metabolomics
Author names : Thomas Moyon, Fabien Le Marec, El Mostafa Qannari, Evelyne Vigneau, Aurélie
Le Plain, Frédérique Courant, Jean-Philippe Antignac, Patricia Parnet, Marie-Cécile Alexandre-
Gouabau.
* To whom correspondence should be addressed : Thomas Moyon, INRA, UMR1280, Physiologie des Adaptations Nutritionnelles, CHU Hôtel-Dieu, Place Alexis Ricordeau, HNB1, 44093 NANTES
CEDEX 1, France ; Phone : 33 2 53 48 20 02 ; Fax : 33 2 53 48 20 03 ; E- mail: [email protected]
Supplementary Tables Table 1. Simplified data matrices of (i) relative spot volumes of the 172 proteins, and (ii) abundances of the 2265 metabolites of male offspring from the three perinatal nutritional status at
PND16.
Perinatal Matrix of the 172 proteinsa (%vol) Matrix of the 2265 metabolitesb (abundances) nutritional status 2942 … 3065 M138T368 … M407T177 (observations) CC1 0.4616 0.0629 1485796.6546 124990.3322 CC2 0.4838 0.0784 1410045.0211 85296.6592 CC3 0.4418 0.1286 2041633.6264 130093.6317 CC4 0.6170 0.1344 730011.5881 35995.1674 CC5 0.5739 0.1564 776594.6227 134296.9869 CC6 0.5057 0.1758 1103538.6506 464353.6675 CC7 0.4293 0.2838 391988.1442 183377.3244 CC8 0.4740 0.1356 670114.7572 426653.3480 RC1 0.5727 0.0946 774571.9774 362410.0800 RC2 0.6500 0.1878 1699158.1945 247570.2969 RC3 0.7153 0.1367 697181.2980 78670.0282 RC5 0.7822 0.0725 958769.2947 57547.2770 RC7 0.4086 0.2287 1646368.1246 101166.1984 RC8 0.6183 0.1704 1703032.4422 377241.9479 RR1 0.2624 0.0359 4203071.8536 1382214.4999 RR2 0.2637 0.0924 5111247.5847 1802374.3164 RR3 0.1897 0.0782 3305651.2484 1119848.5413 RR4 0.3075 0.0671 3263833.8536 1178806.8150 RR5 0.3394 0.2304 4646594.9031 1412946.2534 RR6 0.2737 0.0671 3145759.4865 1543025.4617 RR7 0.2965 0.0869 2999464.0652 1528929.6674 RR8 0.3148 0.0619 2911288.9491 940302.4933 a Matrix of the proteins: column names are spot identifiants. b Matrix of the metabolites: column names are mass(M)-retention time(T) pairs. Data from two protein spots and two metabolite mass- retention time pairs are shown, as example.
Table 2. Plasma annotated metabolites at PND16 and subjected to integration methods. Retention Time Metabolites [M+H]+ observed Compound and metabolism class (seconds)
Leucine a, b 216 132.10175 BCAA involved in fats pathway Valine a, b 162 118.08611 BCAA involved in carbohydrates pathway Tyrosine a, b 310 182.0812 EAA and precursor for the dopamine Proline a, b 143 116.07021 NEAA synthesized from glutamic acid Arginine a 131 175.1187 EAA Phenylalanine a, b 708 166.08633 EAA and precursor for the tyrosine Tryptophan a, b 1080 205.09728 EAA and precursor of serotonin EAA-intermediate in transmethylation reactions and Methionine a, b 180 150.05827 precursor for cysteine. Carnitine a, b 130 162.11234 NEAA-involved in fatty acid metabolism O-acetyl-carnitine a 178 204.12298 oxidation of fatty acids a Identity confirmed with authentic standards and b with bibliography data
Table 3. Hypothalamus proteins identified at PND16 and subjected to integration methods. Spot Id. Protein Name Gene Name
A. Proteins involved in oxidative stress 3492 14-3-3 protein zeta/delta Ywhaz 3525 Cytochrom b-c1 complex subunit Rieske (Bcs1L) Uqcrfs1 3533 Thioredoxin-dependent peroxide reductase (PrdX3) Prdx3 3286 Translation factor eIF-2B subunit alpha (eIF2B) eIF2B
B. Proteins involved in energy & protein metabolism 2942 Serum albumin Alb 2946 Serum albumin Alb 3065 D-3-phosphoglycerate dehydrogenase (GAPDH) Gapdh 3297 Fructose biphosphate aldolase C (AldolC) AldoC 3536 Apolipoprotein A-I (apo-AI) Apoa1 3506 Proteasome subunit alpha type 6 Psma6 3545 Proteasome subunit beta type 6 Psmb6 3195 Ornithine aminotransferase (OAT) Oat 3235 Aminoacylase-1A (Acy1) Acy1
C. Proteins involved in neuronal migration, axon growth and guidance 2993 Dihydropyriminidase related protein 5 (DRP5) DpysI5 3204 Mitogen-activated protein kinase-kinase 1(MAPKK1) Map2k1 3211 Actin cytoplasmic 1 Actb 3091 Fascin Fscn1
Supplementary Figures a
Phenylalanin peak
b
Phenylalanin ions
Fig. 1 (a) Overall Liquid Chromatography of plasma rat maintained in restriction during pregnancy and lactation. Retention time (in minutes) is onto the x axis, abundance onto the y axis. (b) Determination of the chemical structure of the Phenylalanin Liquid Chromatography peak by spectral mass analysis. MW (kDa)
150
15 pI
3 10
Fig. 2 Overview of hypothalamic proteins altered in response to perinatal protein restriction. Total proteins extracted of the whole hypothalamus of rats were separated by Two Dimension gel
Electrophoresis, according to their isoelectric point (pI) in the first dimension and to their molecular weight (MW) in the second dimension. 0 . 1 8 . 0 6 . n 0 o i t a l e r r o 4 C . 0 2 . 0 0 . 0
1 2 3 4 5 6 7 8 9 11 13 15 17 19 21
Canonical variates
Fig. 3 Plot of the correlations between the two sets of canonical variates for the RCCA model based on the 585 selected metabolites and the 103 selected proteins. RR1 0 .
a RR2 b 1 P3302 P3099P3096
P3087 P2987P3177 RR5 P3246P3216Fscn1 P3443 P3209 P3197 M392T357 DpyP3272 sI5
5 P3013P3086P3242
. M248T202 P3181 M303T1823AldoC 5 M310T1547M127T167M250T1143M168T454 0 . M58T135 0 M293T367 M257T133M308T1029M282T274M249T199M364T126 P3341 P2941 M141T133M140T134M96T133M155T943M339T132M202T1051M216T1050M284T126M120T125M216T943M201T944M202T974M322T944M515T125M126T398 M521T2092P3270 M271T137M206T179M253T134M75T151M144T178M187T850M155T1051M201T1052M294T136M204T223M154T1032M322T1050P3370 M390T1991 M85T178M255T715M118T1198M153T1198M407T177M123T1198M114T1203M97T1198M213T1198M245T180M212T1198M269T180M130T1050M109T1051M441T180M138T368M286T1028P2947M349T2066M475T2092M416T2084M227T1381M302T1797M621T2084P3412M737T2075Oat M143T1030M410T126M211T921M306T127M115T1198 M585T109M541T1408M539T2044 P3247M435T1359P3579 M276T140 ) ) M86T178M229T244M127T1416M320T1198M282T126M205T178M107T1198M259T167M180T127M164T128M233T906M118T608M386T127M225T608M69T610M135T1136M235T132M260T126M222T387M267T180M219T1614M227T129M332T1642M525T2077P3423 M431T1384M633T1359P3458P3588 CC7 M250T142O-acetyCarnitine[M+Na]+M222T123M57T152M152T1416 l-carnitine[M+H]+M205T248M74T133M268T1241M105T315M396T1434M416T1375M418T2019M202T140M306T179M226T177P3005M178T163P3327 % % M87T362M232T904M224T1049M503T2055M399T2149 M427T1385M197T358M399T1355
M493T111M181T191 M218T214 M557T1384M739T2075 P3243 M249T140M366T134M73T163M125T130M434T125M130T193M156T132M189T247M290T186M170T317M105T358M171T831M228T1198M426T2105M422T108M243T1636 P3399M610T2086AcyM401T240 1
1 1 M260T194M304T1198M369T2090M273T99 M424T2068 M416T1221 RR4 RC7 M177T196 M372T2091M123T213M435T2046M303T191M232T192M266T103M428T1923M246T1243M138T769M153T307M153T373P2927M290T1198M289T180M292T1785M294T1820P2973P3068 M668T1359 8 8 M343T182 M247T1240M118T314M247T317M408T2095M302T163 M344T1359P3308M497T1289M489T1310M379T138P3573
. . M304T128 M109T314M225T317P2984M252T1970P3435 Map2k1 O-acety l-carnitine[fM318T1306M307T278M240T263M286T1983M261T1222M346T150 ragments]M233T247M60T178M361T1268M109T357M305T176M179T315M357T98M477T1390M626T2048M564T2087M172T94M147T188P3379M459T1359M110T131Gapdh 9 9 M132T1198M308T274M535T2091M254T214M232T126M134T291M118T355M622T2084M435T1785M364T247M283T144 P2911M258T132M262T354M119T213M148T176M117T196M322T137M137T1198M607T2082P3009M203T356M641T2039M553T2046M259T305M380T126M218T141 ( ( M187T152M218T253M415T1544M467T2149M659T2094M225T356 M512T2056M427T153M185T2017M313T138M133T290M313T1161
M555T2139M510T2048 M346T1427 M442T2299M447T2021M581T1371_2M445T1371 M441T1311M441T1312P3397M297T139Psmb6P3494
2 2 M138T191P3127 M447T1080M198T310 0 M209T2032P3010 M415T2056M446T1371M604T2055M329T202 M232T249M91T310
M689T2078 CC8 . M584T2218M178T830 M307T116M449T1371 t t M203T165P3487M519T2090M734T2075P3556M320T1996P3564P3021M91T140P3591M358T179M278T140M407T1320M160T141M286T1482M123T246M119T246P3581M123T310M119T310M184T310P3524 0 M369T959 P3570M217T95M547T2074M369T1299 n 0 n M740T2075M285T1401M171T2225M518T2090M436T1785M581T1371_1M336T1081M389T1991M476T1310P3580TyP3516 rosine[f ragments] . RR3 M137T768M685T2036M338T2388M107T213M92T219P3138 M218T1160P3583TyM161T139Ty rosine[f rosine[M+H]+P3577M363T310M183T310 ragments.] e e M565T2090M330T1350M438T2035M201T260M376T2046M581T1676M335T149P2939 M180T1320M170T761M475T1310M487T1311M86T289P3575M274T127M136T246M311T907M377T1368 0 CC4 M291T770M305T349M208T371M437T1785M295T192M714T2033M304T191M272T163M338T140P2940M253T1080M474T1310M283T1417M165T246M363T238
n n M534T2091M434T2216 M269T769P3212M563T2087M620T2047M199T97M163T162M250T295M505T1311M609T2087M465T126M243T101M468T1310M350T98P3146P2961M254T140M316T179M291T138P3551M139T139M256T139M182T246M183T247M300T1552Prdx3M311T955
o o Carnitine[M+H]+ M207T127M196T2096M182T213P3323Psma6 M134T127 CC6 M217T1030 M292T1544M318T312M275T170P2945M577T2004M366T1357M611T2056M173T142P3498M487T179Proline[fProlineEifM261T1103M138T141P3537M184T233 2b ragments] [M+H]+ p p P3114 M670T2036M729T2033M181T133M130T1080M212T1387M250T124M237T182P3523M170T1080M367T124M243T1080ActbM493T247M135T178 P3036M59T134M526T2003M284T1970M554T2044M299T96M782T2072M533T1670M598T2042M579T2214M237T1632M298T1631M582T2042M153T145M164T1497M283T2093Methionine[fMethionine[fM137T312M221T1080M415T184Methionine[M+H]+M231T182M152T181M151T180M56T180M134T180M306T134 ragments.] ragments]
m m M490T2102 P3069M261T1178M249T336 M359T164M152T793M284T2092M270T769M520T2090Uqcrf s1P3340M713T2033M282T181M310T137M644T2081M412T1724M183T98M160T601P3474 M215T182M143T1080M211T1447 o RC2 RC1 CC5 CC3 o M566T2090 P3538M642T2039M240T115M499T1311M254T1970M147T1080M168T1082M262T1167 RR7 M231T1240 M537T769M576T2047M194T1632M499T1310M498T1310M392T1359M462T716Leucine[fLeucine[M+H]+M379T707M332T128M133T216M141T170 ragments]
C C M368T128M318T230 M216T182M87T215 M268T887 M222T205M472T2428M757T2030M342T1400M172T1381M207T1080M267T1382M296T711Alb.M472T180 RR8 RC5 CC2 M471T2428M263T2056M599T2130 M665T2081M349T1430M408T1321P3549Try ptophane[M+Na]+M229T1078M356T716M461T711M385T712M220T163M331T163M281T1586M160T1080 M424T2094M325T191 M249T1382TryP3535M275T1081M314T127M192T677M245T1382 ptophane[M+H]+M188T1080M190T1080M206T1080M409T1080M127T1585M335T1080M183T1585M264T711M471T180P3070M204T708M181T708M296T1273M370T716 M183T213P3312M597T2043M152T1585YwhazPhenyM189T1080PhenyM159T1080M265T717M265T156M244T180Valine[fM266T179M108T708 lalanine[fM167T708M103T709 lalanine[M+H]+M149T709M369T710M292T133M121T708M211T127M295T1274M133T110 ragments] ragments] M578T2088P3256M137T196 P3488M251T1429M252T1429M268T1592M319T1080M146T1080M144T1080M180T1143M334T1358M534T710M336T128M148T709M331T708M79T708M69T206 P3621 M354T203 P3548M161T1592M359T127M303T1080M145T1592Alb M218T1586M335T1358M155T121 5 M359T2328M376T2312P3472 M126T131M172T1592M231T1592M317T1591M222T1592M156T1592M99T1592M132T1585M311T118M107T712M84T111M130T111 . M623T2084M469T2106M381T2340M382T2341 M210T1591M136T1592Arginine[M+H]+M179T128 0 M138T315 M146T174Apoa1 RC8 - P3072 CC1 M134T220M511T1312
5 M308T176 . RC3 0 - 0 RR6 . 1 -
-1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0
Component 1 ( 12.84 %) Component 1 ( 12.84 %)
Fig. 4 UPCA scores plot (a) for the first two components. UPCA correlation circle plot (b) for the first two components: the 103 selected proteins are labelled in black (labels consist of spot numbers for the unidentified proteins), the 585 selected metabolites are labelled in blue (labels consist of the m/z value and the retention time for the unidentified metabolites). a RR1 b RR1 2 2 RR2 RR2 2
t 2
n t e n n e o n p o 1 CC7 1 CC7 p m o m
c RR5 o RR5
l c a l RC7 RC7 c i a RR4 RR4 c n i o n
RR3 n RR3 o a n c a
: c
0 RC2 0 RC2
CC8 CC8 s
: RC1 CC4 RC1 CC4 e
s RC5 RC5 CC6 t CC6 i
n RR7 l RR7 i o e RR8 CC5 CC5 t b RR8 a o r t e P CC2 CC2 M
1 CC3 1 CC3 - RC8 - RC8 RC3 RC3 CC1 CC1 RR6 RR6
-2 -1 0 1 2 -2 -1 0 1 2 Proteins: canonical component 1 Metabolites: canonical component 1
c 0 d 0 . . 1 1
P3099 P3099 P3096P3302 P3096P3302 Fscn1P2987 Fscn1P2987 P3087P3246P3177 P3087P3246P3177 P3181 P3209P3216 P3181 P3209P3216 P3197 P3197 M58T135 M127T167 P3242 P3272 M58T135 M127T167M392T357P3242 P3272 2 5 M392T357P3086 P3443 5 P3086 P3443
. M248T202 P3013 . M248T202 P3013 DpysI5 t DpysI5 2 0 0
M364T126 M250T1143 M364T126 M250T1143 M271T137M141T133M253T134M308T1029M282T274M310T1547P3370 n M271T137M141T133M253T134M308T1029M282T274M310T1547P3370 t M140T134M75T151M96T133M257T133M294T136P2947 M140T134M75T151M96T133M257T133M294T136P2947 M216T943M349T2066 M303T1823M168T454AldoC e M206T179M216T943M349T2066 M303T1823M168T454AldoC n M206T179M155T943M339T132M201T944M284T126M204T223M154T1032M249T199M126T398 M155T943M339T132M202T1051M216T1050M201T944M284T126M204T223M154T1032M249T199M126T398 M269T180M202T1051M216T1050M120T125M286T1028M293T367 M390T1991 n M269T180M120T125M286T1028M293T367 M390T1991 e M107T1198M57T152M255T715M144T178M155T1051M201T1052M187T850M441T180M202T974M322T1050M322T944M515T125M227T129M178T163P3412P3341M521T2092 M107T1198M57T152M255T715M407T177M144T178M97T1198M155T1051M201T1052M187T850M130T1050M441T180M202T974M322T1050M322T944M515T125M227T129M178T163P3412P3341M521T2092 M153T1198M407T177M97T1198M245T180M130T1050M259T167M219T1614M475T2092 M621T2084 o M153T1198M118T1198M114T1203M245T180M259T167M219T1614M475T2092 M621T2084 n M85T178M118T1198M123T1198M114T1203M303T191M213T1198M212T1198M410T126M211T921M109T1051M180T127 M539T2044 P3270 M85T178M123T1198M303T191M213T1198M212T1198M410T126M211T921M109T1051M180T127M306T127 M539T2044 P3270 M250T142M493T111M115T1198M282T126M205T248M306T127M585T109P3423M227T1381M435T1359P2941 p M250T142M493T111M115T1198M205T248M282T126M585T109P3423M227T1381M435T1359P2941
o M143T1030M233T906 M416T2084M431T1384P3247Oat Carnitine[M+Na]+M143T1030M233T906M503T2055M416T2084M431T1384P3247Oat M249T140O-acetyl-carnitine[M+H]+Carnitine[M+Na]+M222T123M240T263M127T1416M181T191M229T244M87T362M320T1198M205T178M428T1923M118T608M225T608M386T127M69T610M135T1136M266T103M138T368M164T128M224T1049M503T2055M267T180M416T1375M396T1434M202T140M418T2019M332T1642 M737T2075M633T1359 M249T140O-acetyl-carnitine[M+H]+M222T123M240T263M181T191M229T244M127T1416M87T362M320T1198M205T178M428T1923M118T608M69T610M225T608M386T127M135T1136M74T133M138T368M266T103M164T128M224T1049M267T180M396T1434M416T1375M202T140M418T2019M332T1642 M737T2075M633T1359 p M152T1416M74T133M105T315M399T2149M306T179M525T2077 M276T140 M152T1416M105T315M268T1241M399T2149M306T179M525T2077 M276T140 M156T132M268T1241P2927 P3327 m M366T134M86T178M125T130M156T132M235T132P2927M422T108 P3327 M366T134M86T178M125T130M232T904M290T186M235T132M153T307M222T387M260T126M426T2105M422T108M243T1636M226T177M292T1785M427T1385M302T1797M399T1355 M177T196M232T904M290T186M153T307M222T387M260T126M426T2105M243T1636M226T177M292T1785M427T1385M302T1797M399T1355 M177T196M260T194M218T253P2911M372T2091M434T125M189T247M170T317M105T358M153T373M218T214P3005P2973M557T1384 o M260T194M73T163M218T253P2911M372T2091M434T125M189T247M170T317M105T358M153T373M218T214P3005P2973M557T1384 m M343T182M73T163M132T1198M286T1983M435T2046M304T1198M322T137M369T2090M254T214M138T769P3009M273T99P2984M252T1970M541T1408P3399M344T1359M610T2086M668T1359M379T138M416T1221M401T240 M343T182M132T1198M286T1983M435T2046M304T1198M322T137M369T2090M254T214M138T769P3009M273T99P2984M252T1970M541T1408P3399M344T1359M610T2086M668T1359M379T138M416T1221M401T240 M535T2091M247T1240M302T163M564T2087 c O-acetyl-carnitine[fragments]M318T1306M535T2091M247T1240M302T163M564T2087 o O-acetyl-carnitine[fragments]M318T1306M246T1243M232T192M294T1820M424T2068P3068 M497T1289M489T1310P3458P3588 M246T1243M232T192M294T1820M424T2068P3068 M497T1289M489T1310P3458 M209T2032M346T150M123T213M130T193M118T314M247T317M171T831M477T1390 M739T2075M459T1359Acy1P3579 M209T2032M346T150M123T213M130T193M118T314M247T317M171T831M477T1390M739T2075Acy1P3579P3588 M203T165M307T278M233T247M361T1268M119T213M109T314M225T317M228T1198M626T2048M512T2056P3435M197T358 l M203T165M307T278M233T247M361T1268M119T213M109T314M225T317M228T1198M626T2048M512T2056P3435M459T1359M197T358 c M304T128M584T2218M261T1222M734T2075M60T178M117T196M232T126M659T2094M109T357M134T291M357T98M289T180M313T138M218T141 M304T128M584T2218M261T1222M60T178M734T2075M117T196M232T126M659T2094M109T357M134T291M357T98M289T180 M313T138M218T141 M346T1427M308T274M467T2149M607T2082M290T1198M278T140M553T2046P3379GapdhM110T131 P3243 a M346T1427M308T274M467T2149M555T2139M607T2082M290T1198M278T140M553T2046P3379GapdhM110T131 P3243 l M187T152M258T132M555T2139M148T176M415T2056M118T355M203T356M225T356M305T176M179T315M408T2095M622T2084M435T1785M581T1371_2M307T116M427T153M283T144 M187T152M258T132M148T176M415T2056M118T355M203T356M225T356M305T176M179T315M622T2084M408T2095M435T1785M307T116M581T1371_2M427T153M283T144 M510T2048M445T1371M147T188M133T290 c P3010 M510T2048M445T1371M147T188M133T290 i a P3010M689T2078M262T354M442T2299M320T1996M641T2039M446T1371M364T247M449T1371M185T2017M313T1161M297T139 M689T2078M262T354M442T2299M320T1996M641T2039M446T1371M364T247M449T1371M185T2017M313T1161 0 M620T2047P3564 M172T94M380T126M441T1311M441T1312 0 M620T2047P3564 M172T94M380T126M441T1311M441T1312M297T139 c n . M369T959M740T2075M178T830M565T2090P3127P2939M604T2055M407T1320M160T141M447T1080 . M369T959M740T2075M178T830P3127P2939M604T2055M407T1320M160T141 i M685T2036M137T768P3487M437T1785M519T2090M295T192M138T191M171T2225P3570M581T1371_1M259T305M329T202P3308M232T249M198T310M91T310Map2k1 M685T2036M137T768P3487M437T1785M519T2090M295T192M565T2090M138T191M171T2225P3570M581T1371_1M259T305M329T202M447T1080P3308M198T310Map2k1 o 0 M291T770M338T2388P3212M415T1544M107T213M92T219 M476T1310M369T1299M123T246M184T310 0 M338T2388P3212M415T1544M107T213M330T1350M92T219 M476T1310M369T1299M232T249M91T310 n Carnitine[M+H]+M526T2003 M207T127M330T1350M137T1198M272T163M436T1785M447T2021M180T1320M505T1311P2940M389T1991M218T1160M161T139M119T246P3397M119T310M123T310P3573 Carnitine[M+H]+M526T2003M291T770M207T127M137T1198M272T163M436T1785M447T2021M180T1320M505T1311P2940 M161T139M123T246M119T246M123T310M119T310M184T310P3573 M269T769M299T96M275T170M434T2216M199T97P3021P3138M336T1081M217T95M475T1310M286T1482Psmb6 n M299T96M275T170M434T2216M199T97P3021P3138M336T1081M217T95M389T1991M218T1160M286T1482P3397Psmb6 o P3036 M318T312M782T2072M714T2033M581T1676M366T1357M338T140M170T761M474T1310M487T1311M254T140M316T179M291T138Tyrosine[fragments.]Tyrosine[fragments]M165T246Tyrosine[M+H]+M136T246M377T1368M183T310M363T310 P3036M269T769M318T312M782T2072M714T2033M581T1676M366T1357M338T140M170T761M474T1310M475T1310M487T1311M254T140M291T138M316T179Tyrosine[fragments.]Tyrosine[fragments]Tyrosine[M+H]+M136T246M377T1368M363T310M183T310 M217T1030M305T349M285T1401M201T260M358T179M91T140 M274T127M311T907 a M217T1030M305T349M285T1401M201T260M91T140M358T179 M165T246M311T907 n M284T1970M438T2035P3556M304T191P2945M611T2056M173T142M468T1310M547T2074M283T1417M139T139M256T139M182T246M183T247M86T289M363T238 M284T1970M438T2035P3556M304T191P2945M611T2056M173T142M468T1310M547T2074M283T1417M139T139M274T127M256T139M182T246M86T289M183T247M363T238 M576T2047M563T2087M196T2096M163T162M182T213M518T2090M181T133P3591M164T1497M253T1080M138T141M311T955 c M576T2047M563T2087M196T2096M163T162M182T213M518T2090P3591M164T1497M253T1080M138T141M311T955 a M250T295 M300T1552 M250T295M181T133 M300T1552 M368T128M554T2044M534T2091M376T2046M237T1632M412T1724M335T149P3323M465T126ProlineM367T124M184T233M135T178M134T127 [M+H]+P3494 M368T128M554T2044M534T2091M376T2046M237T1632M412T1724M335T149P3323M465T126M367T124M184T233M135T178M134T127 M520T2090M152T793 M298T1631M153T145Proline[fragments]Methionine[M+H]+M415T184 : M520T2090M152T793 M298T1631 Proline [M+H]+P3494 c M263T2056M270T769M208T371M533T1670M644T2081M310T137M577T2004M582T2042M261T1178Methionine[fragments.]Methionine[fragments]M243T101M487T179M237T182M243T1080M152T181M151T180M261T1103M56T180M493T247P3581P3524 M270T769M263T2056M208T371M533T1670M644T2081M310T137M577T2004M153T145Methionine[fragments.]M243T101Methionine[fragments]M487T179Proline[fragments]Methionine[M+H]+M415T184M237T182M261T1103M152T181M151T180M56T180P3581P3524
M713T2033 M130T1080M137T312M231T182M134T180 M59T134 M713T2033 M582T2042M130T1080M261T1178M243T1080M493T247 M231T1240M268T887M359T164M59T134P3114M490T2102M598T2042M670T2036M579T2214M729T2033Psma6M221T1080M350T98M250T124M170T1080M306T134P3577 s M231T1240M359T164P3114 M490T2102M670T2036M598T2042M579T2214M729T2033Psma6M221T1080M137T312M350T98M231T182M250T124M170T1080M134T180P3577 : M282T181M499T1310M498T1310M499T1311M160T601M462T716M609T2087M215T182M249T336P2961P3580P3516 M268T887 M499T1311M160T601M462T716M609T2087M215T182M249T336P2961M306T134P3580P3516 M424T2094Uqcrfs1M292T1544M566T2090M194T1632 M379T707M212T1387M143T1080M211T1447P3583P3575 e M424T2094Uqcrfs1M292T1544M566T2090M194T1632M282T181M499T1310M498T1310M212T1387M143T1080M211T1447P3583P3575 s M342T1400M240T115 M240T115M379T707 M318T230 M385T712Leucine[M+H]+M133T216P3146 Prdx3 t M318T230M342T1400Leucine[M+H]+M133T216P3146 Prdx3 M284T2092M471T2428 M147T1080M183T98M356T716M254T1970M296T711M331T163M472T180Leucine[fragments]P3537 i M284T2092M471T2428 M183T98M147T1080M296T711M385T712M331T163M472T180Leucine[fragments]P3537 n M537T769P3340 M267T1382M245T1382M168T1082M461T711M281T1586M216T182Alb.P3498M262T1167M87T215Eif2bP3551 l P3340 M267T1382M168T1082M356T716M254T1970M461T711M281T1586Alb.M216T182M87T215Eif2bP3551 i M222T205M152T1585M349T1430M249T1382M207T1080M642T2039M229T1078P3538M127T1585M264T711M183T1585P3474M283T2093ActbP3523M141T170 M537T769M222T205M152T1585M349T1430M249T1382M245T1382M207T1080M642T2039M229T1078P3538M127T1585P3474P3498M262T1167M283T2093ActbP3523M141T170 M578T2088 M599T2130M597T2043M172T1381Tryptophan[M+Na]+M757T2030M275T1081M471T180M160T1080M181T708 o M578T2088 M599T2130M597T2043M172T1381M757T2030M275T1081M264T711M183T1585 e M137T196M354T203M376T2312P3312M665T2081M408T1321M268T1592Phenylalanine[fragments]Phenylalanine[M+H]+M192T677M190T1080M392T1359M244T180M409T1080M265T717M220T163M108T708M167T708M204T708M211T127P3069M370T716M332T128 M137T196M354T203M376T2312P3312M665T2081M408T1321M268T1592Tryptophan[M+Na]+Phenylalanine[M+H]+M190T1080M392T1359M244T180M409T1080M471T180M220T163M108T708M160T1080M181T708M167T708M204T708P3069M370T716M332T128 t M359T2328 Tryptophan[M+H]+M180T1143M335T1080Valine[fragments]M266T179M331T708M369T710M296T1273 b M359T2328 Tryptophan[M+H]+Phenylalanine[fragments]M192T677M265T717M369T710M211T127M296T1273 P3256M469T2106M381T2340M382T2341M325T191M183T213 M161T1592M252T1429M231T1592M222T1592M319T1080M145T1592M99T1592M189T1080M188T1080M206T1080M159T1080M265T156M336T128M534T710M148T709M334T1358M149T709M103T709M121T708M292T133M79T708M295T1274 P3256M469T2106M381T2340M382T2341M325T191M183T213 M161T1592M319T1080M145T1592M189T1080M206T1080M159T1080M188T1080M180T1143M335T1080M265T156Valine[fragments]M266T179M336T128M148T709M534T710M331T708M334T1358M149T709M103T709M121T708M292T133M79T708M295T1274 a o M251T1429M359T127M472T2428M172T1592M210T1591M317T1591M156T1592M146T1080M314T127P3549AlbM218T1586 M251T1429M472T2428M359T127M252T1429M172T1592M231T1592M317T1591M156T1592M222T1592M146T1080M99T1592P3549M314T127AlbM218T1586 r P3072P3621 M136T1592M303T1080M144T1080M335T1358P3535M107T712M133T110 t P3072P3621 M136T1592M210T1591M303T1080M144T1080M335T1358P3535M107T712M133T110 5 M126T131M132T1585YwhazM69T206 5 M126T131M132T1585Ywhaz e M69T206 P . M311T118M84T111 . M311T118 M623T2084M138T315 Arginine[M+H]+M179T128P3070M130T111M155T121 M623T2084 Arginine[M+H]+M179T128M84T111P3070M130T111 0 M146T174 0 M138T315 M146T174M155T121 M - M134T220M308T176P3548P3488Apoa1 - M134T220M308T176P3548P3488Apoa1 M511T1312P3472 M511T1312P3472 0 0 . . 1 1 - -
-1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 Proteins: canonical component 1 Metabolites: canonical component 1
Fig. 5 RCCA scores plot for the first two components of the proteins matrix (a), and for the first two components of the metabolites matrix (b). RCCA correlation circle plot for the first two components of the proteins matrix (c), and for the first two components of the metabolites matrix (d): the 103 selected proteins are labelled in black (labels consist of spot numbers for the unidentified proteins), the 585 selected metabolites are labelled in blue (labels consist of the m/z value and the retention time for the unidentified metabolites). Fig. 6 Fig. m the of consist (labels blue in labelled are metabolites selected 585 the proteins), unidentified the for numbers spot of consist (labels black in labelled are proteins selected 103 the 3: and 1 components / a z z Component 3 ( 5.94 %) value and the retention time for the unidentified metabolites). unidentified timeforthe and thevalue retention BL crspo a o h opnns1ad3 3. and 1 components the for (a) plot scores MBPLS -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 -0.8 CC3 CC1 -0.6 CC2 CC6 CC5 -0.4 CC4 CC7 CC8 RC3 Component 112.14 ( %) -0.2 RC1 RC7 RC5 RC8 0.0 RC2 RR5 0.2 RR8 0.4 RR2 RR1 RR4 0.6 RR3 RR6 RR7 b
Component 3 ( 5.94 %)
-1.0 -0.5 0.0 0.5 1.0 hn aaiefragments] lalanine[f Pheny ehoiefragments.] Methionine[f -1.0 ehoiefragments] Methionine[f hn lalanine[M+H]+ Pheny Arginine[M+H]+ M130T111 yrsn[ ragments.] rosine[f Ty ragments] Valine[f yrsn[ ragments] rosine[f Ty ecn[ ragments] Leucine[f M103T709 M295T1274 M370T716 M296T1273 M79T708 M141T170 M121T708 M107T712 M211T127 M369T710 M534T710 M179T128 Methionine[M+H]+ M306T134 M211T1447 M334T1358 M149T709 M155T121 M56T180 M335T1358 rln[ ragments] Proline[f M181T708 M204T708 M331T708 M300T1552 M218T1586 M108T708 M292T133 M69T206 M134T180 M167T708 M148T709 yrosine[M+H]+ Ty M135T178 M336T128 M311T118 M119T310 M311T955 M493T247 M123T310 M461T711 M87T215 M134T127 M146T174 M132T1585 Proline [M+H]+ [M+H]+ Proline Leucine[M+H]+ M471T180 M266T179 M91T310 M84T111 M152T181M151T180 M143T1080 M472T180 M133T110 M183T1585M127T1585 M296T711 M363T310 M363T238 M377T1368 M184T310M183T310 r ptophan[M+H]+ Try M462T716 M281T1586 M119T246 M256T139 M180T1143 M232T249 M133T216 ptophan[M+Na]+ Try M249T336 M198T310 M367T124 Apoa1 M243T1080 M170T1080 M156T1592 M123T246 M244T180 M164T1497 M145T1592 M311T907 M286T1482 M261T1103 M297T139M136T246 M161T139 M262T1167 M172T1592 M210T1591 M136T1592 M379T707 M231T182 M316T179 M137T312 M409T1080 M359T127 M159T1080 M222T1592 M274T127 M165T246M139T139 M385T712 M183T247 M184T233 M265T156 M190T1080 M335T1080 M317T1591 M415T184 M138T141 M216T182 M264T711M356T716 M332T128 M189T1080M188T1080 M182T246 M265T717 M206T1080 M144T1080 M215T182 MBPLS loadings plot (b) for the for (b) plot loadings MBPLS M245T1382 M146T1080 M160T141 M99T1592 M474T1310 M369T1299 M161T1592 M487T1311 M441T1312 M475T1310 M160T1080 M303T1080 M254T140 M291T138 M221T1080 M261T1178M476T1310 M441T1311 M275T1081M229T1078 M218T141 M86T289 M267T1382 M231T1592 M268T1592 M407T1320 M468T1310 M319T1080 M170T761 M220T163 M237T182 M250T124 Psmb6 P3524 Prdx3 M252T1429 M192T677 M331T163 M314T127 M313T1161 M505T1311 P3581 M283T1417 M251T1429 M498T1310M499T1310 P3577 M499T1311 Alb M379T138 M276T140 M487T179 M489T1310 M416T1221 M408T1321 P3573 M130T1080 M152T1585 M465T126 M133T290 M447T1080 M497T1289 M253T1080 M172T1381 P3243 M173T142 M459T1359 M278T140 P3494 P3537 P3535 M212T1387 M283T2093 M249T1382 M254T1970 P3551 M668T1359 M366T1357 M389T1991 M218T1160 P3523 M380T126 P3575 M207T1080 M392T1359 M313T138 P3070 M449T1371 M168T1082 M349T1430 P3516 Alb. M401T240 P3583 M153T145 M350T98 M581T1371_1 Map2k1 2b Eif M399T1355 Actb Psma6 -0.5 M547T2074 M412T1724 M147T1080 M329T202 M160T601 M609T2087 M110T131 P3069 M126T131 M611T2056 M642T2039 P3588 P3146 Gapdh Ywhaz M445T1371 M180T1320 P3397 M435T1359 M581T1371_2 P3538 M633T1359 M240T115 M511T1312 M757T2030 M521T2092 M342T1400 M308T176 P3197 P3549 M446T1371 P3458 M427T153 M243T101 M582T2042 M181T133 P2961 M338T140 M259T305 P3323 P3488 M298T1631 M644T2081 M344T1359 P3270 P3308 M185T2017 M431T1384 P3272 P2941 M553T2046 M577T2004 M194T1632 M436T1785 P3474 P3580 P3498 M737T2075 M512T2056 P3379 M283T144 1 Acy M237T1632 M665T2081 M310T137 M557T1384 M610T2086 P2945 M581T1676 M427T1385 M472T2428 M138T315 P2940 M282T181 M579T2214 M307T116 M250T295 M604T2055 P3327 M134T220 M336T1081 M739T2075 M490T2102 M597T2043 Component 1 12.14 ( %) M183T98 P3548 M252T1970 M272T163 P3570 P3247 P3209 M222T205 Fscn1 M364T247 M197T358 M178T163 M318T230 Oat M729T2033 M196T2096 P3312 M576T2047 P3579 M183T213 M539T2044 M227T1381 P2939 M621T2084 P3177 M217T95 M163T162 M566T2090 M518T2090 M182T213 M620T2047 M782T2072 M304T191 M533T1670 p sI5 Dpy M147T188 M320T1996 M263T2056 M376T2046 M469T2106M382T2341 M477T1390 M376T2312 M381T2340 P3341 P3412 M713T2033 P3435 M670T2036M598T2042 M424T2068 M359T2328 M437T1785 M434T2216 P3216 P3472 M335T149 M471T2428 P2973 M199T97 M91T140 P2987 P3068 M565T2090 M294T1820 M358T179 M447T2021 M299T96 M318T312 M302T1797 M626T2048 M435T1785 M207T127 M275T170 M368T128 M734T2075 M714T2033 M563T2087 M172T94 M354T203 M537T769 M292T1785 M415T2056 M390T1991 M599T2130 M438T2035 M564T2087 P3564 M332T1642 P3246 P3127 M641T2039 P3138 M295T192 0.0 M330T1350 M740T2075 M554T2044 P3443 M525T2077M541T1408 M534T2091 P3302 M305T349 M201T260 M426T2105 M399T2149 M519T2090 M623T2084 P3399 M526T2003 M325T191 P3340 M475T2092 M503T2055 P3591 s1 Uqcrf M243T1636 M622T2084 M302T163 P3423 M284T1970 P3096 M357T98 M153T373 M422T108 P3009 M273T99 M227T129 M202T140 M408T2095 M92T219 M168T454 M424T2094 M171T2225 M153T307 M138T769 M107T213 M396T1434 M416T1375 M270T769 M607T2082 M137T196 M418T2019 M292T1544 M291T770 M689T2078 M289T180 M134T291 M152T793 M659T2094 M555T2139 M442T2299 M520T2090 M285T1401 M467T2149 M269T769 P3212 P3242 P3005 P2984 P3087 M137T768 M303T1823 P3556 M585T109 M369T959 M306T179 M338T2388 M219T1614 M510T2048 M535T2091 P3021 M290T1198 M138T191 M232T126 M178T830 P3099 M262T354 M685T2036 M225T356 M119T213 M208T371 M416T2084 P3013 AldoC P3370 M305T176 M179T315 M203T356 M322T137 P3086 M218T214 M228T1198 M254T214 M118T355 M250T1143 M349T2066 M137T1198 M117T196 M209T2032 P3114 P3072 M284T2092 M226T177 M369T2090 M268T887 M584T2218 M415T1544 P3181 M171T831 M286T1983 M372T2091 M225T317 M218T253 P3036 P2927 P3487 M203T165 M359T164 P3621 M392T357 M109T357 M578T2088 P3256 M247T317 M232T192 P2947 M148T176 M109T314 P3010 P2911 M240T263 M428T1923 M303T191 M435T2046 M118T314 M266T103 M268T1241 M515T125 M224T1049 M59T134 -ct -antn[ ragments] l-carnitine[f O-acety M246T1243 M247T1240 M361T1268 M260T126 M231T1240 M258T132 M290T186 M304T1198 Carnitine[M+H]+ M267T180 M434T125M127T167 M74T133 M217T1030 M126T398 M346T150 M105T358 M156T132 M170T317 M308T274 M310T1547 M107T1198 M222T387 M364T126 M132T1198 M493T111 M233T247 l-carnitine[M+H]+ O-acety M261T1222 M235T132 0.5 M189T247 M386T127 M123T213 M282T126 M322T1050 M152T1416 M307T278 M135T1136 M346T1427 M322T944 M105T315 M294T136 M130T193 M286T1028 Carnitine[M+Na]+ M293T367 M201T1052 M155T1051 M441T180 M202T1051 M60T178 M410T126 M202T974 M154T1032M109T1051 M164T128 M180T127 M204T223 M225T608 M216T1050 M118T608 M201T944 M306T127 M69T610 M318T1306 M259T167 M187T152 M343T182 M115T1198 M155T943 M127T1416 M282T274 M130T1050 M320T1198 M125T130 M233T906 M205T248 M260T194 M339T132 M216T943 M232T904 M304T128 M212T1198 M138T368 M213T1198 M143T1030 M187T850 M97T1198 M211T921 M366T134 M308T1029 M123T1198 M249T199 M248T202 M57T152 M87T362 M73T163 M255T715 M114T1203 M269T180 M222T123 M153T1198 M205T178 M245T180 M181T191 M177T196 M118T1198 M284T126M120T125 M229T244 M407T177 M86T178 M144T178 M257T133 M58T135 M253T134 M250T142 M85T178 M249T140 M75T151 M140T134 M206T179 M271T137 M141T133 M96T133 1.0