PME8 QTAPAS A LABEL FREE

PME8 QTAPAS A LABEL FREE QUANTIFICATION APPROACH MARTA MENDES, MARÍA FERNANDÉZ AND IGNACIO CASAL CENTRO DE INVESTIGACIONES BIOLÓGICAS MADRID MARCH 2013

PME 8 QTAPAS OVERVIEW TARGETED PROTEOMICS FOR AQUA GLOBAL MASS LIST vs LABEL FREE QUANTIFICATION TOP 10

PME 8 QTAPAS THE METHODS Proxeon nlc coupled to LTQ Orbitrap Velos Pre column: 100 m, 2 cm, 5 m Column: Biosphere C18, 75 m, 15 cm, 3 m, 120 A Buffer A: 0.1% Formic acid/2%acn Buffer B: 0.1% Formic acid in ACN

PME 8 QTAPAS TARGET

PME 8 QTAPAS LABEL FREE QUANTIFICATION

PME 8 QTAPAS DATA ANALYSIS TARGETED PROTEOMICS FOR AQUA LABEL FREE QUANTIFICATION OMICSHUB SKYLINE MAXQUANT AND PERSEUS

PME 8 QTAPAS TARGETED PROTEOMICS FOR AQUA SKYLINE FOR GGPFSDSYR A A B B C C D D E E

PME 8 QTAPAS TARGETED PROTEOMICS FOR AQUA SKYLINE FOR FSTVAGESGSADTVR A A B B C C D D E E

PME 8 QTAPAS TARGETED PROTEOMICS FOR AQUA Tier Peptide Sample A Sample B Sample C Sample D Sample E 1 CAH1/P00915_GGPFSDSYR 1,00 1,00 1,00 1,00 1,00 1 CAH1/P00915_VLDALQAIK 2,00 2,00 2,00 2,00 2,00 CAH2/P00918_AVQQPDGLAVLGIFL K 10,00 10,00 10,00 10,00 10,00 1 1 CAH2/P00918_SADFTNFDPR 50,00 50,00 50,00 50,00 50,00 2 NQO1/P15559_EGHLSPDIVAEQK 1,00 1,00 1,00 1,00 1,00 2 NQO1/P15559_ALIVLAHSER 2,00 2,00 2,00 2,00 2,00 2 CRP/P02741_ESDTSYVSLK 10,00 10,00 10,00 10,00 10,00 2 CRP/P02741_GYSIFSYATK 50,00 50,00 50,00 50,00 50,00 3 PPIA/P62937_FEDENFILK 0,50 0,50 0,50 0,50 0,50 3 PPIA/P62937_VSFELFADK 1,00 1,00 1,00 1,00 1,00 3 PPIA/P62937_TAENFR 2,00 2,00 2,00 2,00 2,00 CATA/P04040_GAGAFGYFEVTHDIT K 0,20 0,20 0,20 0,20 0,20 3 3 CATA/P04040_FSTVAGESGSADTVR 10,00 10,00 10,00 10,00 10,00 3 CATA/P04040_NLSVEDAAR 50,00 50,00 50,00 50,00 50,00 Tier Peptide Sample A Sample B Sample C Sample D Sample E Sample A Sample B Sample C Sample D Sample E 1 CAH1/P00915_GGPFSDSYR 0,99 0,98 1,03 1,01 1,00 0,99 0,98 1,03 1,01 1,01 1 CAH1/P00915_VLDALQAIK 2,09 2,04 1,95 2,02 2,97 2,10 2,04 1,98 2,02 2,98 CAH2/P00918_AVQQPDGLAVLGIFL 1,50 6,23 8,41 7,70 8,97 6,77 1 K 19,09 8,45 7,71 8,97 1 CAH2/P00918_SADFTNFDPR 27,15 42,77 48,80 60,00 59,31 27,58 42,79 49,08 60,01 59,33 2 NQO1/P15559_EGHLSPDIVAEQK 0,74 0,70 0,70 0,69 0,72 0,75 0,71 0,74 0,66 0,70 2 NQO1/P15559_ALIVLAHSER 0,63 0,83 0,90 0,89 0,82 0,62 0,83 0,98 0,94 0,86 2 CRP/P02741_ESDTSYVSLK 1,62 6,15 8,22 9,25 8,92 4,31 6,26 8,77 9,08 9,59 2 CRP/P02741_GYSIFSYATK Heavy only Heavy only Heavy only Heavy only Heavy only Heavy only Heavy only Heavy only Heavy only Heavy only 3 PPIA/P62937_FEDENFILK 0,27 0,30 0,25 0,27 0,27 0,27 0,30 0,24 0,28 0,28 3 PPIA/P62937_VSFELFADK 1,80 0,42 0,13 0,57 0,52 1,76 0,79 0,04 0,21 0,55 3 PPIA/P62937_TAENFR 117,95 97,26 36,01 10,80 4,55 118,68 99,59 33,57 19,83 4,15 CATA/P04040_GAGAFGYFEVTHDIT 0,07 0,25 0,81 0,47 0,22 0,07 3 K 0,25 0,69 0,36 0,26 3 CATA/P04040_FSTVAGESGSADTVR 4,33 4,89 5,61 5,77 5,90 4,33 4,90 5,29 6,63 6,42 3 CATA/P04040_NLSVEDAAR 6,09 2,97 33,24 30,41 34,75 19,76 21,63 26,68 26,42 33,64

PME 8 QTAPAS TARGETED PROTEOMICS FOR LABEL FREE QUANTIFICATION Tier Peptide E/A E/B E/C E/D E/E 1 CAH1/P00915_GGPFSDSYR 86.97 27.59 10.82 4.56 1 1 CAH1/P00915_VLDALQAIK 172.29 41.64 29.61 6.13 1 1 CAH2/P00918_AVQQPDGLAVLGIFLK 1210.96 112.37 160.23 8.29 1 1 CAH2/P00918_SADFTNFDPR 106.62 28.87 549.67 4.45 1 2 NQO1/P15559_EGHLSPDIVAEQK 106.64 35.72 9.03 5.25 1 2 NQO1/P15559_ALIVLAHSER 314.08 49.71 10.29 6.32 1 2 CRP/P02741_ESDTSYVSLK 118.94 31.92 94.24 4.84 1 2 CRP/P02741_GYSIFSYATK 3 PPIA/P62937_FEDENFILK 220.79 34.22 3.27 5.76 1 3 PPIA/P62937_VSFELFADK 5.94 9.34 2.16 6.67 1 3 PPIA/P62937_TAENFR 2.22 1.05 37.20 0.82 1 3 CATA/P04040_GAGAFGYFEVTHDITK 58.95 2.79 0.85 1.38 1 3 CATA/P04040_FSTVAGESGSADTVR 102.23 28.78 61.00 4.59 1 3 CATA/P04040_NLSVEDAAR 101.16 28.91 355.52 4.37 1

PME 8 QTAPAS LABEL FREE QUANTIFICATION MAXQUANT CAH1 (A, B, C, D and E) CAH2 (A, B, C, D and E) NQO1 (A, B, C, D and E) CRP (A, B, C, D and E) PPIA (C, E) CAT (A, B, C, D and E)

A3 B1 B2 B3 C1 C2 C3 D1 D2 D3 E1 E2 CORRELATIONS E3 PEARSON A2 A1 PME 8 QTAPAS LABEL FREE QUANTIFICATION A1 A2 A3 B1 B2 B3 C1 C2 C3 D1 D2 D3 E1 E2 E3

PME 8 QTAPAS LABEL FREE QUANTIFICATION PERSEUS ANOVA CAT CAH1 CAH2 NQO1 CRP CAT CAH1 CAH2 NQO1 CRP CAH1 CA T CAH2 NQO1 CRP CAH1 CAT CAH2 NQO1 CRP PERSEUS T TEST CAH2 CAT CAH1 NQO1 CRP CAH1 CAT CAH2 NQO1 CRP CAH1 CAT NQO1 CAH2 CRP PPIA CAT CAH1 NQO1 CAH2 CRP

PME 8 QTAPAS LABEL FREE QUANTIFICATION Tier Peptide Sample A Sample B Sample C Sample D Sample E 1 CAH1/P00915_GGPFSDSYR 50,0 25,0 10,0 5,0 1,0 1 CAH1/P00915_VLDALQAIK 50,0 25,0 10,0 5,0 1,0 1 CAH2/P00918_AVQQPDGLAVLGIFLK 50,0 25,0 10,0 5,0 1,0 1 CAH2/P00918_SADFTNFDPR 50,0 25,0 10,0 5,0 1,0 2 NQO1/P15559_EGHLSPDIVAEQK 50,0 25,0 10,0 5,0 1,0 2 NQO1/P15559_ALIVLAHSER 50,0 25,0 10,0 5,0 1,0 2 CRP/P02741_ESDTSYVSLK 50,0 25,0 10,0 5,0 1,0 2 CRP/P02741_GYSIFSYATK 50,0 25,0 10,0 5,0 1,0 3 PPIA/P62937_FEDENFILK 50,0 25,0 10,0 5,0 1,0 3 PPIA/P62937_VSFELFADK 50,0 25,0 10,0 5,0 1,0 3 PPIA/P62937_TAENFR 50,0 25,0 10,0 5,0 1,0 3 CATA/P04040_GAGAFGYFEVTHDITK 50,0 25,0 10,0 5,0 1,0 3 CATA/P04040_FSTVAGESGSADTVR 50,0 25,0 10,0 5,0 1,0 3 CATA/P04040_NLSVEDAAR 50,0 25,0 10,0 5,0 1,0

PME 8 QTAPAS LABEL FREE QUANTIFICATION MAXQUANT Peptide E/A E/B E/C E/D E/E CAH1/P00915_GGPFSDSYR 74.70 22.94 10.10 3.87 1 CAH1/P00915_VLDALQAIK 95.17 21.78 8.18 3.09 1 CAH2/P00918_AVQQPDGLAVLGIFLK - 88.46 19.06 8.57 1 CAH2/P00918_SADFTNFDPR 85.39 24.66 9.25 4.34 1 NQO1/P15559_EGHLSPDIVAEQK 164.17 51.78 11.31 6.84 1 NQO1/P15559_ALIVLAHSER - - 14.62 5.83 1 CRP/P02741_ESDTSYVSLK 9282.18 11.45 4.81 1 CRP/P02741_GYSIFSYATK 351.44 67.08 18.66 6.99 1 PPIA/P62937_FEDENFILK - 136.69 14.13 4.49 1 PPIA/P62937_VSFELFADK - - - - - PPIA/P62937_TAENFR - - - - - CATA/P04040_GAGAFGYFEVTHDITK - - - - CATA/P04040_FSTVAGESGSADTVR 97.77 32.09 11.39 4.29 1 CATA/P04040_NLSVEDAAR - - 15.72 3.68 1 Protein E/A E/B E/C E/D E/E CAH1 33.36 22.08 8.52 4.69 1.00 CAH2 32.25 26.92 11.40 4.91 1.00 NQO1 61.65 46.53 12.36 5.58 1.00 CRP 137.75 46.41 13.11 5.80 1.00 PPIA - - 14.41-1.00 CAT 43.21 26.67 12.13 5.89 1.00

PME 8 QTAPAS LABEL FREE QUANTIFICATION Experiments Accession Peptides A B C D E P00915/CAH1 ADGLAVIGVLMK + + + + + ASPDWGYDDK + + + + + ASPDWGYDDKNGPEQWSK + + + + ESISVSSEQLAQFR + + + GGPFSDSYR + + + + + HDTSLKPISVSYNPATAK + + + + + LYPIANGNNQSPVDIK + + + + + NGPEQWSK + + + + + PISVSYNPATAK + + + + SLLSNVEGDNAVPMQHNNR + + + SLLSNVEGDNAVPMQHNNRPTQPLK + + VLDALQAIK + + + + + YSAELHVAHWNSAK + YSSLAEAASK + + + + + P00918/CAH2 AVQQPDGLAVLGIFLK + + + + EPISVSSEQVLK + + + + + GGPLDGTYR + + + + + HNGPEHWHK + + ILNNGHAFNVEFDDSQDK + + + LNFNGEGEPEELMVDNWR + + + + QSPVDIDTHTAK + + + + + SADFTNFDPR + + + + + SHHWGYGK + + + VGSAKPGLQK + + + + + VVDVLDSIK + + + + + YAAELHLVHWNTK + YDPSLKPLSVSYDQATSLR + + + + + YGDFGK + + + + P15559/NQO1 ALIVLAHSER + + + + DPANFQYPAESVLAYK + + + EGHLSPDIVAEQK + + + + + FGLSVGHHLGK + + IQILEGWK + + + + LKDPANFQYPAESVLAYK + + + + SIPTDNQIK + + + + TSFNYAMK + + + + Experiments Accession Peptides A B C D E P02741/CRP APLTKPLK + + + + + ESDTSYVSLK + + + + GYSIFSYATK + + + + + QDNEILIFWSK + YEVQGEVFTK + + + YEVQGEVFTKPQLWP + + + P62937/PPIA EGMNIVEAMER + + + FEDENFILK + + + + IIPGFMCQGGDFTR + + + KITIADCGQLE + + + MVNPTVFFDIAVDGEPLGR + + + + + TEWLDGK + + + P04040/CAT ADVLTTGAGNPVGDK + + + + + AFYVNVLNEEQR + DAQIFIQK + DLFNAIATGK + + + + + DPASDQMQHWK + + + + + DPILFPSFIHSQK + + + DYPLIPVGK + + + + + FNTANDDNVTQVR + + + + + FSTVAGESGSADTVR + + + + + FSTVAGESGSADTVRDPR + + + + HMNGYGSHTFK + + + LCENIAGHLK + + LFAYPDTHR + LGPNYLHIPVNCPYR + LNVITVGPR + + + + + LSQEDPDYGIR + + LVNANGEAVYCK + + + + + NLSVEDAAR + + + VFEHIGK + + + + +

PME 8 QTAPAS LABEL FREE QUANTIFICATION: TARGET PROTEOMICS VS HTP TARGETED PROTEOMICS Tier Peptide E/A E/B E/C E/D E/E 1 CAH1/P00915_GGPFSDSYR 86.97 27.59 10.82 4.56 1 1 CAH1/P00915_VLDALQAIK 172.29 41.64 29.61 6.13 1 1 CAH2/P00918_AVQQPDGLAVLGIFLK 1210.96 112.37 160.23 8.29 1 1 CAH2/P00918_SADFTNFDPR 106.62 28.87 549.67 4.45 1 2 NQO1/P15559_EGHLSPDIVAEQK 106.64 35.72 9.03 5.25 1 2 NQO1/P15559_ALIVLAHSER 314.08 49.71 10.29 6.32 1 2 CRP/P02741_ESDTSYVSLK 118.94 31.92 94.24 4.84 1 2 CRP/P02741_GYSIFSYATK 3 PPIA/P62937_FEDENFILK 220.79 34.22 3.27 5.76 1 3 PPIA/P62937_VSFELFADK 5.94 9.34 2.16 6.67 1 3 PPIA/P62937_TAENFR 2.22 1.05 37.20 0.82 1 3 CATA/P04040_GAGAFGYFEVTHDITK 58.95 2.79 0.85 1.38 1 3 CATA/P04040_FSTVAGESGSADTVR 102.23 28.78 61.00 4.59 1 3 CATA/P04040_NLSVEDAAR 101.16 28.91 355.52 4.37 1 LABEL FREE QUANTIFICATION

PME 8 QTAPAS ACKNOWLEDGEMENTS Laboratorio de Proteómica Funcional, CIB Ignacio Casal Servicio de Proteómica y Genómica, CIB María Fernández Roi Villar Alberto Peláez Rodrigo Barderas Sofía Torres Rubén Bartolomé Irene Palmero Alex Campos Franscesc Canals