1. - TARGETED ANALYSIS - Proteomics Unit. Centro Nacional de Biotecnología sHPP Adán Alpízar Morúa

2. KnownUnknown ProteinAccession numberNameProteinAccession numberName P02795MT2_HUMANMetallothionein-2Q96M29TEKT5_HUMANTektin-5 Q58EX7PKHG4_HUMANPuratrophin-1Q96MC5CP045_HUMANUncharacterized protein C16orf45 Q01726MSHR_HUMAN Melanocyte-stimulating hormone receptor Q14028CNGB1_HUMAN Cyclic nucleotide-gated cation channel beta-1 Q14764MVP_HUMANMajor vault proteinP23975SC6A2_HUMAN Sodium-dependent noradrenaline transporter Q0VD83APOBR_HUMANApolipoprotein B receptorQ8NC67NETO2_HUMANNeuropilin and tolloid-like protein 2 Q6EMK4VASN_HUMANVasorinQ8IZ96CKLF1_HUMAN CKLF-like MARVEL transmembrane domain- containing protein 1 O14983AT2A1_HUMAN Sarcoplasmic/endoplasmic reticulum calcium ATPase 1 P33527MRP1_HUMANMultidrug resistance-associated protein 1 P22695QCR2_HUMAN Cytochrome b-c1 complex subunit 2, mitochondrial P60510PP4C_HUMAN Serine/threonine-protein phosphatase 4 catalytic subunit P55287CAD11_HUMANCadherin-11 Q49A26GLYR1_HUMANPutative oxidoreductase GLYR1 Q4KMP7TB10B_HUMANTBC1 domain family member 10B Q14019COTL1_HUMANCoactosin-like protein Q9NXV2KCTD5_HUMAN BTB/POZ domain-containing protein KCTD5 Q9Y221NIP7_HUMAN 60S ribosome subunit biogenesis protein NIP7 homolog P55259GP2_HUMAN Pancreatic secretory granule membrane major glycoprotein GP2 O75150BRE1B_HUMANE3 ubiquitin-protein ligase BRE1B Q2VPK5CTU2_HUMANCytoplasmic tRNA 2-thiolation protein 2 Q8N9N5BANP_HUMANProtein BANP Assigned Proteins

3. KnownUnknown ProteinAccession numberNameProteinAccession numberName P02795MT2_HUMANMetallothionein-2Q96M29TEKT5_HUMANTektin-5 Q58EX7PKHG4_HUMANPuratrophin-1Q96MC5CP045_HUMANUncharacterized protein C16orf45 Q01726MSHR_HUMAN Melanocyte-stimulating hormone receptor Q14028CNGB1_HUMAN Cyclic nucleotide-gated cation channel beta-1 Q14764MVP_HUMANMajor vault proteinP23975SC6A2_HUMAN Sodium-dependent noradrenaline transporter Q0VD83APOBR_HUMANApolipoprotein B receptorQ8NC67NETO2_HUMANNeuropilin and tolloid-like protein 2 Q6EMK4VASN_HUMANVasorinQ8IZ96CKLF1_HUMAN CKLF-like MARVEL transmembrane domain- containing protein 1 O14983AT2A1_HUMAN Sarcoplasmic/endoplasmic reticulum calcium ATPase 1 P33527MRP1_HUMANMultidrug resistance-associated protein 1 P22695QCR2_HUMAN Cytochrome b-c1 complex subunit 2, mitochondrial P60510PP4C_HUMAN Serine/threonine-protein phosphatase 4 catalytic subunit P55287CAD11_HUMANCadherin-11 Q49A26GLYR1_HUMANPutative oxidoreductase GLYR1 Q4KMP7TB10B_HUMANTBC1 domain family member 10B Q14019COTL1_HUMANCoactosin-like protein Q9NXV2KCTD5_HUMAN BTB/POZ domain-containing protein KCTD5 Q9Y221NIP7_HUMAN 60S ribosome subunit biogenesis protein NIP7 homolog P55259GP2_HUMAN Pancreatic secretory granule membrane major glycoprotein GP2 O75150BRE1B_HUMANE3 ubiquitin-protein ligase BRE1B Q2VPK5CTU2_HUMANCytoplasmic tRNA 2-thiolation protein 2 Q8N9N5BANP_HUMANProtein BANP Methods (MRM Pilot) 1

4. AEAESR 331.7/462.2 331.7/591.3 331.7/488.2 331.7/401.2

5. GPLEYPSAK 530.8/1003.6 530.8/664.4 530.8/793.4 530.8/906.5

6. MCF7 (SDS/PAGE Band 8) P60510 2 685.3/861.4 685.3/960.5 685.3/1073.6 EILVEESNVQR (+2)QITQVYGFYDECLR (+2) QITQVYGFYDECLRK (+3) 896.4/855.4 896.4/1059.5 896.4/1222.5 640.6/983.5 640.6/1130.5 640.6/1187.5

7. Selection Criteria of the Synthetic Peptides Peptide Synthesis 3 3-4 peptides per protein Unique peptides Peptide Length: 9-21 residues No missed cleavages No M and W No Q by N-ter MRM atlas

8. Peptide Synthesis 3

9. Peptide Synthesis (MRM) 3

10. Currently, data depedent analysis yields better results in terms of sensitivity and number of identifications than MRM We have not yet implemented a robust workflow for the routine implementation of MRM methods We have implemented a platform that allows the simultaneous synthesis of a significant (≈ 60) number of peptides Conclusions

11. acknowledgments Fernando Roncal Manuel Lombardía Miguel Marcilla Severine Gharbi Carmen González Rosana Navajas Alberto Paradela Salvador Martínez Alberto Medina