- TARGETED ANALYSIS -Proteomics Unit. Centro Nacional de Biotecnología

sHPPAdán Alpízar Morúa

Known Unknown

Protein Accession number Name Protein Accession number NameP02795 MT2_HUMAN Metallothionein-2 Q96M29 TEKT5_HUMAN Tektin-5Q58EX7 PKHG4_HUMAN Puratrophin-1 Q96MC5 CP045_HUMAN Uncharacterized protein C16orf45Q01726 MSHR_HUMAN Melanocyte-stimulating hormone

receptor Q14028 CNGB1_HUMAN Cyclic nucleotide-gated cation channel beta-1

Q14764 MVP_HUMAN Major vault protein P23975 SC6A2_HUMAN Sodium-dependent noradrenaline transporter

Q0VD83 APOBR_HUMAN Apolipoprotein B receptor Q8NC67 NETO2_HUMAN Neuropilin and tolloid-like protein 2 Q6EMK4 VASN_HUMAN Vasorin Q8IZ96 CKLF1_HUMAN CKLF-like MARVEL transmembrane domain-

containing protein 1 O14983 AT2A1_HUMAN Sarcoplasmic/endoplasmic reticulum

calcium ATPase 1 P33527 MRP1_HUMAN Multidrug resistance-associated protein 1 P22695 QCR2_HUMAN Cytochrome b-c1 complex subunit 2,

mitochondrial P60510 PP4C_HUMAN Serine/threonine-protein phosphatase 4

catalytic subunit P55287 CAD11_HUMAN Cadherin-11 Q49A26 GLYR1_HUMAN Putative oxidoreductase GLYR1 Q4KMP7 TB10B_HUMAN TBC1 domain family member 10B Q14019 COTL1_HUMAN Coactosin-like proteinQ9NXV2 KCTD5_HUMAN BTB/POZ domain-containing protein

KCTD5Q9Y221 NIP7_HUMAN 60S ribosome subunit biogenesis protein

NIP7 homolog P55259 GP2_HUMAN Pancreatic secretory granule membrane

major glycoprotein GP2 O75150 BRE1B_HUMAN E3 ubiquitin-protein ligase BRE1B Q2VPK5 CTU2_HUMAN Cytoplasmic tRNA 2-thiolation protein 2Q8N9N5 BANP_HUMAN Protein BANP

Assigned Proteins

Known Unknown

Protein Accession number Name Protein Accession number NameP02795 MT2_HUMAN Metallothionein-2 Q96M29 TEKT5_HUMAN Tektin-5Q58EX7 PKHG4_HUMAN Puratrophin-1 Q96MC5 CP045_HUMAN Uncharacterized protein C16orf45Q01726 MSHR_HUMAN Melanocyte-stimulating hormone

receptor Q14028 CNGB1_HUMAN Cyclic nucleotide-gated cation channel beta-1

Q14764 MVP_HUMAN Major vault protein P23975 SC6A2_HUMAN Sodium-dependent noradrenaline transporter

Q0VD83 APOBR_HUMAN Apolipoprotein B receptor Q8NC67 NETO2_HUMAN Neuropilin and tolloid-like protein 2 Q6EMK4 VASN_HUMAN Vasorin Q8IZ96 CKLF1_HUMAN CKLF-like MARVEL transmembrane domain-

containing protein 1 O14983 AT2A1_HUMAN Sarcoplasmic/endoplasmic reticulum

calcium ATPase 1 P33527 MRP1_HUMAN Multidrug resistance-associated protein 1 P22695 QCR2_HUMAN Cytochrome b-c1 complex subunit 2,

mitochondrial P60510 PP4C_HUMAN Serine/threonine-protein phosphatase 4

catalytic subunit P55287 CAD11_HUMAN Cadherin-11 Q49A26 GLYR1_HUMAN Putative oxidoreductase GLYR1 Q4KMP7 TB10B_HUMAN TBC1 domain family member 10B Q14019 COTL1_HUMAN Coactosin-like proteinQ9NXV2 KCTD5_HUMAN BTB/POZ domain-containing protein

KCTD5Q9Y221 NIP7_HUMAN 60S ribosome subunit biogenesis protein

NIP7 homolog P55259 GP2_HUMAN Pancreatic secretory granule membrane

major glycoprotein GP2 O75150 BRE1B_HUMAN E3 ubiquitin-protein ligase BRE1B Q2VPK5 CTU2_HUMAN Cytoplasmic tRNA 2-thiolation protein 2Q8N9N5 BANP_HUMAN Protein BANP

Methods (MRM Pilot)1

AEAESR

331.7/462.2

331.7/591.3

331.7/488.2

331.7/401.2

GPLEYPSAK

530.8/1003.6

530.8/664.4

530.8/793.4

530.8/906.5

MCF7 (SDS/PAGE Band 8) P605102

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

Selection Criteria of the Synthetic Peptides

Peptide Synthesis3

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

Peptide Synthesis3

N0 Accession Number Peptide sequence MRM Atlas

1 ACGGPTQELSALR

2 DIAPSEEAINDR

3 EAQSLVHFQLR

4 VDGLLHQLTLQSNQR

5 YISIFYALR

6 YHSIVTLPR

7 ACQHAQGIAR

8 ALQPLEEGEDEEK

9 ELPPGVEELLNK

10 HYCTVANPVSR

11 LFSVPDFVGDACK

12 CGDYHPEGEAPR

13 GNTQEDAADGEQR

14 IVEEEAQEDLEGLR

15 TEEAAESQTAGR

16 ESHVTLASPEETR

17 SLTLGIEPVSPTSLR

18 VPLEPGPK

19 YGLNELPAEEGK

20 GAPEGVIDR

21 VDQSILTGESVSVIK

22 VGEATETALTTLVEK

23 DGAFAEFLR

24 ENILFGCQLEEPYYR

25 HIFENVIGPK

26 TQFEDCTVLTIAHR

27 DPDAANSPIR

28 DTNRPLEPPSEFIVK

29 LHSDIDSGDGNIK

30 VEAANVHIDPK

P33527

P55287

Q58EX7

Q01726

Q14764

Q0VD83

Q6EMK4

O14983

N0 Accession Number Peptide sequence MRM Atlas

31 CQNILQGNFKPDFYLK

32 DLVLGPSGVLQGIRPGK

33 TSFFLGEVGNAAK

34 VEQLKPYHAHK

35 ALAAGADSPK

36 ELLEQNPGK

37 VALVLLR

38 DLAEEGVLEEAEFYNITSLIK

39 LCQADPDLDSDK

40 LNVGGTYFLTTR

41 TSQVPVK

42 LAANISGDK

43 LVSLGTCFGK

44 VYYVSEK

45 YIGENLQLLVDRPDGTYCFR

46 DPNCSSILQTEER

47 NTLSLVNDFIIR

48 SEVPAIDLAR

49 STENSAGSQGCDK

50 ETGEYR

51 GLLLATK

52 ISLEYSELQDK

53 VTSAETK

54 AGDAFCR

55 DCLIEDSDDEAGQS

56 EVAFYNR

57 IPCLLAPSVFK

58 LDLVTNK

59 LDSIEAK

60 LQALEATCK

O75150

Q2VPK5

Q8N9N5

Q49A26

Q4KMP7

Q9NXV2

Q9Y221

P55259

Peptide Synthesis (MRM)3

Identification •Identification of the best transitions by direct infusion of the peptides

Optimization •Optimization of CE for each transition

LC-MS •Determination of the retention time of each peptide

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

acknowledgments

Fernando Roncal

Manuel Lombardía

Miguel Marcilla

Severine Gharbi

Carmen González

Rosana Navajas

Alberto Paradela

Salvador Martínez

Alberto Medina