- targeted analysis - proteomics unit. centro nacional de biotecnología shpp adán alpízar morúa
TRANSCRIPT
- 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 C16orf45
Q01726 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 protein
Q9NXV2 KCTD5_HUMAN BTB/POZ domain-containing protein KCTD5
Q9Y221 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 C16orf45
Q01726 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 protein
Q9NXV2 KCTD5_HUMAN BTB/POZ domain-containing protein KCTD5
Q9Y221 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