CHR 16 - Human Proteome Project

Data dependent and inclusion list results on LTQ – (Orbitrap) Velos

27-28th August 2012La Cristalera - Madrid

PCB – Eliandre de Oliveira - Barcelona

First Multicenter Experiment of Chr16 Spanish Consortium

Chr 16 proteins database

Chr 16 proteins database

List of 06 “unknown” proteins

List of 06 “unknown” proteins

List of proteotypic peptides of the 06 unknown proteins

List of proteotypic peptides of the 06 unknown proteins

ensembl_gene_idexternal_gene_id description strand band

transcript_count

ENSG00000170537 TMC7

transmembrane channel-like 7 [Source:HGNC Symbol;Acc:23000] 1 p12.3 2

ENSG00000140853 NLRC5

NLR family, CARD domain containing 5 [Source:HGNC Symbol;Acc:29933] 1 q13 31

ENSG00000124074 C16orf48

chromosome 16 open reading frame 48 [Source:HGNC Symbol;Acc:25246] -1 q22.1 1

ENSG00000166669 ATF7IP2

activating transcription factor 7 interacting protein 2 [Source:HGNC Symbol;Acc:20397] 1 p13.2 6

ENSG00000205423 TMEM188

transmembrane protein 188 [Source:HGNC Symbol;Acc:26759] 1 q12.1 3

ENSG00000103089 FA2H

fatty acid 2-hydroxylase [Source:HGNC Symbol;Acc:21197] -1 q23.1 2

Sample distribution (proteins extracts)

Francesc Canals: MCF7 epitelial cells (500µg/50µL; 6M Urea, ammonium) bicarbonate 50mM

Ignacio Casal: CCD-18 fibroblast cells (700µg/100µL; 6M Urea, ammonium) bicarbonate 50mM

Manuel Fuentes: Ramos cell line

MCF7 CCD-18 Ramos

SDS-PAGE:- 12% acrilamide-100 µg/ sample- Coloidal Coomassie

- Cutted 15 slides/fractions - manual digestion with trypsin

LC-MSMS conditions

LC systemGradient to 35%ACN in 90min.Column length: 25 cm

Inclusion List MSMS analysis (FAIMS, top 5)Incorporate a list of 131 parent ions or m/z in the High Resolution MS experimentResolution: 60000Maximum inject time MS: Velos 120 msec, FT 250 msecMSMS: Velos 100 msec, FT 300 msecMinimum signal required: 100Number of microscans: 1Precursor isolation width: 2 m/z units

Data dependent MSMS analysis (top 10)Resolution: 60000Maximum inject time MS: Velos 120 msec, FT 250 msecMSMS: Velos 100 msec, FT 300 msecMinimum signal required: 500Number of microscans: 1Precursor isolation width: 2 m/z units

Peptides Proteins Unknown Proteins

Inclusion List 1 49 contaminants 0

DDA >25000 >3020 0

Peptides Proteins Unknown Proteins

Inclusion List 1 49 contaminants 0

DDA >25000 >3020 0

RESULTS of MCF7 sample

Mascot 2.3 Database: DDA, Uniprot Homo sapiens (SwissProt+Trembl)

Inclusion list, 06 unknown proteins + isoforms + CRAPFDR >1% for DDA, minimal 2 peptides/proteinNo FDR for inclusion list, minimal 1 peptide/protein