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CHR 16 - Human Proteome Project Data dependent and inclusion list results on LTQ – (Orbitrap) Velos 27-28th August 2012 La Cristalera - Madrid PCB – Eliandre.

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Presentation on theme: "CHR 16 - Human Proteome Project Data dependent and inclusion list results on LTQ – (Orbitrap) Velos 27-28th August 2012 La Cristalera - Madrid PCB – Eliandre."— Presentation transcript:

1 CHR 16 - Human Proteome Project Data dependent and inclusion list results on LTQ – (Orbitrap) Velos 27-28th August 2012 La Cristalera - Madrid PCB – Eliandre de Oliveira - Barcelona

2 First Multicenter Experiment of Chr16 Spanish Consortium Chr 16 proteins database List of 06 “unknown” proteins List of proteotypic peptides of the 06 unknown proteins ensembl_gene_id external_ gene_iddescriptionstrandband transcript_ count ENSG00000170537TMC7 transmembrane channel-like 7 [Source:HGNC Symbol;Acc:23000] 1p12.32 ENSG00000140853NLRC5 NLR family, CARD domain containing 5 [Source:HGNC Symbol;Acc:29933] 1q1331 ENSG00000124074C16orf48 chromosome 16 open reading frame 48 [Source:HGNC Symbol;Acc:25246] q22.11 ENSG00000166669ATF7IP2 activating transcription factor 7 interacting protein 2 [Source:HGNC Symbol;Acc:20397] 1p13.26 ENSG00000205423TMEM188 transmembrane protein 188 [Source:HGNC Symbol;Acc:26759] 1q12.13 ENSG00000103089FA2H fatty acid 2-hydroxylase [Source:HGNC Symbol;Acc:21197] q23.12

3 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 MCF7CCD-18Ramos SDS-PAGE: - 12% acrilamide -100 µg/ sample - Coloidal Coomassie - Cutted 15 slides/fractions - manual digestion with trypsin

4 LC-MSMS conditions LC system Gradient 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 experiment Resolution: 60000 Maximum inject time MS: Velos 120 msec, FT 250 msec MSMS: Velos 100 msec, FT 300 msec Minimum signal required: 100 Number of microscans: 1 Precursor isolation width: 2 m/z units Data dependent MSMS analysis (top 10) Resolution: 60000 Maximum inject time MS: Velos 120 msec, FT 250 msec MSMS: Velos 100 msec, FT 300 msec Minimum signal required: 500 Number of microscans: 1 Precursor isolation width: 2 m/z units

5 Peptides ProteinsUnknown Proteins Inclusion List 1 49contaminants0 DDA >25000 >30200 Peptides ProteinsUnknown Proteins Inclusion List 1 49contaminants0 DDA >25000 >30200 RESULTS of MCF7 sample Mascot 2.3 Database: DDA, Uniprot Homo sapiens (SwissProt+Trembl) Inclusion list, 06 unknown proteins + isoforms + CRAP FDR >1% for DDA, minimal 2 peptides/protein No FDR for inclusion list, minimal 1 peptide/protein

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