Project PXD002486

PRIDE Assigned Tags:
Biomedical Dataset
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Summary

Title

Target identification for 11q13 amplicon

Description

Proteomic strategy to define therapeutically relevant targets in cell lines that contain the 11q13 amplicon compared to those that do not and to ascertain which genes are amplified at the protein level and, concomitantly, are key drivers for tumor growth and/or maintenance. Furthermore, so called passenger genes that are amplified with driver genes and a manifest on the cell surface can be attractive targets for an antibody – drug conjugate approach (ADC).

Sample Processing Protocol

Cell lines with high or low expression of the 11q123 amplicon across 4 cancer tissue types (head/ neck, breast, oral squamous cell carcinoma and liver) were chosen. In situ labeling of surface proteins by NHS-Sulfo linked biotin, followed by quenching of excess reagent, cell lysis and protein enrichment via streptavidin biotin interactions. Trypsin digestion followed by TMT6 labeling of peptides and subsequent high pH separation and fraction collection. 12 fractions run on the LTQ-Orbitrap-Velos under HCD conditions over a 90 minuet reverse phase gradient.

Data Processing Protocol

Raw data from the LTQ-Orbitrap-Velos were processed with Mascot (vs 2.0.0) using default parameters. The data was searched using trypsin as the enzyme and allowing for up to 2 missed cleavages. The search criteria included peptide mass tolerance (± 15 ppm), fragment mass tolerance (± 0.05 Da), fixed modifications of Carbamidomethyl (C) and variable modifications: Oxidation (M), Phospho (ST), Phospho (Y). Sulfo-NHS-S-S-Biotin-IOA (K), Sulfo-NHS-S-S-Biotin-IOA (N-term), TMT6plex (K), TMT6plex (N-term). Mass values are monoisotopic and protein mass is unrestricted. Mascot results for sample fractions were aggregated and submitted to the PeptideProphet and ProteinProphet algorithms for peptide and protein validation, respectively (ISB/SPC Trans Proteomic Pipeline TPP v4.3 JETSTREAM rev 1, Build 200909091257 (MinGW)). Protein results were then filtered using a false discovery rate of less than 1%.

Contact

Heather Hoover, Novartis
Nancy Finkel, Novartis Isntitutes for Biomedical Research, Cambridge, MA 02139, USA DMP ( lab head )

Submission Date

06/07/2015

Publication Date

04/08/2015

Publication

    Hoover H, Li J, Marchese J, Rothwell C, Borawoski J, Jeffery DA, Gaither LA, Finkel N. Quantitative Proteomic Verification of Membrane Proteins as Potential Therapeutic Targets Located in the 11q13 Amplicon in Cancers. J Proteome Res. 2015 Jul 29 PubMed: 26151158

Assay

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Showing 1 - 10 of 269 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 55220 no assay title provided (mzIdentML) 5 44 8 1014 33
2 54997 no assay title provided (mzIdentML) 297 2630 716 6434 2269
3 55221 no assay title provided (mzIdentML) 128 2052 272 6417 1825
4 54998 no assay title provided (mzIdentML) 13 21 18 606 20
5 55222 no assay title provided (mzIdentML) 6 22 9 861 18
6 54999 no assay title provided (mzIdentML) 15 65 21 1670 53
7 55223 no assay title provided (mzIdentML) 12 60 14 2416 53
8 55224 no assay title provided (mzIdentML) 45 140 65 2506 126
9 55225 no assay title provided (mzIdentML) 6 30 12 2376 30
10 55226 no assay title provided (mzIdentML) 108 430 211 4365 386