Project PXD000065

PRIDE Assigned Tags:
Technical Dataset Biomedical Dataset

Summary

Title

HiRIEF deep proteomics of A431 human vulvar cancer cell line and N2A mouse neuroblastoma cell line

Description

Here we present high resolution data from two cancer cell lines. A human cell line, A431 was treated with a drug at different time points and then fractionated into its different subcellular compartments (nuclear, organellar, soluble,whole cell). Protein extracts were trypsinized, peptides separated by HiRIEF (high resolution isoelectric focusing) and analysed by LC-MS. From the mouse cell line N2A proteins were extracted and subjected to the same HiRIEF-LC-MS procedure. All MS/MS spectra were searched by Sequest/Percolator under the software platform Proteome Discoverer (PD, v1.3.0.339, Thermo Scientific) using a target-decoy strategy. The reference databases used were the human protein subset of Ensembl 63 and the mouse protein subset of Ensembl 64. Precursor mass tolerance of 10 ppm and product mass tolerances of 0.02 Da for HCD-FTMS and 0.8 Da for CID-ITMS were used. Additional settings were: trypsin with 1 missed cleavage; Lys-Pro and Arg-Pro not considered as cleavage sites; carbamidomethylation on cysteine and iTRAQ-8plex on lysine and N-terminal as fixed modifications; and oxidation of methionine and phosphorylation on serine, threonine or tyrosine as variable modifications. Quantitation of iTRAQ-8plex reporter ions was done using an integration window tolerance of 20ppm. Peptides found at 1% FDR (false discovery rate) were used by the protein grouping algorithm in PD to infer protein identities.

Sample Processing Protocol

See details in reference(s) : 24240322

Data Processing Protocol

See details in reference(s) : 24240322

Contact

Rui Branca, Clinical Proteomics Unit, Dep. of Oncology-Pathology

Submission Date

06/11/2012

Publication Date

27/09/2013

Publication

    Branca RM, Orre LM, Johansson HJ, Granholm V, Huss M, Pérez-Bercoff A, Forshed J, Käll L, Lehtiö J; HiRIEF LC-MS enables deep proteome coverage and unbiased proteogenomics., Nat Methods, 2013 Nov 17, PubMed: 24240322