Dataset Information

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

ABSTRACT: 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.

OTHER RELATED OMICS DATASETS IN: MSV000079945

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Rui Branca

LAB HEAD: Rui Branca

PROVIDER: PXD000065 | Pride | 2013-09-27

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	A431-Gef_0-2-6-24h_iTRAQ8_200ugIPG37-49_nuc-heavy_fr01-72_seq-perc_ensembl63_phos.msf	Msf
	A431-Gef_0-2-6-24h_iTRAQ8_200ugIPG37-49_nuc-heavy_fr01-72_seq-perc_ensembl63_phos.pep.xml	Pepxml
	A431-Gef_0-2-6-24h_iTRAQ8_200ugIPG37-49_nuc-heavy_fr01-72_seq-perc_ensembl63_phos.prot.xml	Xml
	A431-Gef_0-2-6-24h_iTRAQ8_200ugIPG37-49_nuc-heavy_fr01.raw	Raw
	A431-Gef_0-2-6-24h_iTRAQ8_200ugIPG37-49_nuc-heavy_fr02.raw	Raw

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Publications

HiRIEF LC-MS enables deep proteome coverage and unbiased proteogenomics.

Branca Rui M M RM Orre Lukas M LM Johansson Henrik J HJ Granholm Viktor V Huss Mikael M Pérez-Bercoff Åsa Å Forshed Jenny J Käll Lukas L Lehtiö Janne J

Nature methods 20131117 1

We present a liquid chromatography-mass spectrometry (LC-MS)-based method permitting unbiased (gene prediction-independent) genome-wide discovery of protein-coding loci in higher eukaryotes. Using high-resolution isoelectric focusing (HiRIEF) at the peptide level in the 3.7-5.0 pH range and accurate peptide isoelectric point (pI) prediction, we probed the six-reading-frame translation of the human and mouse genomes and identified 98 and 52 previously undiscovered protein-coding loci, respectivel ...[more]

PMID: 24240322

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