Dataset Information

Large scale qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immuno-affinity purification and stable isotope dimethyl labeling

ABSTRACT: Triplex stable isotope dimethyl labeling of phosphotyrosine peptides after EGF stimulation

INSTRUMENT(S): LTQ Orbitrap, instrument model

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Paul Boersema

PROVIDER: PRD000123 | Pride | 2010-07-09

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	PRIDE_Exp_Complete_Ac_9777.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_9777.pride.mztab.gz	Mztab
	PRIDE_Exp_Complete_Ac_9777.xml.gz	Xml
	PRIDE_Exp_Complete_Ac_9779.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_9779.pride.mztab.gz	Mztab

Items per page:

1 - 5 of 9

Publications

In-depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity purification and stable isotope dimethyl labeling.

Boersema Paul J PJ Foong Leong Yan LY Ding Vanessa M Y VM Lemeer Simone S van Breukelen Bas B Philp Robin R Boekhorst Jos J Snel Berend B den Hertog Jeroen J Choo Andre B H AB Heck Albert J R AJ

Molecular & cellular proteomics : MCP 20090921 1

Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffi ...[more]

PMID: 19770167

Similar Datasets

Project description:Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated.