Robust phosphoproteomic profiling of tyrosine phosphorylation sites from human T cells using immobilized metal affinity chromatography and tandem mass spectrometry.

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Brill LM, Salomon AR, Ficarro SB, Mukherji M, Stettler-Gill M, Peters EC

Robust phosphoproteomic profiling of tyrosine phosphorylation sites from human T cells using immobilized metal affinity chromatography and tandem mass spectrometry.

Anal Chem. 2004 May 15;76(10):2763-72.

PubMed ID
15144186 [ View in PubMed
]
Abstract

Protein tyrosine phosphorylation cascades are difficult to analyze and are critical for cell signaling in higher eukaryotes. Methodology for profiling tyrosine phosphorylation, considered herein as the assignment of multiple protein tyrosine phosphorylation sites in single analyses, was reported recently (Salomon, A. R.; Ficarro, S. B.; Brill, L. M.; Brinker, A.; Phung, Q. T.; Ericson, C.; Sauer, K.; Brock, A.; Horn, D. M.; Schultz, P. G.; Peters, E. C. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 443-448). The technology platform included the use of immunoprecipitation, immobilized metal affinity chromatography (IMAC), liquid chromatography, and tandem mass spectrometry. In the present report, we show that when using complex mixtures of peptides from human cells, methylation improved the selectivity of IMAC for phosphopeptides and eliminated the acidic bias that occurred with unmethylated peptides. The IMAC procedure was significantly improved by desalting methylated peptides, followed by gradient elution of the peptides to a larger IMAC column. These improvements resulted in assignment of approximately 3-fold more tyrosine phosphorylation sites, from human cell lysates, than the previous methodology. Nearly 70 tyrosine-phosphorylated peptides from proteins in human T cells were assigned in single analyses. These proteins had unknown functions or were associated with a plethora of fundamental cellular processes. This robust technology platform should be broadly applicable to profiling the dynamics of tyrosine phosphorylation.

DrugBank Data that Cites this Article

Polypeptides
NameUniProt ID
T-cell surface glycoprotein CD3 epsilon chainP07766Details
Microtubule-associated protein 1AP78559Details
Tyrosine-protein kinase ZAP-70P43403Details
Tyrosine-protein kinase ITK/TSKQ08881Details
T-cell surface glycoprotein CD3 delta chainP04234Details
T-cell surface glycoprotein CD3 zeta chainP20963Details
Microtubule-associated protein 4P27816Details
Receptor-type tyrosine-protein phosphatase betaP23467Details
SRSF protein kinase 2P78362Details
AfadinP55196Details
Sodium bicarbonate cotransporter 3Q9Y6M7Details
Breakpoint cluster region proteinP11274Details
Nucleolar and coiled-body phosphoprotein 1Q14978Details
TRAF2 and NCK-interacting protein kinaseQ9UKE5Details
Serine/threonine-protein kinase LATS1O95835Details
Receptor-interacting serine/threonine-protein kinase 1Q13546Details
1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2P16885Details