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Article

Robust Phosphoproteomic Profiling of Tyrosine Phosphorylation Sites from Human T Cells Using Immobilized Metal Affinity Chromatography and Tandem Mass Spectrometry

Supporting Info

Laurence M. Brill,*^† Arthur R. Salomon,^† Scott B. Ficarro,^† Mridul Mukherji,^‡ Michelle Stettler-Gill,^† and Eric C. Peters^†

Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, and Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037

Anal. Chem., 2004, 76 (10), pp 2763–2772

DOI: 10.1021/ac035352d

Publication Date (Web): April 17, 2004

To whom correspondence should be addressed. Phone: (858) 812-1974 (office). Fax: (858) 812-1918. E-mail: lbrill@gnf.org.

†

Genomics Institute of the Novartis Research Foundation.

‡

The Scripps Research Institute.

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 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.