Journal Article

Profiling of Tyrosine Phosphorylation Pathways in Human Cells Using Mass Spectrometry

Phung, Qui T., Schultz, Peter G., Brinker, Achim, Brill, Laurence M., Sauer, Karsten, Brock, Ansgar, Salomon, Arthur R., Horn, David M., Peters, Eric C., Ericson, Christer, Ficarro, Scott B.

2003

Overview

The reversible phosphorylation of tyrosine residues is an important mechanism for modulating biological processes such as cellular signaling, differentiation, and growth, and if deregulated, can result in various types of cancer. Therefore, an understanding of these dynamic cellular processes at the molecular level requires the ability to assess changes in the sites of tyrosine phosphorylation across numerous proteins simultaneously as well as over time. Here we describe a sensitive approach based on multidimensional liquid chromatography/mass spectrometry that enables the rapid identification of numerous sites of tyrosine phosphorylation on a number of different proteins from human whole cell lysates. We used this methodology to follow changes in tyrosine phosphorylation patterns that occur over time during either the activation of human T cells or the inhibition of the oncogenic BCR-ABL fusion product in chronic myelogenous leukemia cells in response to treatment with ST1571 (Gleevec). Together, these experiments rapidly identified 64 unique sites of tyrosine phosphorylation on 32 different proteins. Half of these sites have been documented in the literature, validating the merits of our approach, whereas motif analysis suggests that a number of the undocumented sites are also potentially involved in biological pathways. This methodology should enable the rapid generation of new insights into signaling pathways as they occur in states of health and disease.

• Publisher: National Academy of Sciences,National Acad Sciences,The National Academy of Sciences
• Subject: Amino Acid Sequence / Antibodies / Benzamides / Biological Sciences / Cancer / Cell lines / Cells / Chromatography, High Pressure Liquid / Chronic myeloid leukemia / Fusion Proteins, bcr-abl - metabolism / Humans / Imatinib Mesylate / Immunoprecipitation / Jurkat Cells / K562 cells / Leukemia / Liquid chromatography / Mass Spectrometry / Mass spectroscopy / Molecular Sequence Data / Phosphorylation / Piperazines - pharmacology / Protein-Tyrosine Kinases - metabolism / Proteins / Pyrimidines - pharmacology / Receptors / T lymphocytes / Tyrosine - metabolism / ZAP-70 Protein-Tyrosine Kinase