Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry.
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 STI571 (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.
Salomon
Arthur R
AR
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.
Ficarro
Scott B
SB
Brill
Laurence M
LM
Brinker
Achim
A
Phung
Qui T
QT
Ericson
Christer
C
Sauer
Karsten
K
Brock
Ansgar
A
Horn
David M
DM
Schultz
Peter G
PG
Peters
Eric C
EC
D000595:Amino Acid Sequence; D001549:Benzamides; D002851:Chromatography, High Pressure Liquid; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism; D006801:Humans; D000068877:Imatinib Mesylate; D019169:Jurkat Cells; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*; D051746:ZAP-70 Protein-Tyrosine Kinase
D001549:Benzamides; D010879:Piperazines; D011743:Pyrimidines; D014443:Tyrosine; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human