Differential phosphoprotein mapping in cancer cells using protein microarrays produced from 2-D liquid fractionation.
A combination of protein microarrays and two-dimensional liquid-phase separation of proteins has been used for global profiling of the phosphoproteome in human breast cancer cells. This method has been applied to study changes in phosphorylation profile resulting from treatment of the cancer cells with PD173074, a known receptor tyrosine kinase inhibitor. The proteins separated by 2-D liquid-phase separation were arrayed on epoxy-coated glass slides and first screened for phosphorylation using fluorescent Pro-Q Diamond stain. The candidate proteins were then identified using MALDI/ESI MS/MS analysis. Further, validation was achieved by immunoblot analysis using anti-phosphotyrosine antibodies. A dynamic range of approximately 100 was achieved on the microarray when beta-casein was used as a standard protein for obtaining quantitative data. Importantly, the power of this method lies in its ability to identify a large group of proteins in a single experiment that are coregulated in their posttranslational modifications, upon treatment with the inhibitor. Since proteins are known to form interacting circuits that eventually lead to various signaling events, detection of such global phosphorylation profiles might enable delineation of functional pathways that play an important role during cancer initiation and progression.
Pal
Manoj
M
Department of Chemistry, University of Michigan, Ann Arbor, 48109, USA.
Moffa
Allison
A
Sreekumar
Arun
A
Ethier
Stephen P
SP
Barder
Timothy J
TJ
Chinnaiyan
Arul
A
Lubman
David M
DM
D001943:Breast Neoplasms / Q000737:chemistry* / Q000188:drug therapy / Q000378:metabolism; D045744:Cell Line, Tumor; D005591:Chemical Fractionation / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D009363:Neoplasm Proteins / Q000032:analysis*; D010750:Phosphoproteins / Q000032:analysis*; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011743:Pyrimidines / Q000494:pharmacology; D051497:Receptor, Fibroblast Growth Factor, Type 2 / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D012680:Sensitivity and Specificity
D009363:Neoplasm Proteins; C115711:PD 173074; D010750:Phosphoproteins; D011743:Pyrimidines; C496353:FGFR2 protein, human; D051497:Receptor, Fibroblast Growth Factor, Type 2