Profiling protein tyrosine phosphorylation: a quantitative 45-plex peptide-based immunoassay.
Cellular homeostasis and responses to stimuli are mediated by complex signaling network events dominated by changes in protein phosphorylation states. Understanding information flow in the network is essential for correlating signaling changes to cell physiology. Tyrosine phosphorylation constitutes only a small portion of all protein phosphorylation, but its importance is manifested by the significant role it plays in diseases such as cancer. A peptide-based immunoassay microarray, designed to provide site specificity, quantification, broad coverage, and accessibility, is described that profiles 45 tyrosine phosphorylation sites across 34 proteins. Epidermal growth factor-stimulated A431 cells in the absence and presence of kinase inhibitors analyzed by microarrays showed biologically validated tyrosine phosphorylation changes and unanticipated activation of other targets. The approach is scalable for increasing the breadth of content as well as for interrogating other types of protein posttranslational modifications.
Nadler
Timothy K
TK
Epitome Biosystems, Waltham, Massachusetts 02453, USA.
Rauh-Adelmann
Christine
C
Murphy
Cheryl
C
Hall
Amy B
AB
Graham
James R
JR
Yen
Lucy
L
Gordon
Neal F
NF
Radding
Jeffrey A
JA
D000906:Antibodies / Q000737:chemistry; D002460:Cell Line; D045744:Cell Line, Tumor; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D019169:Jurkat Cells; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods; D012015:Reference Standards; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry*; D020298:U937 Cells
D000906:Antibodies; D000939:Epitopes; D010455:Peptides; D014443:Tyrosine