Quantitative multiplexed profiling of cellular signaling networks using phosphotyrosine-specific DNA-tagged SH2 domains.
Deciphering global signaling networks is of great importance for the detailed understanding of cellular signaling processes controlling many important biological functions. Among signaling processes, tyrosine phosphorylation has a central role. At present, adequate techniques for the global characterization of the tyrosine phosphoproteome are lacking, particularly for the analysis of small amounts of protein. By combining the power of PCR amplification with the unique properties of Src homology region 2 (SH2) domains to specifically recognize tyrosine-phosphorylated proteins, we developed a new proteomic approach, termed oligonucleotide-tagged multiplex assay (OTM). For OTM, multiple SH2 domains are labeled by domain-specific oligonucleotide tags, applied as probes to complex protein mixtures in a multiplex reaction and phosphotyrosine-specific interactions are quantified by PCR. Using OTM we reproducibly quantified differential states of tyrosine phosphorylation with high sensitivity and specificity in small amounts of whole cellular extracts as demonstrated for various tumor cell lines and human leukemia samples.
Dierck
Kevin
K
Department of Clinical Chemistry, Center of Clinical Pathology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
Machida
Kazuya
K
Voigt
Anja
A
Thimm
Julian
J
Horstmann
Martin
M
Fiedler
Walter
W
Mayer
Bruce J
BJ
Nollau
Peter
P
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D006801:Humans; D051379:Mice; D010748:Phosphopeptides / Q000737:chemistry; D019000:Phosphotyrosine / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D025941:Protein Interaction Mapping; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D013379:Substrate Specificity; D018909:src Homology Domains
D010748:Phosphopeptides; D010982:Platelet-Derived Growth Factor; C075278:platelet-derived growth factor A; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases