Experiments would be easier if researchers could use crystal balls to predict their results. To profile tyrosine phosphorylation, which is particularly important in growth factor signaling, researchers currently use phosphorylation state-specific antibodies, which requires foreknowledge of specific molecules to test. Now Dierck et al. have developed an alternative, a high-throughput method to profile and quantify tyrosine phosphorylation, reported in a recent article in Nature Methods.
Proteins with Src homology region 2 (SH2) domains bind to tyrosine-phosphorylated proteins. Approximately 120 SH2 domains are present in the human genome, and each binds to a different tyrosine-phosphorylated motif.
The authors created an oligonucleotide-tagged multiplex (OTM) assay that would both identify and quantify SH2 binding. They used streptavidin to link biotinylated SH2 domains to biotinylated oligonucleotide tags. They incubated these multiplexed probes with membranes that had been spotted with purified proteins or cell lysates. Then they eluted and analyzed the bound multiplex probes. PCR amplified the tag sequences and attached digoxygenin labels to the products. The authors incubated the digoxygenin-labeled tags in microtiter plates coated with an array of 'capture' probes complementary to unique tag sequences. They detected captured tags with a horseradish peroxidase-conjugated antibody to the digoxygenin label.
OTM detected small amounts of tyrosine-phosphorylated protein and discriminated between phosphorylated and unphosphorylated protein states. OTM detected as little as 10 fmol of tyrosine-phosphorylated protein in mixed protein samples and showed a 33-fold increase in SH2 binding in cells treated with the tyrosine phosphatase inhibitor pervanadate relative to untreated cells. The platelet-derived growth factor 
(PDGF) receptor has several phosphotyrosine motifs that bind SH2 domains. The authors stimulated cells transfected with wild-type or mutant PDGF receptors with PDGF and did OTM for 5 SH2 domains. The SH2 domains from ras GTPase-activating protein, phospho-inositide 3-kinase and the SH2-containing phosphatase SHP2 bound strongly to activated PDGF receptors, but SH2 domains from the Src kinase LYN and phospholipase C
did not because their binding sites overlapped with other SH2 domains, according to the authors. SH2 binding was similar for PDGF receptors with phosphotyrosine-site mutations that had been activated by PDGF and wild-type PDGF receptors that had not been activated by PDGF.
The authors found distinct patterns of SH2 binding in eight cell lines, suggesting that different types of cells have distinct patterns of tyrosine phosphorylation. Aberrant tyrosine phosphorylation is also involved in some cancers, and the authors found different SH2-binding patterns in patients with acute and chronic myeloid leukemia. These data indicate that OTM will be useful not only in the laboratory to identify tyrosine-phosphorylated proteins and signaling pathways implicated in biological processes, but also in the clinic as a diagnostic tool.