Protein tyrosine kinase characterization based on fully automated synthesis of (phospho) peptide arrays in microplates.
In view of the importance of information transfer mediated throughout the cell by recognition, phosphorylation or dephosphorylation of kinases, their adapters, or substrates, this method was developed. The method provides a potent research tool for rapidly generating and testing these substrates as modeled by synthetic peptide arrays. The peptides or phosphorylated peptides are automatically generated on the inner surfaces of microplate wells, covalently linked to a polylysine polymer so that they are in a sterically favorable conformation, immediately available for in situ testing. Products up to 18 amino acids long have shown excellent mass spectral homogeneity. Thus, determinate peptide libraries can be ready for testing in as little as 2 days after the conception of an experiment. The process can be easily automated using robotic liquid handlers and is extremely rapid, sensitive, and economical. Optionally, the method can be upgraded to a higher throughput level using more powerful workstations with greater capacity, such as the Biomek FX, or any similar robotics capable of transfer-from-file logic to guide synthesis cycles.
Saxinger
W Carl
WC
Center for Cancer Research, National Cancer Institute, Fort Detrick, Frederick, MD, USA.
D000818:Animals; D055910:Antibodies, Immobilized / Q000378:metabolism / Q000494:pharmacology; D045163:Antibodies, Phospho-Specific / Q000378:metabolism / Q000494:pharmacology; D001331:Automation / Q000295:instrumentation / Q000379:methods; D006801:Humans; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods; D010748:Phosphopeptides / Q000032:analysis / Q000096:biosynthesis*; D040081:Protein Array Analysis / Q000295:instrumentation* / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000378:metabolism*
D055910:Antibodies, Immobilized; D045163:Antibodies, Phospho-Specific; D010748:Phosphopeptides; D011505:Protein-Tyrosine Kinases