Determination of the Substrate Specificity of Protein Kinases with Peptide Micro- and Macroarrays.
Elucidation of the key determinants for the phosphorylation site specificities of protein kinases facilitates identification of their physiological substrates, and serves to better define their critical roles in the signaling networks that underlie a multitude of cellular activities. Albeit with some apparent limitations, such as the lack of contextual information for secondary substrate-binding sites, the synthetic peptide-based approach has been adopted widely for the kinase specificity profiling studies, especially when they are used in an array format, which permits the screening of large numbers of potential peptide substrates in parallel. In this chapter, we present detailed protocols for determining protein kinase substrate specificity using an approach that involves both peptide microarrays and macroarrays. In particular, SPOT synthesis on macroarrays can be used to follow up on in silico predictions of protein kinase substrate specificity with predictive algorithms.
Kinexus Bioinformatics Corporation, Suite 1, 8755 Ash Street, Vancouver, British Columbia, Canada, V6P 6T3.
Shenshen
S
Lai
Kinexus Bioinformatics Corporation, Suite 1, 8755 Ash Street, Vancouver, British Columbia, Canada, V6P 6T3.
Dirk F H
DF
Winkler
Kinexus Bioinformatics Corporation, Suite 1, 8755 Ash Street, Vancouver, British Columbia, Canada, V6P 6T3.
Hong
H
Zhang
Kinexus Bioinformatics Corporation, Suite 1, 8755 Ash Street, Vancouver, British Columbia, Canada, V6P 6T3. spelech@kinexus.ca.|Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada. spelech@kinexus.ca.
Steven
S
Pelech
D002482:Cellulose; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D006801:Humans; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D008956:Models, Chemical; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007202:Indicators and Reagents; D008567:Membranes, Artificial; D019151:Peptide Library; D010455:Peptides; D002482:Cellulose; D011494:Protein Kinases
Peptide array; Peptide microarray; Phosphorylation; Protein kinase specificity; SPOT synthesis; Substrate screening