Rapid characterization of in vivo phosphorylation sites and the protein kinases and phosphatases that regulate them by affinity capture
This chapter discusses methodologies for analysis of the phosphoproteome, and describes how affinity captures using naturally occurring small molecules can be utilized to define protein kinases and phosphatases regulating phosphorylation events in vivo. In this light, it offers alternatives to strategies driven purely by molecular biology for defining phosphoregulation of signaling pathways in vivo. Protein phosphorylation is a reversible and widespread mechanism for regulating protein function, so the phosphoproteome is of great interest. There are several advanced mass spectrometric techniques available to the investigator that enable detailed characterization of phosphoproteomes. These techniques typically use cultured cells, and include phosphopeptide capture approaches, precursor ion scanning, isotope-coded affinity (ICAT), and stable isotope labeling with amino acids in cell culture (SILAC). However, the majority of these techniques have several limitations that result in missing important regulatory sites of phosphorylation. This study describes a procedure that has the considerable advantage over purely MS-based approaches to phosphopeptide analysis in that it is highly quantitative and comprehensive. Finally, it deals with rapid protein kinase and phosphatase identification by small molecule affinity capture and high throughput mass spectrometry. © 2010 Elsevier Inc. All rights reserved.
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