Phosphopeptide microarrays for comparative proteomic pro fi ling of cellular lysates

Abstract

Protein phosphorylation is one of the most important and well-studied posttranslational modi fi cations. Aberrant phosphorylation causes a wide spectrum of diseases, including cancers. As a result, many of the proteins involved in these pathways are seen as vital drug targets and biomarkers in treatment and diagnosis. The availability of broad-based platforms that identify changes across cellular states is critical in understanding unique disease characteristics and changes at the proteomic level. To highlight how microarrays can be applied in this regard, we describe here a comparative proteomic pro fi ling method using two-color sample labeling and application on phosphopeptide microarrays, followed by a pull-down strategy and MS-based protein identi fi cation. This strategy has been applied to uncover candidate biomarkers in breast cancer and colon cancer cell lines. Apart from the synthesis of the phosphopeptide libraries and growth/ isolation of cellular lysates, the protocol takes approximately 15 days to complete, once key steps have been optimized, and can be readily extended to other similarly complex biological specimens/samples.