Profiling Glycoproteins on Functionalized Reverse Phase Protein Array.

Abstract

Reverse phase protein array (RPPA), a high-throughput, parallel immunoassay in a dot-blot format, is a powerful tool to quantitatively profile protein expression in multiple samples simultaneously using small amounts of material. Despite its success, analysis of post-translationally modified (PTM) proteins has been limited in RPPA assays, primarily due to relatively low availability of antibodies specific to proteins of PTMs, e.g., glycosylation. Moreover, the high matrix complexity, with tens of thousands of proteins in cell lysates or tissue extracts and the low abundance of proteins with PTMs, makes it extremely challenging to detect these proteins with PTMs. Therefore, there is an urgent need to fill this gap, which would greatly contribute to the analysis of a specific PTM by RPPA. In this chapter, we introduce a novel RPPA platform, termed polymer-based reverse phase glycoprotein array (polyGPA), to measure the variation of glycosylation patterns on a three-dimensionally functionalized RPPA. Without the need of specific antibody towards glycosylation, polyGPA represents a highly sensitive strategy to analyze protein glycosylation in multiple complex biological samples in parallel.