Detection of Protein Phosphorylation by Open-Sandwich Immunoassay

Abstract

Understanding the post-translational modifications of proteins represents a next major challenge in post-genomic era. Intricate cascades of phosphorylation reactions regulate cell proliferation, differentiation, migration and so on. For example, Olsen et al recently applied high-resolution mass spectrometry-based proteomics to phosphoproteome of human cell cycle, quantifying 6,027 proteins and 20,443 unique phosphorylation sites [Olsen, 2010]. Phosphorylation-dependent signals participate in several diseases, such as cancers, immune diseases, and Alzheimer’s. The method to produce phosphorylation-site and phosphorylation state-specific antibodies was established in 1990’s [Nishizawa, 1991; Yano, 1991; Nagata, 2001; Goto, 2007] after a discovery of phosphorylated cytoskeletal proteinspecific antibodies [Sternberger, 1983]. Since then, investigators have successfully observed spatio-temporal dynamics of particular phosphorylation in vitro or in situ using the antibodies. Recently, these antibodies are also proven useful as a live cell imaging probe in vivo [Hayashi-Takanaka, 2009; Kimura, 2010; Hayashi-Takanaka, 2011], clinical diagnosis, and drug screening [Brumbaugh, 2011]. Here we show a couple of applications of phosphorylation-specific antibodies to proteomic studies, more specifically focusing on a novel immunoassay approach, which we call opensandwich immunoassay.