Journal of Proteomics & Bioinformatics
Open Access

Abstract

Phos-tag-Based Microarray Techniques Advance Phosphoproteomics

Eiji Kinoshita, Emiko Kinoshita-Kikuta and Tohru Koike

The reversible phosphorylation of proteins is a dominant post-translational modification that regulates many important cellular processes, including signal transduction, gene expression, and cell cycle progression. Changes in the phosphorylation status of certain proteins have been implicated in a wide range of human diseases. Effective analytical techniques for quantitative determination of levels of protein phosphorylation are therefore essential tools for studies on the proteome, particularly in relation to disease diagnosis and drug discovery. Large-scale identification of phosphoproteins is now possible as a result of dramatic advancements in methods of shotgun proteomics based on mass spectrometry. However, these techniques require expensive instrumentation and involve complicated procedures for sample preparation through enrichment of phosphopeptides by enzymatic digestion. With regard to the analysis of the phosphoproteome, microarray techniques involving peptide or protein arrays, have demonstrated considerable potential as cost-effective, high-throughput, and convenient approaches for defining activities relating to signal transduction by means of phosphorylation reactions. We recently developed a phosphate-affinity probe that is suitable for monitoring changes in levels of phosphorylation of proteins. This affinity probe is known as biotin-pendant Phos-tag {Phos-tag Biotin; Phos-tag=1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex}. We have demonstrated the utility of Phos-tag Biotin as a novel phosphate-affinity probe in a range of techniques, including microarray-based methods, for determining the phosphorylation status of large numbers of peptides and proteins. Here, we review some of the more advanced applications of Phos-tag Biotin. We discuss the impact of Phos-tag-based microarray techniques in relation to the detection of protein phosphorylation multiplexes, and we compare these techniques with conventional probing procedures on microarrays.