質量分析によるプロテオーム解析―定量分析，リン酸化解析，そして創薬研究へ―

Abstract

Protein concentration is one of the most basic and important parameters in functional analysis because the kinetics/dynamics of cellular molecules is described by changes in the concentrations of proteins in particular compartments. Mass spectrometry (MS) plays a central role in proteome studies and allows protein identification and quantitation at the same time. An MS-based method allows simultaneous identification and quantitation of individual proteins and determination of changes in the levels of modifications at specific sites on individual proteins. Accurate quantitation is achieved through the use of whole cell stable isotope labeling, and the seminal work on differential metabolic labeling of proteins by Oda et al. has led to many strategies for quantitative proteomics including quantitative tissue proteome analysis based on culture-derived isotope tag (CDIT). This is a generalized approach and affords a quantitative description of cellular differences at the level of protein expression and modification, thus providing information that is critical to the understanding of complex biological phenomena. In addition to this relative quantitative proteomics, MS-based absolute quantitation is also possible by adding a known amount of standard peptides (e.g. isotope dilution), or reverse isotope dilution (CDIT). Although it has become feasible to rapidly identify proteins from crude cell extracts using mass spectrometry, it can be difficult to elucidate protein kinase substrates in the presence of a large excess of relatively abundant non-phosphoproteins. Therefore, for effective proteome analysis it becomes critical to enrich the sample of phosphorylated proteins. Although enrichment of phosphotyrosine-containing proteins has been achieved through the use of high-affinity anti-phosphotyrosine antibodies, we developed a method for enriching phosphoserine/threonine-containing proteins from crude cell extracts after chemical replacement of the phosphate moieties by affinity tags. Although this is an interesting method, the procedure is complicated and of low efficiency. Therefore, we developed a simple, highly specific enrichment procedure for phosphopeptides, by increasing the specificity of an immobilized metal affinity column (IMAC) without using any chemical reaction. The increase in selectivity was achieved by (a) using a strong acid to discriminate phosphates from carboxyl groups of peptides, (b) using a high concentration of acetonitrile to remove hydrophobic non-phosphopeptides, and (c) desalting the phosphate buffer used for competitive elution from the IMAC. By applying this efficient approach, we were able to identify more than one thousand phosphopeptides.Chemical proteomics is an effective approach to focused proteomics, having the potential to find specific interactors in moderate-scale comprehensive analysis. Unlike chemical genetics, chemical proteomics directly and comprehensively identifies proteins that bind specifically to candidate compounds by means of affinity chromatographic purification using the immobilized candidate, combined with mass spectrometric identification of interacting proteins. This is an effective approach for discovering unknown protein functions, identifying the molecular mechanisms of drug action, and obtaining information for optimization of lead compounds. However, immobilized-small molecule affinity chromatography always suffers from the problem of nonspecific binders. Although several approaches have been reported to reduce nonspecific binding proteins, these are mainly focused on the use of low-binding-affinity beads or insertion of a spacer between the bead and the compound. Stable isotope labeling strategies have proven particularly advantageous for the discrimination of true interactors from many nonspecific binders, including carrier proteins such as serum albumin, and are expected to be valuable for drug discovery.