We found that a dinuclear metal complex of 1,3-bis[bis(pyridin-2-ylmethyl)-amino]propan-2-olato acted as a novel phosphate-binding tag nanomolecule, Phos-tag, in an aqueous solution under near physiological conditions. The metal complex having a vacancy on two metal ions is suitable for the access of a phosphomonoester dianion (R-OPO32−) as a bridging ligand. A dinuclear zinc(II) complex (Zn2+–Phos-tag) strongly binds to a p-nitrophenyl phosphate dianion (Kd = 2.5 × 10−8 M) at a neutral pH. The anion selectivity indexes against SO42−, CH3COO−, Cl−, and the bisphenyl phosphate monoanion at 25°C are 5.2 × 103, 1.6 × 104, 8.0 × 105, and >2 × 106, respectively. The author has been involved in developing a technology by using the Phos-tag molecule and its derivatives to permit the analysis of phosphorylated biomolecules. To date, Phos-tag technology has contributed to the development of several procedures for phosphoproteomics, including a phosphate-affinity chromatography technique for the separation and enrichment of phosphopeptides and phosphoproteins, a wide variety of microarray/on-chip techniques for the detection of protein phosphorylation multiplexes, and a phosphate-affinity electrophoresis technique for the detection of shifts in the mobilities of phosphoproteins. In this article, the author introduces the impact of Phos-tag-based technological advances for phosphoproteomics.
The ribosome is a molecular machine that decodes mRNA sequences and convert their sequences into proteins. While translational regulation is an important aspect of gene expression control, the ribosome itself is traditionally seen as a rather static machine. However, recent evidence suggests that the composition of the ribosome can vary in terms of ribosomal proteins (RPs), associated factors or post-translational modifications (PTMs). It has therefore been suggested that different types of ribosomes can recognize and preferentially translate specific classes of mRNAs. Here, we systematically characterized such “ribosome heterogeneity” using mass spectrometry-based proteomics. In this review, I summarize an overview of multiple layers of gene expression control and review a recent finding about translational regulation through a phosphorylated ribosome.
Protein glycosylation is one of widely occurred post-translational modifications. Methods for large-scale identification of glycosylated proteins were developed in the early days of field of proteomics. At that time, direct analysis of intact glycopeptide was difficult due to the structural factors. Major reasons of the difficulty were that glycan is diverse and heterogeneous, and glycopeptide is a conjugate composed of two oligomers (peptide and glycan) formed by different energy of bonds. Therefore, glycosylated peptides were identified after removal of glycan to specify glycosylation site. To ensure that the site was actually glycosylated, we used stable isotope labeled water (H218O) to label the site (IGOT method) during glycan removal by Peptide-N-glycanase (PNGase) action. Then, if sample glycopeptides are captured by the lectin affinity chromatography, glycan motif on the captured glycopeptides is presumable based on the lectin specificity. Using the lectin well reflecting disease-related glycan alteration, we have captured glycopeptides to identify glycoproteins as biomarker candidates. Currently, such glycoprotein possessing disease-specific glycan, especially membrane glycoprotein, are thought to be a potential pharmaceutical target, thus we developed a new high sensitivity technology to assign intact glycopeptides relying MS1 rather than MS2 (Glyco-RIDGE method). In this review, we introduce development and applications of glycoproteomic technologies we made.