BUNSEKI KAGAKU Abstracts

Shotgun proteomics has been very powerful and a major driving force in protein identification. However, it has been known that shotgun proteomics has a big pitfall, in that a real portrait of a protein is hard to be elucidated. For example, a global picture of native ("in unharmed") proteins phosphorylated is not really understood in their localized areas (where) and time-course (when), as well as structural alternation (how) in relation to their functional dynamics. The aurthrs propose a new field, Indigeomics and Indigenome (“Indigene means that is native or indigenous”), to clarify the detailed expressions and dynamics of a variety of native biomolecules (genes, mRNAs, proteins, and metabolites), which includes modification during translation, posttranslational modification, localization, and dynamics in time-course, and which is distinguished from the existing "genome, transcriptome, proteome, metabolome". A perspective view of Indigeomics is discussed here, including its key technologies, such as sample preparation reserving native states, top-down approaches, protein complex analysis, native phosphorylation analysis, selected-reaction monitoring (SRM) mass spectrometry, AQUA protein technology, and MS-base immunoassays.

Phosphoproteomics has been applied to investigate cellular signal transduction systems. Although several phosphoproteomics approaches have been developed, it is difficult to apply them to minimum amounts of samples collected in vivo due to the low recovery and low sensitivity of these phosphoproteomics approaches. Therefore, we developed a miniaturized LC-MS system with a high-recovery phosphopeptides enrichment protocol. In the sample preparation, protein extraction and digestion were enhanced by adding sodium deoxycholate and sodium lauroyl sarcosinate to the buffer solution. The phosphopeptides enriched by hydroxy acid-modified metal oxide chromatography were directly injected onto cartridge columns by means of a nitrogen-pressure-driven cell, and then phosphopeptides were analyzed by nanoLC-MS/MS using an analytical column of 25 μm inner diameter with silicate frits. By using the developed system, approximately 1000 phosphorylation sites were identified from a single analysis of 10000 HeLa cells (approximately 1 μg of proteins). The system is expected to be useful for the minimum amount of samples collected by a flow cytometer or laser capture microdissection from in vivo.

Reversible protein phosphorylation is a key signaling mechanism for modulating the functional properties of proteins in various cellular processes. More than 500 protein kinases and more than 100 protein phosphatases are known or predicted in the human proteome alone. The numbers clearly reflect the importance of protein phosphorylation. In fact, abnormal phosphorylation by perturbation of the balance of these enzyme reactions is deeply related to a wide range of human diseases, including cancer, diabetes mellitus, neurodegeneration, and immune/inflammatory disorders. Methods for the determination of the phosphorylation status of a certain protein are thus very important with respect to evaluating the basis for understanding the molecular origins of diseases, and for drug design. Recently, we found that a dinuclear metal complex (Phos-tag) of 1,3-bis[bis(pyridin-2-ylmethyl)-amino]propan-2-olato acts as a novel phosphate-binding tag molecule in an aqueous solution under physiological conditions. Phos-tag has a vacancy on two metal ions, which 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 phenyl phosphate dianion (K_d = 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. By utilizing the Phos-tag molecule and its derivatives, we developed three major Phos-tag technologies, and put them into practical use for a phosphoproteome study. Herein, we describe convenient and reliable methods for the detection of phosphorylated proteins, such as affinity electrophoresis using Phos-tag acrylamide, Western blotting using biotinylated Phos-tag, and affinity chromatography using Phos-tag agarose or Phos-tag TOYOPEARL.

D-Amino acids, enantiomers of L-amino acids, are increasingly recognized as being novel physiologically active substances and biomarkers in higher animals, including human beings. Although L-amino acids are abundantly present in the tissues and physiological fluids, the amounts of their enantiomers, D-forms, are extremely small in most cases. Therefore, for the precise analysis of small amounts of D-amino acids, a highly sensitive and selective analytical method is essential. In the present article, a two-dimensional micro-HPLC procedure combining a reversed-phase column and an enantioselective column is described for chiral amino acid metabolomics studies in mammals.

Lipidomics is one of the major approaches in metabolomics, and often uses a liquid chromatography-mass spectrometer (LC-MS) to obtain profiling of lipid metabolites and to detect changing molecules statistically. However, previously reported methods have been technically insufficient in the simultaneous analysis of amphiphilic glycosphingolipids, such as gangliosides, and in the global detection of various polar metabolites from complex lipid mixtures. Here, we report on newly targeted and non-targeted methods to measure these lipid metabolites sensitively. In the targeted approach, we were able to detect individual ganglioside molecular species using the combination of reversed-phase or hydrophilic interaction liquid chromatography and ESI-MS with multiple reaction monitoring (MRM). Meanwhile, in a non-targeted approach, global analysis of various polar metabolites was possible by reverse-phased high-resolution (LC) coupled quadrapole time-of-flight MS and sensitive two-dimensional (2D) profiling. Our methods will be useful for the elucidation of cancers, metabolic syndrome and nervous-system diseases, etc. ; these diagnostic markers may be detectable.

Metallomics and metallome are newly coined terms. Metallomics and research on metallome require analytical techniques that can provide information for the identification and quantification of metal species. This concept has been named speciation, and the acquisition of data according to this concept is performed with hyphenated techniques involving both separation and detection methods. In this paper, the author intended to present a newly developed technique ; multi-mode gel filtration capillary HPLC was evaluated regarding applications to nano level speciation, which is named nano-speciation. In addition, several applications of a complementary use of HPLC-inductively coupled plasma mass spectrometry and HPLC-electrospray ionization tandem mass spectrometry for selenometabolomics were presented.

Array-based technologies, including DNA microarrays and protein-detecting microarrays, have become more attractive as promising high-throughput research tools in “omics” studies. So far, we have designed peptide libraries composed of various secondary structures to be used for microarray technologies, especially in proteome fields and cellomics fields. Here, we describe the development of microarrays using designed peptides toward “omics” studies including genomics, proteomics, glycomics and cellomics. Designed peptides are potent capturing agents for microarray technology because of the following advantages : 1) Peptides are easier to design and synthesize in a short sequence mimicking other complicated biomolecules. 2) Antimicrobial, signaling, and/or transmembrane activities of peptides make them promising control tools for cellular events. Moreover, novel methods for detection technology, which is one of the key technologies for microarrays, were also developed. New fluorescent-label methods utilizing the excluded volume effect of the target molecule or fluorometric/colorimetric change of the detection moiety were achieved. A useful label-free method utilizing an AR (anomalous reflection of gold) system was also constructed and improved. A microarray using designed peptides would be one of the most promising tools for high-throughput and detailed analyses in “omics” researches.

Sensing of sulfhydryl compounds with thermoresponsive gold nanocomposites was reviewed based on research conducted by the present author. The combination of thermoresponsive polymers and gold nanoparticles enabled us to develop not only their inherent functions, but also new functions that would produce novel analytical systems. Aggregated gold nanoparticles in thermoresponsive gold nanocomposites became disassembled by thermal stimuli ; heating followed by cooling, resulting in a change in the solution color from blue-purple to red. Several colorimetric assay systems were fabricated by using gold nanocomposites based on the facts that cysteine prohibited disassembly of the gold nanocomposites while glutathione facilitated the disassembly. The replacement of the other thermoresponsive polymer conjugated with gold nanoparticles allowed us to develop another colorimetric system of the sulfhydryl amino acid, such as homocysteine, with high selectivity. In addition to the sensing systems, spontaneous growth of small gold nanoparticles (ca. 2 nm) conjugated with thermoresponsive polymer was also discussed from the viewpoint of a phase transition induced by heating a solution. Sulfhydryl compounds inhibited growth of the gold nanocomposites, resulting in a suppression of the development of reddish color.

Recently, it has been demonstrated that the fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) method developed by us enables the differential proteome analysis of small brain regions of mouse during development. In this study, to identify the senescence-related brain proteins, we performed differential proteome analysis of rat hippocampus during the senescence process utilizing the FD-LC-MS/MS method. The analysis demonstrated that 8 proteins were significantly altered in the rat hippocampus with aging. The enzymes related to metabolism, such as Cu/Zn-Superoxide dismutase and glutathione S-transferase, increased in the old rat hippocampus. Additionally, the synapsin family protein increased with aging. These results might provide a clue to understand the senescence-related changes in neuronal functions in the brain.

Ionization methods for mass spectroscopy have been developed, and selected depending on the analyte species or experiment conditions. There still remains a problem for detecting some weakly polar molecules using the conventional methods. We developed a novel method to enable us to ionize a lot of weakly polar molecules softly. This method has two important features using a nonpolar solvent and a cationization reagent. First, cations of the cationization reagents are such strong Lewis acid that they attach to weakly polar molecules in nonpolar solvents. Second, the cations associated with the analyte molecules form stable ion pairs with the counter ions. In this paper, we demonstrate the utility of this method for some of weakly polar molecules by using cation(Li,Ag)-tetrakis[3,5-bis(trifluoromethyl)-phenyl]borate [Cation(Li,Ag)-TFPB].