担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The GeLC-MS workflow, which combines low-cost, easy-to-use sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography-mass spectrometry (LC-MS), is very popular in current bottom-up proteomics. However, GeLC-MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 h of sample preparation for LC-MS. In this study, we overcame the limitations of GeLC-MS by developing a rapid digestion workflow for PAGE separation of proteins using N,N'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment. Making use of an established workflow called BAC-DROP (BAC-gel dissolution to digest PAGE-resolved objective proteins), crude proteome samples were fractionated based on molecular weight by BAC cross-linked PAGE. After fractionation, the gel fragments were reductively dissolved in under 5 min, and in-solution trypsin digestion of the protein released from the gel was completed in less than 1 h at 70 °C, equivalent to a 90-95% reduction in time compared to conventional in-gel trypsin digestion. The introduction of the BAC-DROP workflow to the MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed for serum sample preparation to be completed in as little as 5 h, demonstrating successful marker quantification from a 0.5 μL sample of human serum.

DOI： 10.1021/acs.jproteome.0c00749

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.

DOI： 10.1021/acs.jproteome.0c00303

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

A major challenge in proteomics is the absolute accurate quantification of large numbers of proteins. QconCATs, artificial proteins that are concatenations of multiple standard peptides, are well established as an efficient means to generate standards for proteome quantification. Previously, QconCATs have been expressed in bacteria, but we now describe QconCAT expression in a robust, cell-free system. The new expression approach rescues QconCATs that previously were unable to be expressed in bacteria and can reduce the incidence of proteolytic damage to QconCATs. Moreover, it is possible to cosynthesize QconCATs in a highly-multiplexed translation reaction, coexpressing tens or hundreds of QconCATs simultaneously. By obviating bacterial culture and through the gain of high level multiplexing, it is now possible to generate tens of thousands of standard peptides in a matter of weeks, rendering absolute quantification of a complex proteome highly achievable in a reproducible, broadly deployable system.

DOI： 10.1074/mcp.RA117.000284

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BIOMED CENTRAL LTD  

BACKGROUND: Targeted proteomics, which involves quantitative analysis of targeted proteins using selected reaction monitoring (SRM) mass spectrometry, has emerged as a new methodology for discovery of clinical biomarkers. In this study, we used targeted serum proteomics to identify circulating biomarkers for prediction of disease activity and organ involvement in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). METHODS: A large-scale SRM assay targeting 135 biomarker candidates was established using a triple-quadrupole mass spectrometer coupled with nanoflow liquid chromatography. Target proteins in serum samples from patients in the active and remission (6 months after treatment) stages were quantified using the established assays. Identified marker candidates were further validated by enzyme-linked immunosorbent assay using serum samples (n = 169) collected in a large-cohort Japanese study (the RemIT-JAV-RPGN study). RESULTS: Our proteomic analysis identified the following proteins as biomarkers for discriminating patients with highly active AAV from those in remission or healthy control subjects: tenascin C (TNC), C-reactive protein (CRP), tissue inhibitor of metalloproteinase 1 (TIMP1), leucine-rich alpha-2-glycoprotein 1, S100A8/A9, CD93, matrix metalloproteinase 9, and transketolase (TKT). Of these, TIMP1 was the best-performing marker of disease activity, allowing distinction between mildly active AAV and remission. Moreover, in contrast to CRP, serum levels of TIMP1 in patients with active AAV were significantly higher than those in patients with infectious diseases. The serum levels of TKT and CD93 were higher in patients with renal involvement than in those without, and they predicted kidney outcome. The level of circulating TNC was elevated significantly in patients with lung infiltration. AAV severity was associated with markers reflecting organ involvement (TKT, CD93, and TNC) rather than inflammation. The eight markers and myeloperoxidase (MPO)-ANCA were clustered into three groups: MPO-ANCA, renal involvement (TKT and CD93), and inflammation (the other six markers). CONCLUSIONS: We have identified promising biomarkers of disease activity, disease severity, and organ involvement in AAV with a targeted proteomics approach using serum samples obtained from a large-cohort Japanese study. Especially, our analysis demonstrated the effectiveness of TIMP1 as a marker of AAV activity. In addition, we identified TKT and CD93 as novel markers for evaluation of renal involvement and kidney outcome in AAV.

DOI： 10.1186/s13075-017-1429-3

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

Biologists' preeminent toolbox for separating, analyzing, and visualizing proteins is SDS-PAGE, yet recovering the proteins embedded in these polyacrylamide media as intact species is a long-standing challenge for mass spectrometry. In conventional workflows, protein mixtures from crude biological samples are electrophoretically separated at high-resolution within N,N'-methylene-bis-acrylamide cross-linked polyacrylamide gels to reduce sample complexity and facilitate sensitive characterization. However, low protein recoveries, especially for high molecular weight proteins, often hinder characterization by mass spectrometry. We describe a workflow for top-down/bottom-up mass spectrometric analyses of proteins in polyacrylamide slab gels using dissolvable, bis-acryloylcystamine-cross-linked polyacrylamide, enabling high-resolution protein separations while recovering intact proteins over a broad size range efficiently. The inferior electrophoretic resolution long associated with reducible gels has been overcome, as demonstrated by SDS-PAGE of crude tissue extracts. This workflow elutes intact proteins efficiently, supporting MS and MS/MS from proteins resolved on biologists' preferred separation platform.

DOI： 10.1021/acs.analchem.7b00357

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC CHEMISTRY  

Using a wheat germ cell-free protein synthesis system, we developed a high-throughput method for the synthesis of stable isotope-labeled full-length transmembrane proteins as proteoliposomes to mimic the in vivo environment, and we successfully constructed an internal standard library for targeted transmembrane proteomics by using mass spectrometry.

DOI： 10.1039/c4mb00556b

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SOC NEUROSCIENCE  

Photoreceptor cells achieve high sensitivity, reliably detecting single photons, while limiting the spontaneous activation events responsible for dark noise. We used proteomic, genetic, and electrophysiological approaches to characterize Retinophilin (RTP) (CG10233) in Drosophila photoreceptors and establish its involvement in dark-noise suppression. RTP possesses membrane occupation and recognition nexus (MORN) motifs, a structure shared with mammalian junctophilins and other membrane-associated proteins found within excitable cells. We show the MORN repeats, and both the N- and C-terminal domains, are required for RTP localization in the microvillar light-gathering organelle, the rhabdomere. RTP exists in multiple phosphorylated isoforms under dark conditions and is dephosphorylated by light exposure. An RTP deletion mutant exhibits a high rate of spontaneous membrane depolarization events in dark conditions but retains the normal kinetics of the light response. Photoreceptors lacking neither inactivation nor afterpotential C (NINAC) myosin III, a motor protein/kinase, also display a similar dark-noise phenotype as the RTP deletion. We show that NINAC mutants are depleted for RTP. These results suggest the increase in dark noise in NINAC mutants is attributable to lack of RTP and, furthermore, defines a novel role for NINAC in the rhabdomere. We propose that RTP is a light-regulated phosphoprotein that organizes rhabdomeric components to suppress random activation of the phototransduction cascade and thus increases the signaling fidelity of dark-adapted photoreceptors.

DOI： 10.1523/JNEUROSCI.4464-09.2010

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

The fruit fly Drosophila melanogaster is an excellent model organism for studying insect reproductive biology. Although the gene expression profiles of both male and female reproductive organs have been studied in detail, their proteomic profiles and functional characteristics largely remained to be clarified. In this study, we conducted proteome mapping of the male internal reproductive organs using 2-DE. We identified a total of 440 protein components from gels of the male reproductive organs (testis, seminal vesicle, accessory gland, ejaculatory duct, and ejaculatory bulb). A number of proteins associated with odorant/pheromone-binding, lipid metabolism, proteolysis, and antioxidation were expressed tissue specifically in the male reproductive system. Based on our proteomic data set, we constructed reference proteome maps of the reproductive organs, which will provide valuable information toward a comprehensive understanding of Drosophila reproduction.

DOI： 10.1002/pmic.200800795

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The quantification of proteoforms, i.e., all molecular forms in which proteins can be present, by top-down proteomics provides essential insights into biological processes at the molecular level. Isobaric labeling-based quantification strategies are suitable for multidimensional separation strategies and allow for multiplexing of the samples. Here, we investigated cysteine-directed isobaric labeling by iodoTMT in combination with a gel- and gas-phase fractionation (GeLC-FAIMS-MS) for in-depth quantitative proteoform analysis. We optimized the acquisition workflow (i.e., the FAIMS compensation voltages, isolation windows, acquisition strategy, and fragmentation method) using a two-proteome mix to increase the number of quantified proteoforms and reduce ratio compression. Additionally, we implemented a mass feature-based quantification strategy in the widely used deconvolution algorithm FLASHDeconv, which improves and facilitates data analysis. The optimized iodoTMT GeLC-FAIMS-MS workflow was applied to quantitatively analyze the proteome of Escherichia coli grown under glucose or acetate as the sole carbon source, resulting in the identification of 726 differentially abundant proteoforms.

DOI： 10.1021/acs.jproteome.4c00835

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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Frontiers Media SA  

The establishment of a highly sensitive method for obtaining structural information on proteins and protein complexes in vivo has long been a technological challenge in structural biology. In recent years, protein structure analysis approaches using top-down mass spectrometry, native mass spectrometry, and cross-linking mass spectrometry, among others, have been developed, and these techniques have emerged as the most promising methods for obtaining comprehensive structural information on the cellular proteome. However, information obtained by MS alone is derived mainly from protein components that are abundant in vivo, with insufficient data on low abundance components. For the detection of those low abundance components, sample fractionation prior to mass spectrometry is highly effective because it can reduce the complexity of the sample. Polyacrylamide gel electrophoresis (PAGE), which is widely used in biochemical experiments, is an excellent technique for protein separation in a simple straightforward procedure and is also a promising fractionation tool for structural proteomics. The difficulty of recovering proteins in gels has been an obstacle, thus far limiting its application to structural mass spectrometry. With the breakthrough of PEPPI-MS, an exceptionally efficient passive extraction method for proteins in gels that appeared in 2020, various PAGE-based proteome fractionation workflows have been developed, resulting in the rapid integration of structural mass spectrometry and PAGE. In this paper, we describe a simple and inexpensive PAGE-based sample preparation strategy that accelerates the broad use of structural mass spectrometry in life science research, and discuss future prospects for achieving in-depth structural proteomics using PAGE.

DOI： 10.3389/frans.2022.1107183

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Microbes that can recycle one-carbon (C1) greenhouse gases into fuels and chemicals are vital for the biosustainability of future industries. Acetogens are the most efficient known microbes for fixing carbon oxides CO2 and CO. Understanding proteome allocation is important for metabolic engineering as it dictates metabolic fitness. Here, we use absolute proteomics to quantify intracellular concentrations for >1,000 proteins in the model acetogen Clostridium autoethanogenum grown autotrophically on three gas mixtures (CO, CO+H2, or CO+CO2+H2). We detect the prioritization of proteome allocation for C1 fixation and the significant expression of proteins involved in the production of acetate and ethanol as well as proteins with unclear functions. The data also revealed which isoenzymes are likely relevant in vivo for CO oxidation, H2 metabolism, and ethanol production. The integration of proteomic and metabolic flux data demonstrated that enzymes catalyze high fluxes with high concentrations and high in vivo catalytic rates. We show that flux adjustments were dominantly accompanied by changing enzyme catalytic rates rather than concentrations. IMPORTANCE Acetogen bacteria are important for maintaining biosustainability as they can recycle gaseous C1 waste feedstocks (e.g., industrial waste gases and syngas from gasified biomass or municipal solid waste) into fuels and chemicals. Notably, the acetogen Clostridium autoethanogenum is being used as a cell factory in industrial-scale gas fermentation. Here, we perform reliable absolute proteome quantification for the first time in an acetogen. This is important as our work advances both rational metabolic engineering of acetogen cell factories and accurate in silico reconstruction of their phenotypes. Furthermore, this absolute proteomics data set serves as a reference toward a better systems-level understanding of the ancient metabolism of acetogens.

DOI： 10.1128/msystems.00026-22

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process. RESULTS: We report a new approach to the assembly and construction of QconCATs, based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short construct also allows a second cycle of loop assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. CONCLUSIONS: We refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards. ALACAT methodology is a major gain in flexibility of QconCAT implementation as it supports rapid editing and improvement of QconCATs and permits, for example, substitution of one peptide by another.

DOI： 10.1186/s12915-021-01135-9

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DNA is packaged with histones to form chromatin that impinges on all nuclear processes, including transcription, replication and repair, in the eukaryotic nucleus. A complete understanding of these molecular processes requires analysis of chromatin context in vitro. Here, Drosophila four core histones were produced in a native and unmodified form using wheat germ cell-free protein synthesis. In the assembly reaction, four unpurified core histones and three chromatin assembly factors (dNAP-1, dAcf1 and dISWI) were incubated with template DNA. We then assessed stoichiometry with the histones, nucleosome arrays, supercoiling and the ability of the chromatin to serve as a substrate for histone-modifying enzymes. Overall, our method provides a new avenue to produce chromatin that can be useful in a wide range of chromatin research.

DOI： 10.1002/2211-5463.13178

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: We previously identified tissue inhibitor of metalloproteinase 1 (TIMP-1) as a biomarker of disease activity that distinguished mildly or highly active antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) from remission 6 months after the initiation of remission-induction therapy. In the present study, we investigated whether TIMP-1 is clinically useful as a predictor of relapse and sustained remission in AAV patients with microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) during maintenance therapy. METHODS: The relationship between serum TIMP-1 levels and clinical outcomes in AAV patients receiving maintenance therapy was assessed using the follow-up data of a Japanese large-cohort study (the RemIT-JAV-RPGN study) and data collected from AAV patients on maintenance therapy in our hospital (the MAAV-EU study). RESULTS: In the RemIT-JAV RPGN study, serum levels of TIMP-1 were significantly higher in mildly active AAV patients with MPA and GPA 6 months after the initiation of remission-induction therapy than in patients in remission. Regarding maintenance therapy, elevated levels of TIMP-1 in patients in remission were associated with relapse and/or difficulty reducing the glucocorticoid dosage after 6 to 12 months. In the MAAV-EU study, serum levels of TIMP-1 were elevated in relapsed patients 6 months before relapse, earlier than the increase in serum levels of CRP. Analyses of both studies revealed that approximately 30% of patients in remission with a serum TIMP-1 level ≥ 150 ng/mL relapsed after 6 to 12 months, while the majority of patients with a TIMP-1 level < 150 ng/mL sustained remission for at least 12 months. CONCLUSION: We herein demonstrated that TIMP-1 is more useful as a predictive biomarker of sustained remission than as a predictor of relapse in maintenance therapy for AAV. TIMP-1 levels < 150 ng/mL are important for the long-term maintenance of remission and may be an indicator for the tapering or cessation of treatment.

DOI： 10.1186/s13075-021-02471-5

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Although the important roles of glycolysis in T cells have been demonstrated, the regulatory mechanism of glycolysis in activated T cells has not been fully elucidated. Furthermore, the influences of glycolytic failure on the T cell-dependent immune response in vivo remain unclear. We therefore assessed the role of glycolysis in the T cell-dependent immune response using T cell-specific Pgam1-deficient mice. Both CD8 and CD4 T cell-dependent immune responses were attenuated by Pgam1 deficiency. The helper T cell-dependent inflammation was ameliorated in Pgam1-deficient mice. Glycolysis augments the activation of mTOR complex 1 (mTORC1) and the T-cell receptor (TCR) signals. Glutamine acts as a metabolic hub in activated T cells, since the TCR-dependent increase in intracellular glutamine is required to augment glycolysis, increase mTORC1 activity and augment TCR signals. These findings suggest that mTORC1, glycolysis and glutamine affect each other and cooperate to induce T cell proliferation and differentiation.

DOI： 10.1038/s42003-020-01122-w

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BMC  

Background: Glandular trichomes found in vascular plants are called natural cell factories because they synthesize and store secondary metabolites in glandular cells. To systematically understand the metabolic processes in glandular cells, it is indispensable to analyze cellular proteome dynamics. The conventional proteomics methods based on mass spectrometry have enabled large-scale protein analysis, but require a large number of trichome samples for in-depth analysis and are not suitable for rapid and sensitive quantification of targeted proteins. Results: Here, we present a high-throughput strategy for quantifying targeted proteins in specific trichome glandular cells, using selected reaction monitoring (SRM) assays. The SRM assay platform, targeting proteins in type VI trichome gland cells of tomato as a model system, demonstrated its effectiveness in quantifying multiple proteins from a limited amount of sample. The large-scale SRM assay uses a triple quadrupole mass spectrometer connected online to a nanoflow liquid chromatograph, which accurately measured the expression levels of 221 targeted proteins contained in the glandular cell sample recovered from 100 glandular trichomes within 120 min. Comparative quantitative proteomics using SRM assays of type VI trichome gland cells between different organs (leaves, green fruits, and calyx) revealed specific organ-enriched proteins. Conclusions: We present a targeted proteomics approach using the established SRM assays which enables quantification of proteins of interest with minimum sampling effort. The remarkable success of the SRM assay and its simple experimental workflow will increase proteomics research in glandular trichomes.

DOI： 10.1186/s13007-019-0427-7

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Separation technology of proteins from a complex mixture by two-dimensional gel (2D gel) was invented more than 40 years ago. With a good laboratory practice, the 2D gels are likely to be dried and stored at ambient temperature as archived record. Up until the beginning of this century, it had been difficult to identify the protein spots isolated on 2D gels. However, the advent of mass spectrometry-based proteomics protocols combined with genome information enabled us to determine the identity of a protein separated on 2D gels archived decades ago. The protocol will assist researchers to decipher molecular mechanisms involved in the system by identifying and quantifying the protein of interest from archived 2D gels.

DOI： 10.1007/978-1-4939-8793-1_24

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

While menin plays an important role in preventing T-cell dysfunction, such as senescence and exhaustion, the regulatory mechanisms remain unclear. We found that menin prevents the induction of dysfunction in activated CD8 T cells by restricting the cellular metabolism. mTOR complex 1 (mTORC1) signaling, glycolysis, and glutaminolysis are augmented by menin deficiency. Rapamycin treatment prevents CD8 T-cell dysfunction in menin-deficient CD8 T cells. Limited glutamine availability also prevents CD8 T-cell dysfunction induced by menin deficiency, and its inhibitory effect is antagonized by α-ketoglutarate (α-KG), an intermediate metabolite of glutaminolysis. α-KG-dependent histone H3K27 demethylation seems to be involved in the dysfunction in menin-deficient CD8 T cells. We also found that α-KG activates mTORC1-dependent central carbon metabolism. These findings suggest that menin maintains the T-cell functions by limiting mTORC 1 activity and subsequent cellular metabolism.

DOI： 10.1038/s41467-018-05854-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: The global demand for affordable carbon has never been stronger, and there is an imperative in many industrial processes to use waste streams to make products. Gas-fermenting acetogens offer a potential solution and several commercial gas fermentation plants are currently under construction. As energy limits acetogen metabolism, supply of H2 should diminish substrate loss to CO2 and facilitate production of reduced and energy-intensive products. However, the effects of H2 supply on CO-grown acetogens have yet to be experimentally quantified under controlled growth conditions. Results: Here, we quantify the effects of H2 supplementation by comparing growth on CO, syngas, and a high-H2 CO gas mix using chemostat cultures of Clostridium autoethanogenum. Cultures were characterised at the molecular level using metabolomics, proteomics, gas analysis, and a genome-scale metabolic model. CO-limited chemostats operated at two steady-state biomass concentrations facilitated co-utilisation of CO and H2. We show that H2 supply strongly impacts carbon distribution with a fourfold reduction in substrate loss as CO2 (61% vs. 17%) and a proportional increase of flux to ethanol (15% vs. 61%). Notably, H2 supplementation lowers the molar acetate/ethanol ratio by fivefold. At the molecular level, quantitative proteome analysis showed no obvious changes leading to these metabolic rearrangements suggesting the involvement of post-translational regulation. Metabolic modelling showed that H2 availability provided reducing power via H2 oxidation and saved redox as cells reduced all the CO2 to formate directly using H2 in the Wood-Ljungdahl pathway. Modelling further indicated that the methylene-THF reductase reaction was ferredoxin reducing under all conditions. In combination with proteomics, modelling also showed that ethanol was synthesised through the acetaldehyde:ferredoxin oxidoreductase (AOR) activity. Conclusions: Our quantitative molecular analysis revealed that H2 drives rearrangements at several layers of metabolism and provides novel links between carbon, energy, and redox metabolism advancing our understanding of energy conservation in acetogens. We conclude that H2 supply can substantially increase the efficiency of gas fermentation and thus the feed gas composition can be considered an important factor in developing gas fermentation-based bioprocesses.

DOI： 10.1186/s13068-018-1052-9

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Although Bach2 has an important role in regulating the Th2-type immune response, the underlying molecular mechanisms remain unclear. We herein demonstrate that Bach2 associates with Batf and binds to the regulatory regions of the Th2 cytokine gene loci. The Bach2-Batf complex antagonizes the recruitment of the Batf-Irf4 complex to AP-1 motifs and suppresses Th2 cytokine production. Furthermore, we find that Bach2 regulates the Batf and Batf3 expressions via two distinct pathways. First, Bach2 suppresses the maintenance of the Batf and Batf3 expression through the inhibition of IL-4 production. Second, the Bach2-Batf complex directly binds to the Batf and Batf3 gene loci and reduces transcription by interfering with the Batf-Irf4 complex. These findings suggest that IL-4 and Batf form a positive feedback amplification loop to induce Th2 cell differentiation and the subsequent Th2-type immune response, and Bach2-Batf interactions are required to prevent an excessive Th2 response.

DOI： 10.1038/ncomms12596

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC CHEMISTRY  

Quantitative proteomic approaches using selected reaction monitoring (SRM) are currently limited by the difficulty in the preparation of reference standards. In this study, we demonstrat the high-throughput production of a reference peptide library using a wheat germ cell-free synthesis system to develop a large-scale SRM assay for targeted proteomics.

DOI： 10.1039/c6mb00209a

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担当区分：責任著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Protein phosphorylation on Tyr residues is a key post-translational modification in mammals. In plants, recent studies have identified Tyr-specific protein phosphatase and Tyr-phosphorylated proteins in Arabidopsis by phosphoproteomic screenings, implying that plants have a Tyr phosphorylation signal pathway. However, little is known about the protein kinases (PKs) involved in Tyr phosphorylation in plants. Here, we demonstrate that Arabidopsis calcium-dependent protein kinase (CDPK/CPK)-related PKs (CRKs) have high Tyr-autophosphorylation activity and that they can phosphorylate Tyr residue(s) on substrate proteins in Arabidopsis. To identify PKs for Tyr phosphorylation, we examined the autophosphorylation activity of 759 PKs using an Arabidopsis protein array based on a wheat cell-free system. In total, we identified 38 PKs with Tyr-autophosphorylation activity. The CRK family was a major protein family identified. A cell-free substrate screening revealed that these CRKs phosphorylate β-tubulin (TBB) 2, TBB7, and certain transcription factors (TFs) such as ethylene response factor 13 (ERF13). All five CRKs tested showed Tyr-auto/trans-phosphorylation activity and especially two CRKs, CRK2 and CRK3, showed a high ERF13 Tyr-phosphorylation activity. A cell-based transient expression assay revealed that Tyr(16/)Tyr(207) sites in ERF13 were phosphorylated by CRK3 and that Tyr phosphorylation of endogenous TBBs occurs in CRK2 overexpressing cells. Furthermore, crk2 and crk3 mutants showed a decrease in the Tyr phosphorylation level of TBBs. These results suggest that CRKs have Tyr kinase activity, and these might be one of the major PKs responsible for protein Tyr phosphorylation in Arabidopsis plants.

DOI： 10.1074/jbc.M114.617274

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

To increase individual male fitness, males of various species remain near a (potential) mating partner and repel their rivals (mate-guarding). Mate-guarding is assumed to be mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. The genetic/molecular mechanisms underlying how mate presence affects male competitive motivation in a triadic relationship has remained largely unknown. Here we showed that male medaka fish prominently exhibit mateguarding behavior. The presence of a female robustly triggers male-male competition for the female in a triadic relationship (2 males and 1 female). The male-male competition resulted in one male occupying a dominant position near the female while interfering with the other male's approach of the female. Paternity testing revealed that the dominant male had a significantly higher mating success rate than the other male in a triadic relationship. We next generated medaka mutants of arginine-vasotocin (avt) and its receptors (V1a1, V1a2) and revealed that two genes, avt and V1a2, are required for normal mate-guarding behavior. In addition, behavioral analysis of courtship behaviors in a dyadic relationship and aggressive behaviors within a male group revealed that avt mutant males displayed decreased sexual motivation but showed normal aggression. In contrast, heterozygote V1a2 mutant males displayed decreased aggression, but normal mate-guarding and courtship behavior. Thus, impaired mate-guarding in avt and V1a2 homozygote mutants may be due to the loss of sexual motivation toward the opposite sex, and not to the loss of competitive motivation toward rival males. The different behavioral phenotypes between avt, V1a2 heterozygote, and V1a2 homozygote mutants suggest that there are redundant systems to activate V1a2 and that endogenous ligands activating the receptor may differ according to the social context.

DOI： 10.1371/journal.pgen.1005009

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Enzymatic protein digestion in polyacrylamide gel has been used for sample pretreatment in mass spectrometry-based proteomics due to its effectiveness in removing contaminants that interfere with sample ionization. However, the difficulty of recovering the digested peptides from the solid gel matrix has been a drawback of this method. Here we have developed a novel in-gel digestion method to enhance peptide recovery using a dissolvable, bis-acrylylcystamine (BAC)-crosslinked polyacrylamide gel. After enzymatic protein digestion in BAC gel, we completely dissolved the gel by reductive treatment with tris-(2-carboxyethyl) phosphine to release the digested peptides from the gel. Our analysis revealed that the reductive dissolution of the BAC gel enhances the peptide recovery, which has a significantly higher protein identification capability than the conventional method, using an insoluble polyacrylamide gel. In addition, protein samples trapped in dehydrated BAC gel were stable at room temperature and reproducible sample recovery was obtained after storage for one week. These results indicate that the proposed method could be an effective tool for conducting sample pretreatment for mass spectrometry-based protein analysis.

DOI： 10.1016/j.jchromb.2014.07.006

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cisplatin is currently the most effective anti-tumor agent available against bladder cancer. To clarify the mechanism underlying cisplatin resistance in bladder cancer, the present study examined the role of the aldo-keto reductase family 1 member C2 (AKR1C2) protein on chemoresistance using a human bladder cancer cell line. The function of AKR1C2 in chemoresistance was studied using the human HT1376 bladder cancer cell line and the cisplatin-resistant HT1376-CisR subline. AKR1C2 was expressed in HT1376-CisR cells, but not in the parental cells. The effect of small interfering (si) RNAs and an inhibitor targeting AKR1C2 was examined to determine whether cisplatin sensitivity can be rescued by blocking AKR1C2 expression or function. Silencing of AKR1C2 mRNA or inhibition of AKR1C2 by 5β-cholanic acid resulted in a decrease in the survival of cells following cisplatin exposure. Intracellular accumulation of reactive oxygen species (ROS) was determined using a 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent probe. Cisplatin exposure increased the level of intracellular ROS in HT1376 cells in a dose-dependent manner. The ROS levels in HT1376-CisR cells were significantly lower than those in HT1376 cells and knockdown of AKR1C2 mRNA significantly restored ROS levels. Cisplatin exposure did not increase intracellular ROS in HT1376-CisR cells, although the level of intracellular ROS increased in HT1376 cells following cisplatin exposure. Silencing of AKR1C2 mRNA restored the ROS increase response to cisplatin and menadione as an oxidative stressor in HT1376-CisR cells. Menadione has the function of an oxidative stressor. The silencing of AKR1C2 mRNA restored the increased ROS response to cisplatin and menadione in HT1376-CisR cells. These results indicate that induction of AKR1C2 in human bladder cancer cells aids in the development of cisplatin resistance through antioxidative effects. The results of this study indicate that AKR1C2 may be an effective molecular target for restoring cisplatin resistance.

DOI： 10.3892/ol.2013.1768

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

Vascular endothelial growth factor (VEGF) is a major angiogenic factor that activates pro-angiogenic molecules to generate new vessels. Recently, we identified a VEGF-A-induced pro-angiogenic gene, BCL-6 associated zinc finger protein (BAZF), in endothelial cells. BAZF interacts with CBF1, a transcriptional regulator of Notch signaling, and downregulates Notch signaling by inducing the degradation of CBF1. A signal inhibition assay with a combination of chemical inhibitors and siRNA revealed that the protein kinase D (PRKD) family, mainly PRKD2, mediated BAZF gene expression by VEGF-A stimulation. A luciferase reporter assay showed that the promoter activity of the BAZF gene was unchanged by VEGF-A stimulation. However, we found that the stability of BAZF mRNA increased in a VEGF-A/PRKD2-dependent manner. In further studies to investigate the underlying mechanism, we successfully identified heat shock protein 90 beta (HSP90β) as a molecule that interacts with and stabilizes BAZF mRNA following VEGF-A/PRKD2 activation. These data suggest that HSP90β may positively regulate angiogenesis, not only as a protein chaperone, but also as an mRNA stabilizer for pro-angiogenic genes, such as BAZF, in a PRKD2 activity-dependent manner.

DOI： 10.1007/s10456-013-9345-x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cisplatin is currently the most effective antitumor agent available against bladder cancer. However, a majority of patients eventually relapse with cisplatin-resistant disease. Chemoresistance thus remains a major obstacle in bladder cancer therapy. To clarify the molecular mechanisms underlying cisplatin resistance in bladder cancer, we established a cisplatin-resistant subline from the human bladder cancer cell line HT1376 (HT1376-CisR), and conducted large-scale analyses of the expressed proteins using two-dimensional (2D) gel electrophoresis coupled with mass spectrometry (MS). Comparative proteomic analysis of HT1376 and HT1376-CisR cells revealed 36 differentially expressed proteins, wherein 21 proteins were upregulated and 15 were downregulated in HT1376-CisR cells. Among the differentially regulated proteins, adseverin (SCIN), a calcium-dependent actin-binding protein, was overexpressed (4-fold upregulation) in HT1376-CisR, with the increase being more prominent in the mitochondrial fraction than in the cytosol fraction. SCIN mRNA knockdown significantly reduced cell proliferation with mitochondria-mediated apoptosis in HT1376-CisR cells. Immunoprecipitation analysis revealed voltage-dependent anion channels (VDACs) to be bound to SCIN in the mitochondrial fraction. Our results suggest that the VDAC-SCIN interaction may inhibit mitochondria-mediated apoptosis in cisplatin-resistant cells. Targeting the VDAC-SCIN interaction may offer a new therapeutic strategy for cisplatin-resistant bladder cancer.

DOI： 10.1016/j.molonc.2011.12.002

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC HEMATOLOGY  

Angiogenic homeostasis is maintained by a balance between vascular endothelial growth factor (VEGF) and Notch signaling in endothelial cells (ECs). We screened for molecules that might mediate the coupling of VEGF signal transduction with down-regulation of Notch signaling, and identified B-cell chronic lymphocytic leukemia/lymphoma6-associated zinc finger protein (BAZF). BAZF was induced by VEGF-A in ECs to bind to the Notch signaling factor C-promoter binding factor 1 (CBF1), and to promote the degradation of CBF1 through polyubiquitination in a CBF1-cullin3 (CUL3) E3 ligase complex. BAZF disruption in vivo decreased endothelial tip cell number and filopodia protrusion, and markedly abrogated vascular plexus formation in the mouse retina, overlapping the retinal phenotype seen in response to Notch activation. Further, impaired angiogenesis and capillary remodeling were observed in skin-wounded BAZF(-/-) mice. We therefore propose that BAZF supports angiogenic sprouting via BAZF-CUL3-based polyubiquitination-dependent degradation of CBF1 to down-regulate Notch signaling.

DOI： 10.1182/blood-2011-03-345306

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Because of the availability of genome information combined with proteomics techniques, it is possible to determine the identity of a protein which had been isolated many years ago on a two-dimensional gel electrophoresis and stored in a dry state as a data archive. The protocol described in this chapter will assist researchers who want to know the identity of a protein separated decades ago when no techniques were available to determine the identity of the protein.

DOI： 10.1007/978-1-61779-821-4_25

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：LANDES BIOSCIENCE  

The reproductive systems of male and female Drosophila have been well studied genetically and cytologically because of the importance of meiotic mechanisms in understanding inheritance and speciation. Recently, there has been increasing interest in proteomics of Drosophila melanogaster, because detailed and comprehensive gene annotations have made it straightforward to identify proteins analyzed by mass spectrometry. We have applied proteomic analysis to the male reproductive system with the aim of understanding sperm maturation not only during the process of spermatogenesis, but also during the movement of sperm from the seminal vesicle through the male reproductive system and into the uterus following copulation. In this article, we will provide an overview of the principles of recent proteomic technologies and describe a few proteomic profiles of the male reproductive organs.

DOI： 10.4161/fly.4.1.10838

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

Vitreous samples collected in retinopathic surgeries have diverse properties, making proteomics analysis difficult. We report a cluster analysis to evade this difficulty. Vitreous and subretinal fluid samples were collected from 60 patients during surgical operation of non-proliferative diabetic retinopathy, proliferative diabetic retinopathy, proliferative vitreoretinopathy, and rhegmatogenous retinal detachment. For controls we collected vitreous fluid from patients of idiopathic macular hole, epiretinal, and from a healthy postmortem donor. Proteins from these samples were subjected to quantitative proteomics using two-dimensional gel electrophoresis. We selected 105 proteins robustly expressed among ca 400 protein spots and subjected them to permutation test. By using permutation test analysis we observed unique variations in the expression of some of these proteins in vitreoretinal diseases when compared to the control and to each other: 1) the levels of inflammation-associate proteins such as AAT, APOA4, ALB, and TF were significantly higher in all four types of vitreoretinal diseases, and 2) each vitreoretinal disease elevates a unique set of proteins which can be interpreted based on the pathology of retinopathy. Our protocol will be effective for the study of protein expression in other types of clinical samples of diverse property.

DOI： 10.1002/prca.200800017

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Membrane heterogeneity plays a significant role in regulating signal transduction and other cellular activities. We examined the protein and lipid components associated with the detergent-resistant membrane (DRM) fractions from retinal rod outer segment (ROS) disk and plasma membrane-enriched preparations. Proteomics and correlative western blot analysis revealed the presence of alpha and beta subunits of the rod cGMP-gated ion channel and glucose transporter type 1, among other proteins. The glucose transporter was present exclusively in ROS plasma membrane (not disks) and was highly enriched in DRMs, as was the cGMP-gated channel beta-subunit. In contrast, the majority of rod opsin and ATP-binding cassette transporter A4 was localized to detergent-soluble domains in disks. As expected, the cholesterol : fatty acid mole ratio was higher in DRMs than in the corresponding parent membranes (disk and plasma membranes, respectively) and was also higher in disks compared to plasma membranes. Furthermore, the ratio of saturated : polyunsaturated fatty acids was also higher in DRMs compared to their respective parent membranes (disk and plasma membranes). These results confirm that DRMs prepared from both disks and plasma membranes are enriched in cholesterol and in saturated fatty acids compared to their parent membranes. The dominant fatty acids in DRMs were 16 : 0 and 18 : 0; 22 : 6n3 and 18 : 1 levels were threefold higher and twofold lower, respectively, in disk-derived DRMs compared to plasma membrane-derived DRMs. We estimate, based on fatty acid recovery that DRMs account for only approximately 8% of disks and approximately 12% of ROS plasma membrane.

DOI： 10.1111/j.1471-4159.2007.04971.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

The circadian clock in the retina regulates a variety of physiological phenomena such as disc shedding and melatonin release. Although these events are critical for retinal functions, it is almost unknown how the circadian clock controls the physiological rhythmicity. To gain insight into the processes, we performed a proteomic analysis using 2-DE to find proteins whose levels show circadian changes. Among 415 retinal protein spots, 11 protein spots showed circadian rhythmicity in their intensities. We performed MALDI-TOF MS and NanoLC-MS/MS analyses and identified proteins contained in the 11 spots. The proteins were related to vesicular transport, calcium-binding, protein degradation, metabolism, RNA-binding, and protein foldings, suggesting the clock-regulation of neurotransmitter release, transportation of the membrane proteins, calcium-binding capability, and so on. We also found a rhythmic phosphorylation of N-ethylmaleimide-sensitive fusion protein and identified one of the amino acid residues modified by phosphorylation. These findings provide a new perspective on the relationship between the physiological functions of the retina and the circadian clock system.

DOI： 10.1002/pmic.200700272

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

Post-translational methylation of the epsilon-amino group of lysine residues regulates a number of protein functions. Calmodulin, a key modulator of intracellular calcium signaling, is methylated on lysine 115 in many species. Although the amino acid sequence of calmodulin is highly conserved in eukaryotes, it has been shown that lysine 115 is not methylated in Drosophila calmodulin and no other methylation site has been reported. In this study, we characterized in vivo modification states of Drosophila calmodulin using proteomic methodology involving the protein mapping of microdissected Drosophila tissues on 2-D gels. We found that Drosophila calmodulin was highly expressed in methylated forms in the compound eye, whereas its methylation was hardly detected in other tissues. We identified that lysine 94 located in an EF-hand III is the methylation site in Drosophila calmodulin. The predominance of methylated calmodulin in the compound eye may imply the involvement of calmodulin in photoreceptor-specific functions through methylation.

DOI： 10.1002/pmic.200700343

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER PHYSIOLOGICAL SOC  

Peripheral nerve injury is often followed by the development of severe neuropathic pain. Nerve degeneration accompanied by inflammatory mediators is thought to play a role in generation of neuropathic pain. Neuronal cell death follows axonal degeneration, devastating a vast number of molecules in injured neurons and the neighboring cells. Because we have little understanding of the cellular and molecular mechanisms underlying neuronal cell death triggered by nerve injury, we conducted a proteomics study of rat 4th and 5th lumbar (L4 and L5) dorsal root ganglion (DRG) after L5 spinal nerve ligation. DRG proteins were displayed on two-dimensional gels and analyzed through quantitative densitometry, statistical validation of the quantitative data, and peptide mass fingerprinting for protein identification. Among approximately 1,300 protein spots detected on each gel, we discovered 67 proteins that were tightly regulated by nerve ligation. We find that the injury to primary sensory neurons turned on multiple cellular mechanisms critical for the structural and functional integrity of neurons and for the defense against oxidative damage. Our data indicate that the regulation of metabolic enzymes was carefully orchestrated to meet the altered energy requirement of the DRG cells. Our data also demonstrate that ligation of the L5 spinal nerve led to the upregulation in the L4 DRG of the proteins that are highly expressed in embryonic sensory neurons. To understand the molecular mechanisms underlying neuropathic pain, we need to comprehend such dynamic aspect of protein modulations that follow nerve injury.

DOI： 10.1152/physiolgenomics.00255.2006

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BIOMED CENTRAL LTD  

BACKGROUND: The extracellular matrix can have a profound effect upon the phenotype of cancer cells. Previous work has shown that growth of bladder cancer cells on a matrix derived from normal basement membrane suppresses many malignant features that are displayed when the cells are grown on a matrix that has been modified by malignant tumors. This work was undertaken to investigate proteome-level changes as determined by a new commercially available proteome display involving 2-dimensional chromatography for bladder cancer cells grown on different extracellular matrix preparations that modulate the expression of the malignant phenotype. RESULTS: Depending on the matrix, between 1300 and 2000 distinct peaks were detected by two-dimensional chromatographic fractionation of 2.1-4.4 mg of total cellular protein. The fractions eluting from the reversed-phase fractionation were suitable for mass spectrometric identification following only lyophilization and trypsin digestion and achieved approximately 10-fold higher sensitivity than was obtained with gel-based separations. Abundant proteins that were unique to cells grown on one of the matrices were identified by mass spectrometry. Following concentration, peaks of 0.03 AU provided unambiguous identification of protein components when 10% of the sample was analyzed, whereas peaks of 0.05 AU was approximately the lower limit of detection when the entire sample was separated on a gel and in-gel digestion was used. Although some fractions were homogeneous, others were not, and up to 3 proteins per fraction were identified. Strong evidence for post-translational modification of the unique proteins was noted. All 13 of the unique proteins from cells grown on Matrigel were related to MYC pathway. CONCLUSION: The system provides a viable alternative to 2-dimensional gel electrophoresis for proteomic display of biological systems. The findings suggest the importance of MYC to the malignant phenotype of bladder cancer cells.

DOI： 10.1186/1477-5956-4-13

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

In this report we introduce a new concept "proteomic trajectory mapping" for the investigation of a complex phenomenon underlying biological transformation and transition. We define proteomic trajectory to be the kinetic trace of protein expression and present a successful proteomic trajectory mapping of complex molecular events underlying postnatal development of mouse retina. Cluster analysis of the trajectory data using a two-state model identified four proteomic trajectory types: two distinct trajectory types accounting for the decline or the rise of protein molecules actively expressed in the juvenile stage (J-type) or in the adult stage (A-type), a class of transient trajectories that mediate the transformation from the juvenile to the adult stage (T-type), and the steady trajectories throughout the entire process of transformation (C-type). The dominance of particular protein categories expressed in each trajectory characterizes the stage of retinal development. Proteomic trajectory mapping will be a powerful tool to study the systematic changes of protein expression caused by physiological, genetic, or pathological agents and the reverse of such changes to the norm by a treatment. The proteomic trajectory mapping is applicable to any biological transformation and, therefore, will be a powerful tool in biomedical sciences.

DOI： 10.1002/pmic.200500813

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

Structural characterization of glycoproteins remains among the most challenging areas of glycomics due to the requirement of large quantities of samples and laborious biochemical steps involved in the analytical procedure. Here we report the structural characterization of glycoproteins separated on a 2-D gel by using a MALDI-QIT-TOF MS where QIT is quadrupole IT. The combination of MALDI-ion source and QIT appears to generate a unique tendency to cause fragmentation of glycopeptides without collision-induced dissociation. The majority of such fragmentations observed in our study result from the cleavage of sugar linkages, but not of peptide-peptide or peptide-sugar linkages. This unique feature allows us to perform pseudo-MS3 analysis of a fragmented glycopeptide. A small gel spot of a glycoprotein in the abundance range of low picomoles was enough for the mass spectrometer to analyze fragmentation pathway of the sugar linkage and peptide backbone. In this study, we demonstrate direct determination of glycosylation sites and N-linked glycan-sequences of the tryptic glycopeptides of Drosophila glycoproteins. Glycopeptides with various MWs up to approximately 4000 Da were suitable for structural analysis, including its attachment site and the amino acid sequence, of the glycopeptide through multistage mass spectrometric analysis.

DOI： 10.1002/elps.200500324

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

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記述言語：日本語   出版者・発行元：(株)先端医学社  

ANCA関連血管炎(AAV)は、詳細な発症機序が不明で、診断マーカーとなる血清ANCA値は必ずしも病態と相関するわけではなく、活動性の指標としても鋭敏ではない。筆者らは、当院およびRemlT-JAV-RPGN登録患者における治療前と治療6ヵ月後の血清プロテオームの大規模定量解析をおこない、新たな疾患活動性マーカーとしてTIMP-1など8種、腎障害(腎予後予測)マーカーとしてCD93やTransketolaseを見出した。とくにTIMP-1は、CRPやMPO-ANCAよりも優れており、AAVの寛解導入および寛解維持期における寛解判定と再燃予測に有用な活動性マーカーとなることが示唆された。(著者抄録)

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記述言語：日本語   出版者・発行元：(株)先端医学社  

質量分析法の一種である選択反応モニタリング(SRM)を用いれば、夾雑な臨床サンプルから目的の分子を高感度に検出し、信頼度の高い定量情報を迅速に取得できる。近年は生体内高分子であるタンパク質の定量解析への応用も進んでおり、抗体ベースの従来法と比較してスケーラブルで低コストなアッセイ開発が可能である。本稿では、ターゲットプロテオミクスとよばれる、SRMを活用した標的タンパク質群の包括的定量ワークフローを解説し、筆者らが実施したANCA関連血管炎の活動性をモニタリングするための新規バイオマーカー開発におけるターゲットプロテオミクスの活用例を紹介する。(著者抄録)

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

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記述言語：日本語   出版者・発行元：日本電気泳動学会  

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記述言語：日本語   出版者・発行元：日本プロテオーム学会  

&lt;p&gt;質量分析によるタンパク質の高精度な定量解析には，安定同位体標識ペプチドが内部標準として用いられる．プロテオーム動態の相対的な定量化には安価な粗精製ペプチドの利用例が近年増加しているが，目的とするタンパク質の絶対量計測には高品質な精製ペプチドが必要であり，そのプロテオームワイドな入手は困難な課題である．一方，Quantification Concatamer（QconCAT）と呼ばれる内部標準ペプチドの連結体を用いる定量アプローチが提案されているが，その普及にはQconCATの安定した生合成系の確立が大きな課題となっている．本総説では，強力なインビトロ翻訳を可能にするコムギ無細胞合成系を活用したQconCATの合成戦略について紹介する．著者らが開発したコムギ無細胞系におけるQconCATの多重共発現法は，大規模な内部標準ペプチドライブラリーの迅速な構築を可能にし，様々な生命現象におけるプロテオームの定量化に向けた試みを強力に加速するだろう．&lt;/p&gt;

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY  

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記述言語：日本語  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：英語   出版者・発行元：日本比較生理生化学会  

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記述言語：日本語   出版者・発行元：(一社)日本生物物理学会  

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その他リンク： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2016&ichushi_jid=J00737&link_issn=&doc_id=20160509410005&doc_link_id=%2Fec6sebut%2F2016%2F005602%2F010%2F0116-0125%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fec6sebut%2F2016%2F005602%2F010%2F0116-0125%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

記述言語：日本語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.56.116

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記述言語：日本語  

DOI： 10.14952/SEIKAGAKU.2015.870636

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

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記述言語：日本語   出版者・発行元：(公財)日本眼科学会  

臨床所見をもとに病態を説明する仮説を立て,これを実証していくのは臨床研究者としての醍醐味である.この総説では自らの歩みの中で進めてきたいくつかの取り組みを紹介したい.I.角膜感染症と闘う! 個々の時代の医療背景を受け角膜感染症の病原体は変遷を続けている.1970年代は角膜ヘルペスの全盛期であり,著者も,新たな抗ウイルス療法の開発やウイルスワクチンの研究に精力的に携わった.1980年代後半,アシクロビルの登場で鎮静化した角膜ヘルペスと入れ替わるように角膜内皮炎が出現した.進行性の角膜内皮障害,サイレントな前房内炎症,特異な角膜後面沈着物形成を特徴とするが,その病態基盤として,ウイルス抗原に対する前房関連免疫偏位(anterior chamber-associated immune deviation:ACAID)が作動している可能性を家兎モデルにおいて示した.近年,サイトメガロウイルスが本疾患の新たな病原体として注目を集めている.21世紀に入ると緑膿菌とアカントアメーバを代表的病原体とするコンタクトレンズ関連角膜感染症が若年層において急増し,レンズケアの重要性が再認識された.緑膿菌角膜炎については病原因子であるプロテアーゼに着目して研究を進め,macrophage inflammatory protein 2(MIP-2)を阻害するMucDセリンプロテアーゼの欠失株では角膜感染が成立しないことをマウス角膜炎モデルにおいて証明し,発症阻止メカニズムにおける好中球の重要性を再確認した.アカントアメーバに対するアプローチとしてはメチレンブルー光線力学療法(photodynamic therapy:PDT)による抗アカントアメーバ作用に加えて抗シスト薬であるポリヘキサメチレンビグアニド(PHMB)との相乗効果を見出し,将来の治療オプションとしての可能性を示した.II.白内障術後眼内炎に挑戦する! 完成の域に達した近代白内障手術ではあるが,術後眼内炎は重篤な合併症の一つとして今も恐れられている.眼内炎発症の前段階として外眼部常在菌による前房内汚染は重要な位置を占める.脂溶性微粒子懸濁液による前房内の汚染シミュレーション実験により,切開後の前房形成や眼内レンズ挿入など,粘弾性物質を介する操作で汚染が生じやすいことを示した.多くの術後眼内炎が後嚢破損などの重大な術中合併症のない眼にみられるが,これを説明する仮説として後房経由の硝子体バリア破綻の可能性が考えられる.我々はガドニウムMRIを用いた眼内灌流液の可視化手法やside view techniqueを豚眼に応用し,前部硝子体膜裂孔(anterior hyaloid membrane tear:AHT)と命名したバリア破綻がハイドロダイセクションに伴う眼内圧上昇に関連して発生することを示した.術後眼内炎のうち最も視力予後が不良とされる腸球菌眼内炎については,疫学調査の結果,視力予後良好例と不良例に二分されることが明らかとなった.視力予後には腸球菌が産生するプロテアーゼが強く関与しており,特に網膜における急性循環障害を惹起しているものと推測される.III.涙液クリアランスを考える! 涙腺より産生されて眼表面を潤し,涙道から排出される中で,涙液は「lacrimal river」と呼ぶべき流れを作っている.涙液に関連したさまざまな病態を解明していくうえで,その動態や質の変化をベースに考えていくことは重要である.我々は余剰の涙液排出機構の一つであるKrehbiel flowに着目し,poly(methyl methacrylate)(PMMA)粒子による可視化を試みるとともに,前眼部光干渉断層計を用いた負荷涙液クリアランス試験を新たに考案した.涙液層に量的負荷を与えたとき,高齢者のクリアランス効率は若年者に比較して有意に低下していた.また,涙液の質的変化を蛋白質レベルで解析する目的で,高感度絶対定量法による涙液プロテオーム解析システムを開発し,予備的な検討において高年者の涙液に多量の炎症性産物が含まれていることを明らかとした.若年者では新鮮な涙液への交換が常に行われるのに対し,高齢者では古い涙液を再利用せざるを得ない環境となっており,これが外眼部疾患の病態形成に影響を与えている可能性がある.IV.角膜浮腫を見直す! 臨床例での観察によれば,内皮機能障害からの回復過程において角膜浮腫はまず中央部から消退し,周辺部に残存する傾向を示すこと,内皮機能不全に至る場合はその逆で,周辺部から浮腫が生じ最終的に中央部が障害される.この現象は,中央部から周辺部へ向かう水移動の存在を仮定すればうまく説明できる.現在,家兎を用いた内皮障害モデルにおいてこの仮説を支持する結果が得られつつある.(著者抄録)

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

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記述言語：英語   出版者・発行元：(一社)日本癌学会  

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記述言語：日本語  

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記述言語：英語   出版者・発行元：(一社)日本細胞生物学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：英語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   出版者・発行元：西日本泌尿器科学会  

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記述言語：英語   出版者・発行元：日本癌学会  

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記述言語：英語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2011.0.156.0

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：日本比較生理生化学会  

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その他リンク： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2008&ichushi_jid=J02387&link_issn=&doc_id=20081219330001&doc_link_id=10.3330%2Fhikakuseiriseika.25.139&url=https%3A%2F%2Fdoi.org%2F10.3330%2Fhikakuseiriseika.25.139&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

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記述言語：英語   出版者・発行元：(一社)日本生理学会  

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記述言語：日本語   出版者・発行元：(公財)日本眼科学会  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：ASSOC RESEARCH VISION OPHTHALMOLOGY INC  

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