Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ijms.2024.117212

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ijms.2023.117135

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Publisher：Cold Spring Harbor Laboratory  

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock considering that vancomycin is the last resort for controlling methicillin-resistantStaphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, which is typically considered too large for NMR studies, we successfully assigned the main chain using an amino acid-selective unlabeling method. Because we found that the Zn²⁺-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by mutating the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

DOI： 10.1101/2023.10.13.562161

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ijms.2023.117073

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Language：English   Publishing type：Research paper (scientific journal)  

Neutrophil granulocytes play key roles in innate immunity and shaping adaptive immune responses. They are attracted by chemokines to sites of infection and tissue damage, where they kill and phagocytose bacteria. The chemokine CXCL8 (also known as interleukin-8, abbreviated IL-8) and its G-protein-coupled receptors CXCR1 and CXCR2 are crucial elements in this process, and also the development of many cancers. These GPCRs have therefore been the target of many drug development campaigns and structural studies. Here, we solve the structure of CXCR1 complexed with CXCL8 and cognate G-proteins using cryo-EM, showing the detailed interactions between the receptor, the chemokine and Gαi protein. Unlike the closely related CXCR2, CXCR1 strongly prefers to bind CXCL8 in its monomeric form. The model shows that steric clashes would form between dimeric CXCL8 and extracellular loop 2 (ECL2) of CXCR1. Consistently, transplanting ECL2 of CXCR2 onto CXCR1 abolishes the selectivity for the monomeric chemokine. Our model and functional analysis of various CXCR1 mutants will assist efforts in structure-based drug design targeting specific CXC chemokine receptor subtypes.

DOI： 10.1038/s41467-023-39799-2

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acsomega.3c02737

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Language：English   Publishing type：Research paper (scientific journal)  

Histone lysine acylation, including acetylation and crotonylation, plays a pivotal role in gene transcription in health and diseases. However, our understanding of histone lysine acylation has been limited to gene transcriptional activation. Here, we report that histone H3 lysine 27 crotonylation (H3K27cr) directs gene transcriptional repression rather than activation. Specifically, H3K27cr in chromatin is selectively recognized by the YEATS domain of GAS41 in complex with SIN3A-HDAC1 co-repressors. Proto-oncogenic transcription factor MYC recruits GAS41/SIN3A-HDAC1 complex to repress genes in chromatin, including cell-cycle inhibitor p21. GAS41 knockout or H3K27cr-binding depletion results in p21 de-repression, cell-cycle arrest, and tumor growth inhibition in mice, explaining a causal relationship between GAS41 and MYC gene amplification and p21 downregulation in colorectal cancer. Our study suggests that H3K27 crotonylation signifies a previously unrecognized, distinct chromatin state for gene transcriptional repression in contrast to H3K27 trimethylation for transcriptional silencing and H3K27 acetylation for transcriptional activation.

DOI： 10.1016/j.molcel.2023.05.022

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Language：English   Publishing type：Research paper (scientific journal)  

Ion mobility spectrometry-mass spectrometry (IMS-MS) provides m/z values and collision cross sections (CCSs) of gas-phase ions. In our previous study, an intrinsically disordered protein, the H2A-H2B dimer, was analyzed using IMS-MS, resulting in two conformational populations of CCS. Based on experimental and theoretical approaches, this resulted from a structural diversity of intrinsically disordered regions. We predicted that this phenomenon is related to ion heating in the IMS-MS instrument. In this study, to reveal the effect of ion heating from parameters in the IMS-MS instrument on the conformational population of the H2A-H2B dimer, we investigated the arrival time distributions of the H2A-H2B dimer by changing values of three instrumental parameters, namely, cone voltage located in the first vacuum chamber, trap collision energy (trap CE) for tandem mass spectrometry, and trap bias voltage for the entrance of IMS. These results revealed that the two populations observed for the H2A-H2B dimer were due to the trap bias voltage. Furthermore, to evaluate the internal energies of the analyte ions with respect to each parameter, benzylpyridinium derivatives were used as temperature-sensitive probes. The results showed that the trap CE voltage imparts greater internal energy to the ions than the trap bias voltage. In addition, this slight change in the internal energy caused by the trap bias voltage resulted in the structural diversity of the H2A-H2B dimer. Therefore, the trap bias voltage should be set with attention to the properties of the analytes, even if the effect of the trap bias voltage on the internal energy is negligible.

DOI： 10.5702/massspectrometry.A0131

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：The Mass Spectrometry Society of Japan  

DOI： 10.5702/massspectrometry.a0110

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cold Spring Harbor Laboratory  

<title>Abstract</title>The canonical nucleosome, which represents the predominant packaging unit in eukaryotic chromatin, has an octameric core made up of two histone H2A-H2B and H3-H4 dimers with ~147 base-pair (bp) DNA wrapping around it. Non-nucleosome particles with alterative histone stoichiometries and DNA wrapping configurations have been found, and they could profoundly influence genome architecture and function. Here we solved the structure of the H3-H4 octasome, which is a nucleosome-like particle with a core made up of four H3-H4 dimers. Two conformations, open and closed, are determined at 3.9 Å and 3.6 Å resolutions by cryo-electron microscopy, respectively. The H3-H4 octasome, made up of a di-tetrameric core, is wrapped by ~120 bp DNA in 1.5 negative superhelical turns. The symmetrical halves are connected by a unique H4-H4’ interface along the dyad axis. <italic>In vivo</italic> crosslinking of cysteine probes placed at another unique H3-H3’ interface demonstrated the existence of the H3-H4 octasome in cells.

DOI： 10.1073/pnas.2206542119

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Association for the Advancement of Science (AAAS)  

Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including Escherichia coli RseP. Structure-based chemical modification and cross-linking experiments indicated that the RseP domains surrounding the active center undergo conformational changes to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrate entry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet of RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of intramembrane proteases that cleave transmembrane segments.

DOI： 10.1126/sciadv.abp9011

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Language：English   Publishing type：Research paper (scientific journal)  

Cu/Zn-superoxide dismutase (SOD1) is a homodimer with two identical subunits, each of which binds a copper and zinc ion in the native state. In contrast to such a text book case, SOD1 proteins purified in vitro or even in vivo have been often reported to bind a non-stoichiometric amount of the metal ions. Nonetheless, it is difficult to probe how those metal ions are distributed in the two identical subunits. By utilizing native mass spectrometry, we showed here that addition of a sub-stoichiometric copper/zinc ion to SOD1 led to the formation of a homodimer with a stochastic combination of the subunits binding 0, 1, and even 2 metal ions. We also found that the homodimer was able to bind four copper or four zinc ions, implying the binding of a copper and zinc ion at the canonical zinc and copper site, respectively. Such ambiguity in the metal quota and selectivity could be avoided when an intra-subunit disulfide bond in SOD1 was reduced before addition of the metal ions. Apo-SOD1 in the disulfide-reduced state was monomeric and was found to bind only one zinc ion per monomer. By binding a zinc ion, the disulfide-reduced SOD1 became conformationally compact and acquired the ability to dimerize. Based upon the results in vitro, we describe the pathway in vivo enabling SOD1 to bind copper and zinc ions with high accuracy in their quota and selectivity. A failure of correct metallation in SOD1 will also be discussed in relation to amyotrophic lateral sclerosis.

DOI： 10.1016/j.freeradbiomed.2022.03.014

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ijms.2021.116728

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Language：English   Publishing type：Research paper (scientific journal)  

Native mass spectrometry (MS) enables the determination of the molecular mass of protein complexes. Generally, samples for native MS are isolated, purified, and prepared in volatile solutions. However, to understand the function of proteins in living cells, it is essential to characterize the protein complex as is, without isolation/purification of the protein, using the smallest possible amount of the sample. In the present study, we modified the "live single-cell MS" method, which has mainly been used in metabolomics, and applied it to observe hemoglobin directly sampled from human erythrocytes. By optimizing the experimental methods and conditions, we obtained native mass spectra of hemoglobin using only a single erythrocyte, which was directly sampled into a nanoelectrospray ionization emitter using a micromanipulator and microinjector system. That is, our method enables the analysis of ∼0.45 fmol of hemoglobin directly sampled from an erythrocyte. To our knowledge, this is the first report of native MS for endogenous proteins using a single intact human cell.

DOI： 10.1021/acs.analchem.1c00588

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β-propeller proteins are common in nature, where they are observed to adopt 4- to 10-fold internal rotational pseudo-symmetry. This size diversity can be explained by the evolutionary process of gene duplication and fusion. In this study, we investigated a distorted β-propeller protein, an apparent intermediate between two symmetries. From this template, we created a perfectly symmetric 9-bladed β-propeller named Cake, using computational design and ancestral sequence reconstruction. The designed repeat sequence was found to be capable of generating both 8-fold and 9-fold propellers which are highly stable. Cake variants with 2-10 identical copies of the repeat sequence were characterised by X-ray crystallography and in solution. They were found to be highly stable, and to self-assemble into 8- or 9-fold symmetrical propellers. These findings show that the β-propeller fold allows sufficient structural plasticity to permit a given blade to assemble different forms, a transition from even to odd changes in blade number, and provide a potential explanation for the wide diversity of repeat numbers observed in natural propeller proteins. DATABASE: Structural data are available in Protein Data Bank database under the accession numbers 6TJB, 6TJC, 6TJD, 6TJE, 6TJF, 6TJG, 6TJH and 6TJI.

DOI： 10.1111/febs.15347

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

A convenient analytical system for protein-ligand interactions under crude conditions was developed using native mass spectrometry (MS). As a model protein, Escherichia coli (E. coli) dihydrofolate reductase (DHFR) with and without a histidine tag was used for the study. First, overexpressed DHFR with a His-tag was roughly purified with a Ni-sepharose resin and subjected to native mass spectrometry with or without incubation with an inhibitor, Methotrexate (MTX). Even only with the minimum cleanup by the Ni-sepharose resin, intact ions of DHFR-nicotinamide adenine dinucleotide phosphate (NADPH) and DHFR-NADPH-ligand complexes were successfully observed. By optimizing the preparation procedures of the crude sample for native MS, e.g., avoiding sonication for cell lysis, we successfully observed intact ions of the specific DHFR-NADPH-MTX ternary complex starting with cultivation of E. coli in ≤ 25 mL medium. When the crude DHFR sample was mixed with two, four, or eight candidate compounds, only ions of the specific protein-ligand complex were observed. This indicates that the present system can be used as a rapid and convenient method for the rough determination of binding of specific ligands to the target protein without the time-consuming purification of protein samples. Moreover, it is important to rapidly determine specific interactions with target proteins under conditions similar to those in "real" biological systems. Graphical abstract.

DOI： 10.1007/s00216-020-02649-x

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/jasms.9b00145

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Mass Spectrometry Society of Japan  

Post-translational modifications (PTMs) of histone N-terminal tails in nucleosome core particle (NCP), such as acetylation, play crucial roles in regulating gene expression. To unveil the regulation mechanism, atomic-level structural analysis of in-vitro modified NCP is effective with verifying the PTMs of histones. So far, identification of PTMs of NCP originating from living cells has mainly been performed using mass spectrometry (MS) techniques, such as bottom-up approach. The bottom-up approach is the most established method for protein characterization, but it does not always provide sufficient information on the acetylated sites of lysine residues in the histone tails if trypsin digestion is carried out. For histone proteins, which have many basic amino acids, trypsin generates too many short fragments that cannot be perfectly analyzed by tandem MS. In this study, we investigated the in vitro acetylation sites in the histone H3 tail using a top-down sequence analysis, matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) experiment, in combination with aminopeptidase digestion. Aminopeptidase can cleave peptide bonds one-by-one from the N-terminus of peptides or proteins, generating N-terminally truncated peptides and/or proteins. As a result, it was identified that this method enables sequence characterization of the entire region of the H3 tail. Also, application of this method to H3 in in-vitro acetylated NCP enabled assigning acetylation sites of H3. Thus, this method was found to be effective for obtaining information on in-vitro acetylation of NCP for structural biology study.

DOI： 10.5702/massspectrometry.A0090

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s13361-019-02304-5.

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Facilitates chromatin transcription (FACT) is a histone chaperone, which accomplishes both nucleosome assembly and disassembly. Our combined cryo-electron microscopy (EM) and native mass spectrometry (MS) studies revealed novel key steps of nucleosome reorganization conducted by a Mid domain and its adjacent acidic AID segment of human FACT. We determined three cryo-EM structures of respective octasomes complexed with the Mid-AID and AID regions, and a hexasome alone. We discovered extensive contacts between a FACT region and histones H2A, H2B, and H3, suggesting that FACT is competent to direct functional replacement of a nucleosomal DNA end by its phosphorylated AID segment (pAID). Mutational assays revealed that the aromatic and phosphorylated residues within pAID are essential for octasome binding. The EM structure of the hexasome, generated by the addition of Mid-pAID or pAID, indicated that the dissociation of H2A-H2B dimer causes significant alteration from the canonical path of the nucleosomal DNA.

DOI： 10.1038/s41598-019-46617-7

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An antibody fragment that recognizes the tertiary structure of a target protein with high affinity can be utilized as a crystallization chaperone. Difficulties in establishing conformation-specific antibodies, however, limit the applicability of antibody fragment-assisted crystallization. Here, we attempted to establish an alternative method to promote the crystallization of target proteins using an already established anti-tag antibody. The monoclonal antibody NZ-1 recognizes the PA tag with an extremely high affinity. It was also established that the PA tag is accommodated in the antigen-binding pocket in a bent conformation, compatible with an insertion into loop regions on the target. We, therefore, explored the application of NZ-1 Fab as a crystallization chaperone that complexes with a target protein displaying a PA tag. Specifically, we inserted the PA tag into the β-hairpins of the PDZ tandem fragment of a bacterial Site-2 protease. We crystallized the PA-inserted PDZ tandem mutants with the NZ-1 Fab and solved the co-crystal structure to analyze their interaction modes. Although the initial insertion designs produced only moderate-resolution structures, eliminating the solvent-accessible space between the NZ-1 Fab and target PDZ tandem improved the diffraction qualities remarkably. Our results demonstrate that the NZ-1-PA system efficiently promotes crystallization of the target protein. The present work also suggests that β-hairpins are suitable sites for the PA insertion because the PA tag contains a Pro-Gly sequence with a propensity for a β-turn conformation.

DOI： 10.1002/pro.3580

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Construction of an artificial protein needle (PN), which includes the membrane puncturing needle domain of bacteriophage T4 conjugated to Mn carbonyl (MnCO) complexes, is reported. The responsiveness to visible light of the MnCO complex makes it useful as a photoinduced in vivo magnetic resonance imaging contrast reagent (MRI CR), because the PN carrier has the potential to deliver the MnCO complex into mouse tumors with retention of coordination structure within the in vivo environment. Moreover, the composite has higher relaxivity and longer circulation as an MRI CR than the corresponding MnCO complex. These results demonstrate construction of a responsive in vivo MRI CR by using an artificial metalloprotein.

DOI： 10.1002/chem.201802445

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acs.analchem.8b01649

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Bioscience Biotechnology and Agrochemistry  

DOI： 10.1080/09168451.2018.1436433

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DOI： 10.1126/science.aak9867

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Orchestration of the multiple enzymes engaged in O-mannose glycan synthesis provides a matriglycan on α-dystroglycan (α-DG) which attracts extracellular matrix (ECM) proteins such as laminin. Aberrant O-mannosylation of α-DG leads to severe congenital muscular dystrophies due to detachment of ECM proteins from the basal membrane. Phosphorylation at C6-position of O-mannose catalyzed by protein O-mannosyl kinase (POMK) is a crucial step in the biosynthetic pathway of O-mannose glycan. Several mis-sense mutations of the POMK catalytic domain are known to cause a severe congenital muscular dystrophy, Walker-Warburg syndrome. Due to the low sequence similarity with other typical kinases, structure-activity relationships of this enzyme remain unclear. Here, we report the crystal structures of the POMK catalytic domain in the absence and presence of an ATP analogue and O-mannosylated glycopeptide. The POMK catalytic domain shows a typical protein kinase fold consisting of N- and C-lobes. Mannose residue binds to POMK mainly via the hydroxyl group at C2-position, differentiating from other monosaccharide residues. Intriguingly, the two amino acid residues K92 and D228, interacting with the triphosphate group of ATP, are donated from atypical positions in the primary structure. Mutations in this protein causing muscular dystrophies can now be rationalized.

DOI： 10.1111/gtc.12480

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DOI： 10.1007/s00216-016-9777-4

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DOI： 10.1007/s11095-015-1845-5

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DOI： 10.1111/gtc.12339

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DOI： 10.1039/c6sc00153j

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DOI： 10.1002/pro.2673

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DOI： 10.1021/ac503689w

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DOI： 10.1016/j.jsb.2014.11.006

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DOI： 10.1007/s13361-014-0959-z

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DOI： 10.1074/jbc.M114.564526

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DOI： 10.1021/bi4005655

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DOI： 10.1021/ac400395j

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Language：Japanese   Publisher：Japan Association of Aerosol Science and Technology  

Recent development of ion mobility mass spectrometry (IM-MS) with electrospray ionization (ESI) has enabled the characterization of protein assemblies which play important roles in various events in living cells. Some proteins change their conformations by target recognition. Therefore, the conformational analysis with a small amount of proteins is helpful to understand the mechanism of biological events. By IM-MS, proteins with the same masses but different conformations can be separated, and the information on the shape of analyte ions can be obtained even for the protein assemblies. In this article, an overview of traveling-wave IM-MS for biomolecules is described, showing an example of IM-MS for a globular protein, bovine Ubiquitin. In addition, application of IM-MS to characterize the structure of an intrinsically disordered protein complex, Swi5-Sfr1, is demonstrated.<br>

DOI： 10.11203/jar.28.119

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Other Link： https://jlc.jst.go.jp/DN/JALC/10021049391?from=CiNii

Language：Japanese   Publisher：The Mass Spectrometry Society of Japan  

DOI： 10.5702/massspec.S13-04

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Other Link： https://jlc.jst.go.jp/DN/JALC/10026812150?from=CiNii

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DOI： 10.1039/c2an35878f

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DOI： 10.1016/j.jmb.2012.07.012

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DOI： 10.1093/nar/gks162

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DOI： 10.1002/rcm.5320

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.52.S101_4

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DOI： 10.1074/jbc.M111.303339

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DOI： 10.1111/j.1742-4658.2011.08309.x

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DOI： 10.1107/S0907444910051711

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Other Link： http://orcid.org/0000-0001-7412-3722

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DOI： 10.1002/jms.1778

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DOI： 10.1038/nbt0710-654b

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DOI： 10.1074/jbc.M109.092403

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DOI： 10.1074/jbc.M109.058909

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DOI： 10.1107/S1744309109046181

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DOI： 10.1002/smll.200900667

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DOI： 10.1021/ac802354j

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Language：Japanese   Publisher：The Crystallographic Society of Japan  

DOI： 10.5940/jcrsj.51.104

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DOI： 10.1073/pnas.0801032106

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DOI： 10.2142/biophys.49.S150_8

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DOI： 10.1002/rcm.3517

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DOI： 10.1110/ps.073258108

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DOI： 10.1016/j.jmb.2008.01.020

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DOI： 10.1021/bi702037p

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DOI： 10.1016/j.jmb.2007.05.013

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DOI： 10.1002/rcm.2995

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DOI： 10.2142/biophys.47.S27_3

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DOI： 10.1002/jms.1069

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DOI： 10.1002/med.20051

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DOI： 10.1016/j.jasms.2005.12.016

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DOI： 10.1002/rcm.2603

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DOI： 10.1248/yakushi.126.915

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DOI： 10.1002/rcm.2541

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DOI： 10.1002/prot.20647

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DOI： 10.1016/j.jasms.2004.09.021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Mass Spectrometry Society of Japan  

The laser spray developed in our laboratory was applied to the analysis of biological samples. Because a 10.6 mm laser excites hydrogen bonds of water solvent, vaporization of aqueous samples takes place effectively by the laser irradiation. This makes laser spray superior to ESI for aqueous sample solution. The sensitivity of laser spray is approximately ten times higher than that of ESI for a large number of samples. The increase of signal intensities for both laser spray and ESI was observed by the addition of ammonium acetate to the sample solutions. This result was interpreted by an electrical double layer model proposed by Enke <i>et al</i>. The concentration of analyte ions in laser-sprayed droplets is estimated to be 10 times higher than that in the electrosprayed droplets. Noncovalent complexes were dissociated selectively to subunits by laser irradiation without dissociating the covalent bonds.

DOI： 10.5702/massspec.53.100

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DOI： 10.1002/jms.677

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Mass Spectrometry Society of Japan  

The structure of melittin in the presence of dioleoylphosphatidylcholine (DOPC) was investigated using hydrogen-deuterium (H/D) exchange in conjunction with collision induced dissociation (CID) in an rf-only hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS). The deuterium incorporation into backbone amide hydrogens of melittin in the presence of DOPC was analyzed at different time points examining the mass of each fragment ion produced by hexapole-CID. The percentage of deuterium incorporation into the fragments of melittin was less than 45% at initial 10 s of isotope exchange. It increased rapidly as the exchange period was prolonged. After 300 s of incubation in D₂O about 85% of amide hydrogens were exchanged with deuterium. When melittin was incubated in D₂O in the presence of dodecylphosphocholine (DPC), the rate of isotope exchange was reduced at every time point. In the case of melittin alone, more than 80% amide hydrogens were exchanged with deuterium within 10 s. By comparing these time courses, it seems that the contact with DOPC did not induce melittin to change its conformation. DOPC possibly just shielded the melittin molecule while DPC induced melittin to form some stable conformation, such as helical structure. It was revealed that H/D exchange and MS analysis enabled to study such structural changes of a peptide brought about by the interaction with two types of phospholipid even in the presence of 50-fold amount of lipid.

DOI： 10.5702/massspec.50.67

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/S1044-0305(01)00314-2

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1006/jmbi.2000.4416

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publisher：The Mass Spectrometry Society of Japan  

In order to understand the protein function in detail, it is important to have information on tertiary structure of the protein. Protein tertiary structure has generally been studied using X-ray crystallography and NMR. However, these techniques require a large scale sample preparation. On the other hand, mass spectrometry requires a smaller sample amount and is much faster than X-ray crystallography or NMR. These are great advantages of MS in the structural characterization of a protein. In the present commentary, it will be described how to study the tertiary structure of proteins using hydrogen-deuterium exchange in conjunction with mass spectrometry.

DOI： 10.5702/massspec.48.94

CiNii Books

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Other Link： https://jlc.jst.go.jp/DN/JALC/00087531756?from=CiNii

Language：English   Publisher：The Mass Spectrometry Society of Japan  

For the elucidation of the biosynthetic relationship between nontoxic peptides and hepatotoxic peptides produced together with hepatotoxic peptides, we applied the high-energy collision FAB MS/MS method to the confirmation and the determination of the peptide structure isolated from cyanobacteria. The structures of the cyanobacterial peptides including the cyclic peptides were definitely elucidated by complementary use of the NMR and MS/MS methods. Furthermore, the MS/MS method is very effective for the structural determination of closely related peptides derived from the difference of a part of the constituent amino acids, and their structures were determined by MS/MS experiments and amino acid analysis without NMR techniques. Additionally, the charge-remote fragmentation was observed in the cases of peptides containing Arg and the sulfate group which facilitated the interpretation of the resulting product ion spectrum. From the present study, the MS/MS method was found to be practicable for the confirmation and determination of the structures of cyanobacterial peptides.

DOI： 10.5702/massspec.48.56

CiNii Books

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Other Link： https://jlc.jst.go.jp/DN/JALC/00087245714?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Mass Spectrometry Society of Japan  

This paper presents the results of electrospray ionization mass spectrometry (ESIMS) applied to an enzyme-inhibitor complex, using papain and cystatin with partly lagged N-terminus. It has been reported that the inhibitory activity of cystatin, a thiol protease inhibitor, toward papain decreases in several orders of magnitude when the N-terminal seven or eight residues are lost. In the absence of papain, multiply charged full-length cystatin was mainly observed accompanied by the signals of minor components, N-terminally truncated forms. When cystatin was mixed with equimolar quantity of papain, the relative intensity of the free full-length cystatin dramatically decreased. It might be caused by the higher binding affinity of the intact cystatin for papain than those of the truncated forms. The present study indicates the potential of ESIMS for the investigation of the structure-activity relationship without isolation of each inhibitor species carrying different level of inhibitory activity.

DOI： 10.5702/massspec.48.346

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Other Link： https://jlc.jst.go.jp/DN/JALC/00089429655?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publisher：The Mass Spectrometry Society of Japan  

Hydrogen/deuterium exchange (H/D) was performed on ubiquitin, cytochrome <I>c</I> and myoglobin, and deuterium incorporation was investigated by electrospray ionization mass spectrometry (ESIMS) under several conditions, with different pH and different content of organic solvent. Circular dichroism (CD) was employed to observe conformational changes in proteins under the conditions for H/D exchange and ESIMS measurements. Comparing the results of H/D exchange experiments, ubiquitin was found to be the most stable among the three proteins against acid and organic solvent. Cytochrome <I>c</I> seemed to have interconvertible conformers under acidic condition with the addition of acetonitrile, though it was not indicated in CD spectra. Myoglobin appeared to be folded very loosely in mildly acidic condition and it was difficult to observe its H/D exchange rate by ESIMS, though its CD spectra showed little change under the conditions used for ESIMS analyses. It was possible to investigate slight conformational changes in proteins by utilizing H/D exchange and ESIMS analyses complementary to CD spectra.

DOI： 10.5702/massspec.46.75

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Other Link： https://jlc.jst.go.jp/DN/JALC/00084640734?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

DOI： 10.1006/bbrc.1997.7668

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1006/abio.1997.2122

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Language：Japanese   Publisher：The Mass Spectrometry Society of Japan  

With the development of soft ionization techniques and the advances in instruments, mass spectrometry has come to be an essential tool for the characterization of proteins. It has been used successfully for the investigation of the amino acid sequences of large macromolecules, such as IgG. In addition, it is effective for the characterization of post-translational modifications of proteins.<br>The tertiary structure of proteins has generally been investigated using X-ray crystallography and NMR. Precise information on the tertiary structure can be obtained by these techniques. However, these analyses are time consuming and require a large scale sample preparation. Accordingly, it is impossible to apply these methods directly to a small amount of proteins derived from the living organisms. On the other hand, mass spectrometry requires only a small amount of sample, such as several pico-mole, for the determination of the molecular weight of a protein and/or a peptide in a routine analysis. Consequently, studies on the tertiary structure and/or the surface structure of the protein molecules have come to be carried out by mass spectrometry, in order to characterize the tertiary and/or surface structure of the protein with a small amount of sample.<br>This paper discusses the characterization not only of the primary structure but also of the tertiary structure of proteins by mass spectrometry.

DOI： 10.5702/massspec.45.1

CiNii Books

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Other Link： https://jlc.jst.go.jp/DN/JALC/00083450730?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/1044-0305(96)80517-4

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Language：English   Publisher：The Mass Spectrometry Society of Japan  

A detection of non-covalent complexes of Ras·GDP and Ras·GppNp (guanosine-5′-(β,γ-imido)-triphosphate; GTP analogue) using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS) is reported. The Ras proteins are bound to GDP or GppNp, non-covalently, with ionic bonds. The GDP-bound Ras is inactive, while the GTP-bound Ras is active. This paper shows the possibility of the detection of the active and inactive form of a regulatory protein, which is non-covalently bound with a nucleotide, with a small sample amount by MALDI-TOFMS analyses. It was also suggested that MALDI-TOFMS is promising for the estimation of the ratio of nucleotide bound Ras protein and free apo-Ras protein.

DOI： 10.5702/massspec.44.269

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Other Link： https://jlc.jst.go.jp/DN/JALC/00082521875?from=CiNii

Language：English   Publishing type：Research paper (international conference proceedings)  

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/oms.1210281225

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DOI： 10.2116/analsci.5.759

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J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)  

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DOI： 10.1002/jms.1853

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Language：Japanese  

CiNii Books

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Language：Japanese   Publisher：The Mass Spectrometry Society of Japan  

This commentary describes the advantages of laser spray, which was developed by Hiraoka <i>et al.,</i> in the analysis of binding affinities of biomolecular complexes, such as DNA-protein and DNA-drug complexes. Since laser spray enables dissociation of the complexes without fragmentation of labile compounds such as DNA, a good correlation was found between the binding affinities and applied laser power for dissociation. We found that laser spray is promising for rapid characterization of binding affinities of complexes with small differences in structure.

DOI： 10.5702/massspec.56.155

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Language：English  

DOI： 10.1002/rcm.2603

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Language：Japanese   Publishing type：Book review, literature introduction, etc.  

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Language：Japanese  

CiNii Books

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Language：Japanese   Publisher：ジャスコレポート社  

CiNii Books

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DOI： 10.5702/massspec.48.346

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Event date： 2023.5

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