Hiroki Kuyama

Publications (49) View all

• Article: Enriching C-terminal peptide from endopeptidase ArgC digest for protein C-terminal analysis.

  Hiroki Kuyama, Chihiro Nakajima, Koichi Tanaka
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  ABSTRACT: This Letter describes a method for enriching C-terminal peptide of protein for C-terminal sequence analysis. This method employs endopeptidase ArgC digestion and C-terminal peptide enrichment using m-aminophenylboronic acid-agarose as an arginine-capture material. The selectively recovered C-terminal peptide incorporates no artificial derivatization. Therefore, the widely used functional groups (e.g. α-NH(2) and α-COOH) can be used for any necessary transformation. In this research, a TMPP mass tag was attached to the α-NH(2) group to clarify the amino acid sequence of the C-terminal peptide.
  Bioorganic & medicinal chemistry letters 09/2012; · 2.65 Impact Factor
• Article: C-terminal sequencing of protein by MALDI mass spectrometry through the specific derivatization of the α-carboxyl group with 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide.

  Chihiro Nakajima, Hiroki Kuyama, Takashi Nakazawa, Osamu Nishimura
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  ABSTRACT: We present here an approach to C-terminal sequencing of proteins by the procedure consisting of the following: (1) derivatization of the C-terminal α-carboxyl group with 3-aminopropyltris(2,4,6-trimethoxyphenyl)-phosphonium bromide (TMPP-propylamine) through oxazolone chemistry, (2) enzymatic proteolysis of the TMPP-derivatized protein, and (3) MALDI-MS/MS analysis of the peptide mixture, in which the C-terminal peptide incorporating the TMPP group is preferably detected. In this protocol, it is possible to choose any endoproteinase such as trypsin, GluC, and AspN for digestion so that a C-terminal peptide with length appropriate for mass spectrometric sequencing could be generated. The peptide labeled with TMPP-propylamine at the C terminus tends to exhibit y-type ions in MS/MS spectra, allowing manual sequence interpretation with the simplified fragmentation pattern. The efficacy of the method was verified with five proteins, which demonstrated that the C-terminal peptides were readily distinguishable by their peak intensity and characteristic mass signature peak in MALDI-PSD analysis.
  Analytical and Bioanalytical Chemistry 06/2012; 404(1):125-32. · 3.78 Impact Factor
• Article: Development of iodoacetic acid-based cysteine mass tags: detection enhancement for cysteine-containing peptide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

  Takashi Shimada, Hiroki Kuyama, Taka-Aki Sato, Koichi Tanaka
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  ABSTRACT: We developed and characterized 6 new cysteine mass tags for high-sensitivity peptide analysis. The structural features are: (1) iodoacetyl group for thiol tagging, (2) hydrophilic character for reducing sample loss, (3) tertiary amino, quaternary ammonium, or guanidino group for high proton affinity, and (4) no amide bonding for minimizing fragmentation of tag moiety in collision-induced dissociation. By using these tags, 2- to 200-fold MS sensitivity was achieved, compared to control peptide with carbamydomethylation.
  Analytical Biochemistry 12/2011; 421(2):785-7. · 3.00 Impact Factor
• Article: A novel type of prophenoloxidase from the kuruma prawn Marsupenaeus japonicus contributes to the melanization of plasma in crustaceans.

  Taro Masuda, Ryosuke Otomo, Hiroki Kuyama, Kyosuke Momoji, Masashi Tonomoto, Shota Sakai, Osamu Nishimura, Tatsuya Sugawara, Takashi Hirata
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  ABSTRACT: Melanization is one of the major immune responses in arthropods. Prophenoloxidases (proPOs) catalyze the oxidation of mono- or o-diphenols, a reaction that is the key initial step of melanin formation. Well-characterized proPOs from crustaceans are synthesized in haemocytes and are released into plasma in response to microbial attack. However, PO activity does exist in the plasma of haemolymph without pathogenic infections. Here, we demonstrate that a novel type of proPO contributes to such PO activity in the plasma fraction of haemolymph of crustaceans. The novel enzyme, which was purified from the plasma of the kuruma prawn (Marsupenaeus japonicus), possessed strong and specific monophenol and o-diphenol oxidation activity compared with that of known haemocyte-type proPO. Amino acid sequence analyses indicated that this enzyme was distinct from the known proPO. The cDNA sequence and deduced amino acid sequence of this enzyme has a putative binuclear copper center, and showed approximately 30% and 20% identity with the primary structures of reported proPO and haemocyanin sequences of the kuruma prawn, respectively. Reverse transcription PCR analysis showed that this enzyme was synthesized in the hepatopancreas rather than in haemocytes. Although the primary structure and enzymatic properties of this novel enzyme suggested that it is a phenoloxidase, its biogenesis, tissue distribution, and oligomeric state resemble those of haemocyanin, which belongs to the same protein family (type III copper protein). This novel proPO enzyme may share a role with the already characterized version, itself a major component of the innate immune system in crustaceans.
  Fish &amp Shellfish Immunology 10/2011; 32(1):61-8. · 3.32 Impact Factor
• Article: Selective isolation of N-blocked peptide by combining AspN digestion, transamination, and tosylhydrazine glass treatment.

  Kazuhiro Sonomura, Hiroki Kuyama, Ei-Ichi Matsuo, Susumu Tsunasawa, Shiroh Futaki, Osamu Nishimura
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  ABSTRACT: Many eukaryotic proteins are blocked at the α-amino group of their N-terminal with various modifications, thereby making it difficult to determine their N-terminal sequence by protein sequencer. We propose a novel method for selectively isolating the blocked N-terminal peptide from the peptide mixture generated by endoproteinase AspN digestion of N-blocked protein. This method is based on removal of all peptides other than the N-terminal one (non-N-terminal peptides) through their carbonyl group introduced by a chemical transamination reaction. The transamination reaction converts the free α-amino group of the non-N-terminal peptides to a carbonyl group, whereas the blocked N-terminal peptide, which lacks only the free α-amino group, remains unchanged. Silica functionalized with the tosylhydrazino group effectively captures non-N-terminal peptides through their carbonyl group; thus, the blocked N-terminal peptide is selectively recovered in the supernatant. This method was applied to several model proteins, and their N-terminal peptides were successfully isolated and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, the method was extended to N-terminal analysis of N-free protein by artificially blocking the free α-amino group of its N-terminal with N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide reagent.
  Analytical Biochemistry 03/2011; 410(2):214-23. · 3.00 Impact Factor