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新規シアル酸含有糖ペプチドの抽出法の確立

Research Project

Project/Area Number 18710178
Research Category

Grant-in-Aid for Young Scientists (B)

Allocation TypeSingle-year Grants
Research Field Living organism molecular science
Research InstitutionHokkaido University

Principal Investigator

黒河内 政樹  Hokkaido University, 大学院・先端生命科学研究院, 特任助教 (30422009)

Project Period (FY) 2006 – 2007
Project Status Completed (Fiscal Year 2007)
Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2007: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2006: ¥2,400,000 (Direct Cost: ¥2,400,000)
Keywordsシアル酸糖鎖抽出 / 選択的酸化 / アミノオキシ担体 / 糖ペプチド / 質量分析計 / アミノ酸配列分析 / 糖鎖構造解析 / 糖タンパク質
Research Abstract

本年度は、シアル酸含有糖ペプチドの調整並びに混合物の試料を用いてシアル酸糖ペプチドの解析を行った。
1)単一糖タンパク質(AFP, Firinogen, EPO)由来のシアル酸含有糖ペプチドの効率的捕捉と切断(遊離)。
糖タンパク質のトリプシン消化物を1mM過ヨウ素酸ナトリウム溶液を用いて氷冷下で15分間酸化した。次に、反応液をポリアクリル酸型のアミノオキシ担持樹脂を用いて、シアル酸含有糖ペプチドのみを選択的に単離収集した。捕捉した糖ペプチドは、3%TFA溶液を用いてシアル酸部位で切断を行い、糖ペプチドを回収し、MALDI TOF-MSによる解析を行った。
3)血清中のシアル酸含有糖ペプチドの選択的捕捉と切断(遊離)。
マウス疾患モデルマウスの血清試料のトリプシン消化物を1mM過ヨウ素酸ナトリウム溶液を用いて氷冷下で15分間酸化した。次に、反応液をポリアクリル酸型のアミノオキシ担持樹脂を用いて、シアル酸含有糖ペプチドのみを選択的に単離し回収した。捕捉した糖ペプチドは、3% TFA溶液を用いてシアル酸部位で切断を行い、糖ペプチドを回収し、MALDI TOF-MSによる解析を行った。
本プロジェクトにおいて得られた成果は、Angew. Chem. Int. Ed (2007)に発表させて頂きました。
本法は、非還元末端にシアル酸を有する複合糖質の網羅的なエンリッチが可能であり、かつペプチドおよび糖鎖の配列を同時かつ大規模に取得可能な唯一の方法論であることから、グライコプロテオミクスにおける基盤技術になることが期待される。

Report

(2 results)
  • 2007 Annual Research Report
  • 2006 Annual Research Report
  • Research Products

    (17 results)

All 2008 2007 2006 Other

All Journal Article (13 results) (of which Peer Reviewed: 9 results) Book (1 results) Remarks (3 results)

  • [Journal Article] Glycosylation status of haptoglobin in sera of patients with prostate cancer vs. benign prostate disease or normal subjects2008

    • Author(s)
      Fujimura T.
    • Journal Title

      Intl. J. Cancer 122

      Pages: 39-49

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] BlotGlycoABC^<TM>: An integrated glycoblotting technique for rapid and large-scale clinical glycomics2008

    • Author(s)
      Miura Y.
    • Journal Title

      Mol. Cell. Proteomics (in press)

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Alteration of N-glycans related to articular cartilage deterioration after anterior cruciate ligament transection in rabbits.2008

    • Author(s)
      Matsuhashi T.
    • Journal Title

      Osteoarthritis Cartilage. (in press)

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] A comprehensive approach to structural and functional glycomics based on chemoselective glycoblotting and sequential tag conversion2008

    • Author(s)
      Furukawa J-I.
    • Journal Title

      Anal. Chem. (in press)

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Reverse glycoblotting allows rapid enrichment glycoproteomics of biopharmaceuticals and disease-related biomarkers2007

    • Author(s)
      Kurogochi M.
    • Journal Title

      Angew. Chem. Int. Ed. 46

      Pages: 8808-8813

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Increased al, 6-fucosylation of N-glycan in serum glycoprotein of db/db mice and humans with type 2 diabetes2007

    • Author(s)
      Itoh N.
    • Journal Title

      Am. J. Physiol.-Endocrinol. Metab. 293

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Molecular Basis of Guanine Nucleotide Dissociation Inhibitor Activity of Human Neuroglobin by Chemical Cross-Linking and Mass Spectrometry2007

    • Author(s)
      Kitatsuji C.
    • Journal Title

      J. Mol. Biol. 368

      Pages: 150-160

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] One-Pot Solid-Phase Glycoblotting and Probing by Transoximization for High-Throughput Glycomics and Glycoproteomics2007

    • Author(s)
      Shimaoka H.
    • Journal Title

      Chem. Eur. J. 13

      Pages: 1664-1673

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Highly efficient synthesis of versatile proteoglycan core structures from 1,6-anhydro-β-lactose as a key starting material2007

    • Author(s)
      Shimawaki K.
    • Journal Title

      Angew. Chem. Int. Ed. 46

      Pages: 3074-3079

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Construction of Highly Glycosylated Mucin-Type Glycopeptides Based on Microwave-Assisted Solid Phase Synthesis and Enzymatic Modifications2006

    • Author(s)
      Matsushita Takahiko
    • Journal Title

      The Journal of Organic Chemistry 71

      Pages: 3051-3063

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Unusual N-glycan structures in a-mannosidase II/IIx double null embryos, identified by a systematic glycomic approach based on 2D-LC mapping and MDSF method in MALDI-TOF/TOF mass spectrometry2006

    • Author(s)
      Hato Megumi
    • Journal Title

      Molecular & Cellular Proteomics 5

      Pages: 2146-2157

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Construction and structural characterization of versatile lactosaminoglycan-related compound library for the synthesis of complex glycopeptides and phingoglycolipids2006

    • Author(s)
      Naruchi Kentarou
    • Journal Title

      The Journal of Organic Chemistry 71

      Pages: 9609-9621

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Mechanism-based inhibitors to probe transitional states of glycoside hydrolases2006

    • Author(s)
      Hinou Hiroshi
    • Journal Title

      Methods in Enzymology 415

      Pages: 202-212

    • Related Report
      2006 Annual Research Report
  • [Book] 糖鎖のMALDI-TOFMSスペクトルデータブック2007

    • Author(s)
      黒河内 政樹
    • Total Pages
      201
    • Publisher
      三共出版
    • Related Report
      2007 Annual Research Report
  • [Remarks]

    • URL

      http://altair.sci.hokudai.ac.jp/g4/

    • Related Report
      2007 Annual Research Report
  • [Remarks]

    • URL

      http://www.lfsci.hokudai.ac.jp/innovahome/

    • Related Report
      2007 Annual Research Report
  • [Remarks]

    • URL

      http://www.gfrg.org/

    • Related Report
      2007 Annual Research Report

URL: 

Published: 2006-04-01   Modified: 2016-04-21  

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