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Capillary HPLC/electrospray ionization mass spectrometry for structural analysis of micro-quantities of proteins

Capillary HPLC/electrospray ionization mass spectrometry for structural analysis of micro-quantities of proteins
毛细管 HPLC/电喷雾电离质谱法用于微量蛋白质的结构分析
批准号:
06680584
负责人:
TAKAO Toshifumi
金额:
$1.28万
依托单位:
依托单位国家:
日本
项目类别:
Grant-in-Aid for General Scientific Research (C)
财政年份:
1994
资助国家:
日本
项目状态:
已结题
起止时间:
1994 至 1995

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中文摘要
翻译
通过改进电喷雾离子源,将毛细管高效液相色谱(HPLC)与磁扇形质谱仪相结合。它可以在HPLC/ESI-MS中实现灵敏、准确的质量测量。因此,在毛细管柱(直径300毫米)分离后,可以有效地测量混合样品中涉及的所有组分,例如酶解蛋白质。对蛋白质混合物和蛋白质水解消化液的分析表明,毛细管高效液相色谱和磁扇形质谱联用测定皮摩尔质量的准确度优于0.01%。它可以成功地应用于dna衍生蛋白序列的阅读框以及重组蛋白或天然蛋白中存在的修饰位点和类型的测定。微印迹仪被设计成与毛细管高效液相色谱相结合,用于收集从毛细管柱洗脱到聚偏二氟乙烯膜上的小体积样品溶液,并被证明可以对毛细管柱分离的每种成分进行分选,回收率高(超过90%),可以很容易地进行氨基酸组成或序列分析。毛细管高效液相色谱/ESI-MS和微量印迹系统的结合将为数量有限的肽和蛋白质的微量分析提供一种很好的方法。
英文摘要
Capillary high-performance liquid chromatography (HPLC) has been combined with a magnetic sector mass spectrometer by improving the electrospray ion source. It allows the sensitive and accurate mass measurement in the HPLC/ESI-MS.As a result, all the components involved in a mixture sample such as an enzymatic digest of a protein could be efficiently measured following separation by a capillary column (300 mm diameter). Analyzes of a protein mixture and proteolytic digest indicate that the combination of a capillary HPLC and magnetic sector mass spectrometer is capable of attaining better than 0.01 % accuracy in mass determination on picomole quantities. It could successfully be applied to determination of the reading frames of DNA-derived protein sequence and both sites and types of modifications present in recombinant or natural proteins. A micro-blotting apparatus, which had been designed to be coupled with a capillary HPLC for collecting small volumes of sample solution eluted from a capillary column onto a polyvinylidene fluoride membrane, was demonstrated to fractionate each component separated by a capillary column with the high recovery (more than 90 %), which could readily be subjected to amino acid composition or sequence analyzes. The combination of a capillary HPLC/ESI-MS and micro-blotting system would go a good way for micro-analysis of peptides and proteins that are available only in limited quantities.
期刊论文(35)
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会议论文
Yoshino, K.: "Characterization of Highly Toxic, Large Molecular Size Heat-Stable Enterotoxin Produced by a Clinical Isolate of Yersinia enterocolitica" FEBS Lett.362. 319-322 (1995)
Yoshino, K.:“小肠结肠炎耶尔森菌临床分离株产生的高毒性、大分子热稳定肠毒素的表征”FEBS Lett.362。
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Matsuda,T.: "Characterization of Interactions between Transducin α/Br-Subunits and Lipid Membrans." J.Biol.Chem. 269. 30358-30363 (1994)
Matsuda, T.:“转导蛋白 α/Br 亚基与脂质膜之间相互作用的表征。”J.Biol.Chem. 269. 30358-30363 (1994)
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Agawara,K.: "Activation Peptide of Human Factor IX Has Oligosaccharides O-Glycosidically Linked to Threonine residues at 159 and 169" Biochemistry. 33. 5167-5171 (1994)
Akawara,K.:“人因子 IX 的激活肽具有与 159 和 169 处苏氨酸残基 O-糖苷连接的寡糖”生物化学。
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共 25 条
    Mass Spectrometry of Metalloproteins and High-Molecular-Weight Protein Complexes
    • 批准号:
      22370040
    • 项目类别:
      Grant-in-Aid for Scientific Research (B)
    • 资助金额:
      $11.65万
    • 财政年份:
      2010
    • 负责人:
      TAKAO Toshifumi
    • 依托单位:
    Dynamism analysis of posttranslational modifications by mass spectrometry.
    • 批准号:
      14380293
    • 项目类别:
      Grant-in-Aid for Scientific Research (B)
    • 资助金额:
      $6.08万
    • 财政年份:
      2002
    • 负责人:
      TAKAO Toshifumi
    • 依托单位:
    Development of the method for high-sensitivity structural analysis of carbohydrate moieties of a glycoprotein by mass spectrometry
    • 批准号:
      12558077
    • 项目类别:
      Grant-in-Aid for Scientific Research (B)
    • 资助金额:
      $4.35万
    • 财政年份:
      2000
    • 负责人:
      TAKAO Toshifumi
    • 依托单位:
    Development of a high-energy collision-induced dissociation device for high-sensitivity and high-resolution MS/MS/MS
    • 批准号:
      10558099
    • 项目类别:
      Grant-in-Aid for Scientific Research (B)
    • 资助金额:
      $8.0万
    • 财政年份:
      1998
    • 负责人:
      TAKAO Toshifumi
    • 依托单位:
    海外基金