ＰＶＤＦ膜を利用した高感度・ハイスループットＭＡＬＤＩ－ＴＯＦ　ＭＳによるプロテオーム解析法の開発と細菌細胞膜タンパク質解析への応用

• 武内 桂吾

  筑波大学生物科学系 (独)産業技術総合研究所

• 根本 直

  (独)産業技術総合研究所

• 山根 國男

  筑波大学生物科学系

書誌事項

タイトル別名

• Rapid, Simple and Sensitive "On Membrane Digestion" Method Using PVDF Membrane for MALDI-TOF MS and Its Application to a Bacterial Membrane Protein Proteomics
• PVDF マク オ リヨウ シタ コウカンド ハイスループット MALDI TOF MS ニ ヨル プロテオーム カイセキホウ ノ カイハツ ト サイキン サイボウマク タンパクシツ カイセキ エ ノ オウヨウ

抄録

To improve sample preparation method for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) with the aim of proteomics, proteins separated by Tris/tricine SDS-PAGE were electroblotted onto PVDF membrane by a semi-dry discontinuous buffer system, visualized by staining with Coomassie Brilliant Blue, excised, digested with trypsin or lysC in the presence of 80% acetonitrile. Digested proteins were analyzed with MALDI-TOF MS. This method has several advantages over “in gel digestion” in terms of simple handling, sensitivity and time. We analyzed 105 fmol of Bacillus subtilis SecA protein and 300 to 500 fmol of standard proteins. We also analyzed three submembrane fractions of B. subtilis which had been solubilized stepwise using different mixtures of detergents from a washed cell membrane that was insoluble in 134 mM non-detergent sulfobetain solution. After two different 1D- and three different 2D-PAGEs followed by MALDI-TOF MS analysis, we identified 637 different proteins. Among them, 357 were predicted to be membrane proteins having certain or putative transmembrane segments (TMS) using the analysis by TopPred II algorithm, but 280 were soluble proteins having no TMS. On the basis of a bioinformatics analysis on B. subtilis genome data-base, 38 ABC trasnporter solute-binding proteins were predicted and 30 of the 38 were identified by this analysis. However, this improved method still have a limitation for the analysis of cell membrane proteins having more than 4 TMSs in their amino acid sequences.

収録刊行物

• 質量分析

  質量分析 51 (5), 530-537, 2003

  一般社団法人 日本質量分析学会

キーワード

• PVDF membrane
• On membrane digestion
• 80% acetonitrile
• 2D Polyacrylamide gel electrophoresis
• Membrane protein proteomics
• MALDI-TOF MS

詳細情報

• CRID: 1390001206494900480
• NII論文ID: 10011671775
• NII書誌ID: AN0010555X
• DOI: 10.5702/massspec.51.530
• COI: 1:CAS:528:DC%2BD3sXpsFajs7c%3D
• ISSN: 18804225
• ISSN: 18843271
• ISSN: 13408097
• ISSN: http://id.crossref.org/issn/13408097
• NDL書誌ID: 6725779
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I6725779
• Web Site: http://www.jstage.jst.go.jp/article/massspec/51/5/51_5_530/_pdf
• 本文言語コード: ja
• データソース種別:
  • JaLC
  • NDL
  • Crossref
  • CiNii Articles
• 抄録ライセンスフラグ: 使用不可