Rapid, Simple and Sensitive "On Membrane Digestion" Method Using PVDF Membrane for MALDI-TOF MS and Its Application to a Bacterial Membrane Protein Proteomics

DOI Web Site Web Site 1 Citations 19 References
  • BUNAI Keigo
    Institute of Biological Sciences, University of Tsukuba National Institute of Industrial Science and Technology
  • NEMOTO Tadashi
    National Institute of Industrial Science and Technology
  • YAMANE Kunio
    Institute of Biological Sciences, University of Tsukuba

Bibliographic Information

Other Title
  • 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
  • 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

Search this article

Abstract

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.

Journal

Citations (1)*help

See more

References(19)*help

See more

Keywords

Details 詳細情報について

Report a problem

Back to top