Identification and characterization of hydrophobic Escherichia coli virulence proteins by liquid chromatography-electrospray ionization mass spectrometry

被引:12
作者
Hess, S
Cassels, FJ
Pannell, LK
机构
[1] NIDDKD, Struct Mass Spectrometry Facil, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA
[2] Walter Reed Army Inst Res, Dept Enter Infect, Div Communicable Dis & Immunol, Silver Spring, MD 20902 USA
基金
美国国家卫生研究院;
关键词
liquid chromatography-mass spectrometry; hydrophobic protein; virulence proteins; colonization factor; hexafluoroisopropanol; cyanogen bromide; peptide mapping; electrospray ionization mass spectrometry;
D O I
10.1006/abio.2001.5534
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Virulence of enterotoxicogenic Escherichia coli is mediated by rodlike, rigid, highly hydrophobic proteins designated fimbriae or colonization factors (CFs). More than 20 different colonization factors have been described so far using predominantly immunological and genetic methods. To characterize these hydrophobic proteins by liquid chromatography-mass spectrometry (LC-MS), different methodologies were explored. A novel LC-MS method was developed using hexafluoroisopropanol to maintain the hydrophobic proteins in solution. In addition, these proteins were digested with cyanogen bromide and peptide mapping by LC-MS was established. This technique was particularly useful in identification of closely related CFs. Both LC-MS and peptide mapping methodologies were found to be useful in characterizing highly hydrophobic CFs of E. coli. To search for molecular weights of mature proteins in the National Center for Biotechnology Information (NCBI) database, a new feature was developed and its applicability tested. The identification of a class of pathogenic virulence proteins, either intact or digested, is possible with molecular weight database searching.
引用
收藏
页码:123 / 130
页数:8
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