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Initial Proteome Analysis of Model Microorganism Haemophilus influenzae Strain Rd KW20

BIATECH, Bothell, Washington 98011,¹ National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894,² Institute for Systems Biology, Seattle, Washington 98103,³ Department of Computer Science,⁴ Department of Management Science, University of Washington, Seattle, Washington 98195,⁵ Department of Molecular Microbiology and Immunology, University of Missouri-Columbia, Columbia, Missouri 65212,⁶ Department of Bioengineering, University of California at San Diego, La Jolla, California 92093,⁷ Seattle Biomedical Research Institute, Seattle, Washington 98109⁸

Received 30 January 2003/ Accepted 25 April 2003

The proteome of Haemophilus influenzae strain Rd KW20 was analyzed by liquid chromatography (LC) coupled with ion trap tandem mass spectrometry (MS/MS). This approach does not require a gel electrophoresis step and provides a rapidly developed snapshot of the proteome. In order to gain insight into the central metabolism of H. influenzae, cells were grown microaerobically and anaerobically in a rich medium and soluble and membrane proteins of strain Rd KW20 were proteolyzed with trypsin and directly examined by LC-MS/MS. Several different experimental and computational approaches were utilized to optimize the proteome coverage and to ensure statistically valid protein identification. Approximately 25% of all predicted proteins (open reading frames) of H. influenzae strain Rd KW20 were identified with high confidence, as their component peptides were unambiguously assigned to tandem mass spectra. Approximately 80% of the predicted ribosomal proteins were identified with high confidence, compared to the 33% of the predicted ribosomal proteins detected by previous two-dimensional gel electrophoresis studies. The results obtained in this study are generally consistent with those obtained from computational genome analysis, two-dimensional gel electrophoresis, and whole-genome transposon mutagenesis studies. At least 15 genes originally annotated as conserved hypothetical were found to encode expressed proteins. Two more proteins, previously annotated as predicted coding regions, were detected with high confidence; these proteins also have close homologs in related bacteria. The direct proteomics approach to studying protein expression in vivo reported here is a powerful method that is applicable to proteome analysis of any (micro)organism.

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