Proteomics Analyses of the Opportunistic PathogenBurkholderia vietnamiensisUsing Protein Fractionations and Mass Spectrometry

Abstract

The main objectives of this work were to obtain a more extensive coverage of theBurkholderia vietnamiensisproteome than previously reported and to identify virulence factors using tandem mass spectrometry. The proteome ofB. vietnamiensiswas precipitated into four fractions to as extracellular, intracellular, cell surface and cell wall proteins. Two different approaches were used to analyze the proteins. The first was a gel-based method where 1D SDS-PAGE was used for separation of the proteins prior to reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS). The second method used MudPIT analysis (Multi dimensional Protein Identification Technique), where proteins are digested and separated using cation exchange and reversed phase separations before the MS/MS analysis (LC/LC-MS/MS). Overall, gel-based LC-MS/MS analysis resulted in more protein identifications than the MudPIT analysis. Combination of the results lead to identification of more than 1200 proteins, approximately 16% of the proteins coded from the annotated genome ofBurkholderiaspecies. Several virulence factors were detected including flagellin, porin, peroxiredoxin and zinc proteases.