Wickramasekara Samanthi, Neilson Julie, Patel Naren, Breci Linda, Hilderbrand Amy, Maier Raina M, Wysocki Vicki
Department of Chemistry and Biochemistry, University of Arizona, 1306 E University Bolevard, Tucson, AZ 85721, USA.
J Biomed Biotechnol. 2011;2011:701928. doi: 10.1155/2011/701928. Epub 2011 Dec 1.
The main objectives of this work were to obtain a more extensive coverage of the Burkholderia vietnamiensis proteome than previously reported and to identify virulence factors using tandem mass spectrometry. The proteome of B. vietnamiensis was 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 of Burkholderia species. Several virulence factors were detected including flagellin, porin, peroxiredoxin and zinc proteases.
这项工作的主要目标是获得比之前报道的更广泛的越南伯克霍尔德菌蛋白质组覆盖范围，并使用串联质谱法鉴定毒力因子。越南伯克霍尔德菌的蛋白质组被沉淀为四个部分，即细胞外、细胞内、细胞表面和细胞壁蛋白。采用了两种不同的方法来分析蛋白质。第一种是基于凝胶的方法，在进行反相液相色谱串联质谱（LC-MS/MS）分析之前，使用一维SDS-PAGE分离蛋白质。第二种方法使用多维蛋白质鉴定技术（MudPIT）分析，在进行串联质谱（MS/MS）分析（LC/LC-MS/MS）之前，通过阳离子交换和反相分离对蛋白质进行消化和分离。总体而言，基于凝胶的LC-MS/MS分析比MudPIT分析鉴定出更多的蛋白质。综合结果导致鉴定出1200多种蛋白质，约占伯克霍尔德菌属注释基因组编码蛋白质的16%。检测到了几种毒力因子，包括鞭毛蛋白、孔蛋白、过氧化物还原酶和锌蛋白酶。
