Department of Microbiology and Immunology, Laboratory of Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL 36688-0002, USA.
Appl Environ Microbiol. 2011 Jul;77(14):4712-8. doi: 10.1128/AEM.05140-11. Epub 2011 Jun 3.
The obligate intracellular growth of Rickettsia prowazekii places severe restrictions on the analysis of rickettsial gene expression. With a small genome, predicted to code for 835 proteins, identifying which proteins are differentially expressed in rickettsiae that are isolated from different hosts or that vary in virulence is critical to an understanding of rickettsial pathogenicity. We employed a liquid chromatography (LC)-linear trap quadrupole (LTQ)-Orbitrap mass spectrometer for simultaneous acquisition of quantitative mass spectrometry (MS)-only data and tandem mass spectrometry (MS-MS) sequence data. With the use of a combination of commercially available algorithms and in-house software, quantitative MS-only data and comprehensive peptide coverage generated from MS-MS were integrated, resulting in the assignment of peptide identities with intensity values, allowing for the differential comparison of complex protein samples. With the use of these protocols, it was possible to directly compare protein abundance and analyze changes in the total proteome profile of R. prowazekii grown in different host backgrounds. Total protein extracted from rickettsiae grown in murine, tick, and insect cell lines or hen egg yolk sacs was analyzed. Here, we report the fold changes, including an upregulation of shock-related proteins, in rickettsiae cultivated in tissue culture compared to the level for rickettsiae harvested from hen yolk sacs. The ability to directly compare, in a complex sample, differential rickettsial protein expression provides a snapshot of host-specific proteomic profiles that will help to identify proteins important in intracellular growth and virulence.
普氏立克次体的专性细胞内生长对分析立克次体基因表达有严格的限制。该菌基因组较小，预计编码 835 种蛋白质，鉴定从不同宿主中分离出或毒力不同的立克次体中哪些蛋白质表达存在差异，对于了解立克次体的致病性至关重要。我们采用液相色谱（LC）-线性阱四极杆（LTQ）-轨道阱质谱仪，同时获取定量质谱（MS）仅数据和串联质谱（MS-MS）序列数据。通过结合使用商业上可获得的算法和内部软件，将定量 MS 仅数据和来自 MS-MS 的综合肽覆盖率进行整合，从而确定具有强度值的肽身份，从而可以对复杂的蛋白质样品进行差异比较。使用这些方案，可以直接比较蛋白质丰度并分析在不同宿主背景下生长的普氏立克次体的总蛋白质组图谱的变化。从在鼠、蜱和昆虫细胞系或鸡胚卵黄囊中生长的立克次体中提取总蛋白进行分析。在这里，我们报告了在组织培养中培养的立克次体与从鸡卵黄囊中收获的立克次体相比的折叠变化，包括与休克相关的蛋白质的上调。能够在复杂的样本中直接比较差异的立克次体蛋白质表达，提供了宿主特异性蛋白质组图谱的快照，有助于确定在细胞内生长和毒力中重要的蛋白质。
