School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, 2052, NSW, Australia.
J Proteome Res. 2010 Feb 5;9(2):664-76. doi: 10.1021/pr9007865.
Proteomic studies have proven useful for studying the Antarctic archaeon Methanococcoides burtonii; however, little has been learned about the hydrophobic and membrane proteins, despite knowledge of their biological importance. In this study, new methods were developed to analyze and maximize the coverage of the hydrophobic proteome. Central to the analysis was a differential solubility fractionation (DSF) procedure using n-octyl-beta-D-glucopyranoside. The study achieved a significant increase (330) in the total number of known expressed proteins. From 612 identified, 185 were predicted to contain transmembrane domains or be associated with the membrane and 190 to be hydrophobic. The DSF procedure increased the efficacy of identifying membrane proteins by up to 169% and was economical, requiring far fewer runs (12% of machine time) to analyze the proteome compared to procedures without DSF. The analysis of peptide spectral counts enabled the assessment of growth temperature specific proteins. This semiquantitative analysis was particularly useful for identifying low abundance proteins unable to be quantified using labeling strategies. The proteogenomic analysis of the newly identified proteins revealed many cellular processes not previously associated with adaptation of the cell. This DSF-based approach is likely to benefit proteomic analyses of hydrophobic proteins for a broad range of biological systems.
蛋白质组学研究已被证明对研究南极古菌 Methanococcoides burtonii 非常有用；然而，尽管人们已经了解了它们的生物学重要性，但对于疏水性和膜蛋白的了解却很少。在这项研究中，开发了新的方法来分析和最大限度地覆盖疏水性蛋白质组。分析的核心是使用正辛基-β-D-吡喃葡萄糖苷的差异溶解度分级（DSF）程序。该研究实现了已知表达蛋白总数的显著增加（330 个）。在鉴定的 612 个中，有 185 个被预测含有跨膜结构域或与膜相关，190 个是疏水性的。DSF 程序将鉴定膜蛋白的效果提高了高达 169%，而且经济实惠，与没有 DSF 的程序相比，分析蛋白质组所需的运行次数（机器时间的 12%）要少得多。肽谱计数的分析能够评估特定生长温度的蛋白质。这种半定量分析对于鉴定使用标记策略无法定量的低丰度蛋白质特别有用。对新鉴定蛋白质的蛋白质基因组学分析揭示了许多以前与细胞适应无关的细胞过程。这种基于 DSF 的方法可能会有益于广泛的生物学系统中疏水性蛋白质的蛋白质组学分析。
