Assiddiq Bobby F, Snijders Ambrosius P L, Chong Poh Kuan, Wright Phillip C, Dickman Mark J
Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD, United Kingdom.
J Proteome Res. 2008 Jun;7(6):2253-61. doi: 10.1021/pr7006472. Epub 2008 May 9.
We have identified and characterized the proteome of Sulfolobus solfataricus P2 using multidimensional liquid phase protein separations. Multidimensional liquid phase chromatography was performed using ion exchange chromatography in the first dimension, followed by reverse-phase chromatography using 500 microm i.d. poly(styrene-divinylbenzene) monoliths in the second dimension to separate soluble protein lysates from S. solfataricus. The 2DLC protein separations from S. solfataricus protein lysates enabled the generation of a 2D liquid phase map analogous to the traditional 2DE map. Following separation of the proteins in the second dimension, fractions were collected, digested in solution using trypsin and analyzed using mass spectrometry. These approaches offer significant reductions in labor intensity and the overall time taken to analyze the proteome in comparison to 2DE, taking advantage of automation and fraction collection associated with this approach. Furthermore, following proteomic analysis using 2DLC, the data obtained was compared to previous 2DE and shotgun proteomic studies of a soluble protein lysate from S. solfataricus. In comparison to 2DE, the results show an overall increase in proteome coverage. Moreover, 2DLC showed increased coverage of a number of protein subsets including acidic, basic, low abundance and small molecular weight proteins in comparison to 2DE. In comparison to shotgun studies, an increase in proteome coverage was also observed. Furthermore, 187 unique proteins were identified using 2DLC, demonstrating this methodology as an alternative approach for proteomic studies or in combination with 2DE and shotgun workflows for global proteomics.
我们已利用多维液相蛋白质分离技术对嗜热栖热菌P2的蛋白质组进行了鉴定和表征。多维液相色谱分析中，第一维采用离子交换色谱，第二维则使用内径为500微米的聚（苯乙烯 - 二乙烯基苯）整体柱进行反相色谱，以分离嗜热栖热菌的可溶性蛋白质裂解物。嗜热栖热菌蛋白质裂解物的二维液相色谱蛋白质分离能够生成类似于传统二维电泳图谱的二维液相图谱。在第二维分离蛋白质后，收集各组分，用胰蛋白酶在溶液中进行消化，并采用质谱分析。与二维电泳相比，这些方法显著降低了劳动强度，并且大大缩短了分析蛋白质组所需的总时间，这得益于该方法所具备的自动化和组分收集功能。此外，在使用二维液相色谱进行蛋白质组分析后，将所得数据与先前对嗜热栖热菌可溶性蛋白质裂解物进行的二维电泳和鸟枪法蛋白质组学研究数据进行了比较。与二维电泳相比，结果显示蛋白质组覆盖率总体有所提高。此外，与二维电泳相比，二维液相色谱在包括酸性、碱性、低丰度和小分子蛋白质在内的多个蛋白质亚组的覆盖率上有所增加。与鸟枪法研究相比，也观察到蛋白质组覆盖率有所提高。此外，使用二维液相色谱鉴定出了187种独特蛋白质，这表明该方法可作为蛋白质组学研究的一种替代方法，或者与二维电泳和鸟枪法工作流程结合用于全局蛋白质组学研究。
