Identification and characterization of sulfolobus solfataricus P2 proteome using multidimensional liquid phase protein separations.
Identification and characterization of sulfolobus solfataricus P2 proteome using multidimensional liquid phase protein separations.
复制标题
使用多维液相蛋白质分离鉴定和表征硫磺菌 P2 蛋白质组。
DOI：
10.1021/pr7006472
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发表时间：
2008
影响因子：
4.4
通讯作者：
Assiddiq BF
中科院分区：
文献类型：
--
作者：
Assiddiq BF
我们已经确定和特点的蛋白质组硫磺硫化叶菌P2使用多维液相蛋白质分离。在第一维中使用离子交换色谱进行多维液相色谱，然后使用500 µ m i.d.聚（苯乙烯-二乙烯基苯）整料在第二维分离可溶性蛋白质裂解物从S。太阳神从S. solfataricus蛋白裂解物能够产生类似于传统2DE图谱的2D液相图谱。在第二维中分离蛋白质后，收集级分，使用胰蛋白酶在溶液中消化并使用质谱法分析。与2DE相比，这些方法显著降低了劳动强度和分析蛋白质组所需的总时间，利用了与这种方法相关的自动化和馏分收集。此外，在使用2DLC进行蛋白质组学分析之后，将获得的数据与来自S的可溶性蛋白裂解物的先前2DE和鸟枪蛋白质组学研究进行比较。太阳神与2DE相比，结果显示蛋白质组覆盖率总体增加。此外，与2DE相比，2DLC显示出许多蛋白质子集的覆盖率增加，包括酸性、碱性、低丰度和小分子量蛋白质。与鸟枪法研究相比，还观察到蛋白质组覆盖率增加。此外，使用2DLC鉴定了187种独特的蛋白质，证明这种方法作为蛋白质组学研究的替代方法或与2DE和鸟枪工作流程结合用于全球蛋白质组学。
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 µm 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.