Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Proteomics. 2011 May;11(10):2019-26. doi: 10.1002/pmic.201000722. Epub 2011 Apr 18.
In this study, we evaluated a concatenated low pH (pH 3) and high pH (pH 10) reversed-phase liquid chromatography strategy as a first dimension for two-dimensional liquid chromatography tandem mass spectrometry ("shotgun") proteomic analysis of trypsin-digested human MCF10A cell sample. Compared with the more traditional strong cation exchange method, the use of concatenated high pH reversed-phase liquid chromatography as a first-dimension fractionation strategy resulted in 1.8- and 1.6-fold increases in the number of peptide and protein identifications (with two or more unique peptides), respectively. In addition to broader identifications, advantages of the concatenated high pH fractionation approach include improved protein sequence coverage, simplified sample processing, and reduced sample losses. The results demonstrate that the concatenated high pH reversed-phased strategy is an attractive alternative to strong cation exchange for two-dimensional shotgun proteomic analysis.
在这项研究中，我们评估了一种串联的低 pH（pH3）和高 pH（pH10）反相液相色谱策略，作为胰蛋白酶消化的人 MCF10A 细胞样本的二维液相色谱串联质谱（“shotgun”）蛋白质组分析的第一维。与更传统的强阳离子交换方法相比，串联高 pH 反相液相色谱作为第一维分级策略，分别使肽和蛋白质鉴定的数量增加了 1.8 倍和 1.6 倍（具有两个或更多独特肽）。除了更广泛的鉴定外，串联高 pH 分级方法的优点还包括改善蛋白质序列覆盖度、简化样品处理和减少样品损失。结果表明，串联高 pH 反相策略是强阳离子交换用于二维 shotgun 蛋白质组分析的一种有吸引力的替代方法。
