Dwivedi Ravi C, Spicer Vic, Harder Michael, Antonovici Mihaela, Ens Werner, Standing Kenneth G, Wilkins John A, Krokhin Oleg V
Manitoba Centre for Proteomics and Systems Biology, University of Manitoba, 799 JBRC, 715 McDermot Avenue, Winnipeg, MB, R3E 3P4, Canada.
Anal Chem. 2008 Sep 15;80(18):7036-42. doi: 10.1021/ac800984n. Epub 2008 Aug 8.
We describe the practical implementation of a new RP (pH 10 - pH 2) 2D HPLC-ESI/MS scheme for large-scale bottom-up analysis in proteomics. When compared to the common SCX-RP approach, it provides a higher separation efficiency in the first dimension and increases the number of identified peptides/proteins. We also employed the methodology of our sequence-specific retention calculator (SSRCalc) and developed peptide retention prediction algorithms for both LC dimensions. A diverse set of approximately 10,000 tryptic peptides from the soluble protein fraction of whole NK-type cells gave retention time versus hydrophobicity correlations, with R (2) values of 0.95 for pH 10 and 0.945 for pH 2 (formic acid) separation modes. The superior separation efficiency and the ability to use retention prediction to filter out false-positive MS/MS identifications gives promise that this approach will be a method of choice for large-scale proteomics analyses in the future. Finally, the "semi-orthogonal" separation selectivity permits the concatenation of fractions in the first dimension of separation before the final LC-ESI MS step, effectively cutting the analysis time in half, while resulting in a minimal reduction in protein identification.
我们描述了一种用于蛋白质组学大规模自下而上分析的新型RP（pH 10 - pH 2）二维HPLC-ESI/MS方案的实际应用。与常见的SCX-RP方法相比，它在第一维提供了更高的分离效率，并增加了鉴定出的肽段/蛋白质的数量。我们还采用了序列特异性保留计算器（SSRCalc）的方法，并为液相色谱的两个维度开发了肽段保留预测算法。从全NK型细胞的可溶性蛋白组分中选取的约10,000种胰蛋白酶肽段的多样集合，给出了保留时间与疏水性的相关性，在pH 10分离模式下R²值为0.95，在pH 2（甲酸）分离模式下为0.945。卓越的分离效率以及利用保留预测来滤除假阳性MS/MS鉴定结果的能力，预示着这种方法在未来将成为大规模蛋白质组学分析的首选方法。最后，“半正交”分离选择性允许在最终的LC-ESI MS步骤之前，在分离的第一维中连接各组分，有效地将分析时间减半，同时蛋白质鉴定的减少量最小。
