反相高效液相色谱双梯度洗脱分离肽混合物及质谱分析

Reversed-phase liquid chromatography with double gradient elution for the separation and mass spectrometric analysis of peptides

复杂肽段混合物的有效分离是高覆盖率地鉴定蛋白质混合物的前提。"鸟枪法"(Shotgun)蛋白质组学研究策略通常采用蛋白酶切、二维液相色谱-串联质谱分析肽段混合物从而鉴定蛋白质,其中高效率地分离肽段混合物是关键步骤之一。本文通过pH梯度结合有机溶剂梯度的反相高效液相色谱(RP-HPLC)进行一维液相色谱分离,按等时间间隔收集馏分并将一个梯度的前段的一个馏分与后段一个馏分混合,然后进行纳升级液相色谱-质谱联用(nanoRPLC-MS/MS)分析。将该方法应用于酵母蛋白质的分离和鉴定,实验结果为:与常规的强阳离子色谱-反相液相色谱-质谱分离鉴定方法相比,采用pH梯度结合有机相梯度的RP-HPLC-RPLC-MS分离鉴定方法多鉴定到567个酵母蛋白质(簇,含有3 035个唯一肽段);其中鉴定到肽段的pI分布范围为3.42～12.01,相对分子质量范围为587.67～3 499.79;蛋白质的pI分布范围为3.82～12.19,相对分子质量范围为3 446.55～432 905。该结果表明这种方法在复杂体系蛋白质组分离鉴定中具有明显的优势,在蛋白质组学研究中有较好的应用前景。

Highly effective separation of complex peptide mixture is a prerequisite for protein identification with high coverage in proteomics. Currently, peptide mixture is separated by two-dimensional liquid chromatography （2D-LC）, ion exchange chromatography as the first dimension and reversed-phase chromatography as the second dimension, in Shotgun proteomics. Though the 2D-LC is now widely used, its separation efficiency still needs further improve- ment. In this work, the first dimensional separation was performed by the pH and organic solvent double-gradient elution. And then the two fractions, one from the early eluted section and the other from the later eluted section （ with equal time intervals） were pooled and analyzed by MS/MS. The experimental results from the protein mixture of saccharomyces cerevisiae lysate showed that the separation by pH and organic solvent double-gradient elution, RP-HPLC-nanoRPLC coupled with MS/MS identified 567 more yeast protein groups （3 035 peptides） over strong cation exchange chromatography-nanoRPLC coupled with MS/MS. The pI values and relative molecular masses of identified peptides ranged from 3.42 to 12.01, and from 587.67 to 3 499.79, respectively. The pI values and relative molecular masses of identified proteins ranged from 3.82 to 12. 19 and from 3 446.55 to 432 905, respectively. These results indicated that this new 2DLC-MS method has the advantages over the conventional Shotgun method, and were expected to be applied in the separation of complex samples for proteomic studies in the future.