色谱2011，Vol.29Issue(3)：205-211,7.DOI:10.3724/SP.J.1123.2011.00205
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
摘要
Abstract
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 dimen-sion 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 improvement. 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.关键词
反相高效液相色谱/pH梯度洗脱/有机溶剂梯度洗脱/纳升级反相高效液相色谱-串联质谱/肽混合物/酵母Key words
reversed-phase liquid chromatography (RPLC)/ pH gradient elution/ organic solvent gradient elution/ nano reversed-phase liquid chromatography-tandem mass spectrometry (nanoRPLC-MS/MS) / peptides/ saccharomyces cerevisiae分类
化学化工引用本文复制引用
马岩,张万军,卫军营,牛明,林虹君,秦伟捷,张养军,钱小红..反相高效液相色谱双梯度洗脱分离肽混合物及质谱分析[J].色谱,2011,29(3):205-211,7.基金项目
国家高技术研究发展计划(2006AA02A308)、国家自然科学基金(30621063,20635010,20735005,20875101,20905077)和"973"计划(2007CB914104,20lOCB912701,2011CB910603)项目. （2006AA02A308）
