Zhou Hu, Dai Jie, Sheng Quan-Hu, Li Rong-Xia, Shieh Chia-Hui, Guttman Andras, Zeng Rong
Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Shanghai, PR China.
Electrophoresis. 2007 Dec;28(23):4311-9. doi: 10.1002/elps.200700463.
The conventional 2-D LC-MS/MS setup for global proteome analysis was based on online and offline salt gradients (step and continuous) using strong-cation-exchange chromatography in conjunction with RP chromatography and MS. The use of the online system with step salt elution had the possibility of resulting in peptide overlapping across fractions. The offline mode had the option to operate with continuous salt gradient to decrease peak overlap, but exhibited decreased robustness, lower reproducibility, and sample loss during the process. Due to the extensive washing requirement between the chromatography steps, online continuous gradient was not an option for salt elution. In this report, a fully automated, online, and continuous gradient (pH continuous online gradient, pCOG) 2-D LC-MS/MS system is introduced that provided excellent separation and identification power. The pH gradient-based elution provided more basic peptides than that of salt-based elution. Fraction overlap was significantly minimized by combining pH and continuous gradient elutions. This latter approach also increased sequence coverage and the concomitant confidence level in protein identification. The salt and pH elution-based 2-D LC-MS/MS approaches were compared by analyzing the mouse liver proteome.
用于全球蛋白质组分析的传统二维液相色谱-串联质谱（2-D LC-MS/MS）设置基于在线和离线盐梯度（分步和连续），使用强阳离子交换色谱结合反相色谱和质谱。采用分步盐洗脱的在线系统有可能导致肽段在各组分间重叠。离线模式可以选择采用连续盐梯度操作以减少峰重叠，但在该过程中显示出稳健性降低、重现性较差以及样品损失。由于色谱步骤之间需要大量冲洗，在线连续梯度不适用于盐洗脱。在本报告中，介绍了一种全自动、在线且连续梯度（pH连续在线梯度，pCOG）的二维液相色谱-串联质谱系统，该系统具有出色的分离和鉴定能力。基于pH梯度的洗脱比基于盐的洗脱提供了更多碱性肽段。通过结合pH和连续梯度洗脱，组分重叠显著减少。后一种方法还增加了序列覆盖率以及蛋白质鉴定中的伴随置信水平。通过分析小鼠肝脏蛋白质组，比较了基于盐洗脱和pH洗脱的二维液相色谱-串联质谱方法。
