Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, United States.
Laboratory of Analytical Biochemistry and Bio-Separation, State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Talanta. 2018 Mar 1;179:588-593. doi: 10.1016/j.talanta.2017.11.060. Epub 2017 Nov 28.
We develop a new two-dimensional (2D) high performance liquid chromatography (HPLC) approach for intact protein analysis. Development of 2D HPLC has a bottleneck problem - limited second-dimension (second-D) separation speed. We solve this problem by incorporating multiple second-D columns to allow several second-D separations to be proceeded in parallel. To demonstrate the feasibility of using this approach for comprehensive protein analysis, we select ion-exchange chromatography as the first-dimension and reverse-phase chromatography as the second-D. We incorporate three second-D columns in an innovative way so that three reverse-phase separations can be performed simultaneously. We test this system for separating both standard proteins and E. coli lysates and achieve baseline resolutions for eleven standard proteins and obtain more than 500 peaks for E. coli lysates. This is an indication that the sample complexities are greatly reduced. We see less than 10 bands when each fraction of the second-D effluents are analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), compared to hundreds of SDS-PAGE bands as the original sample is analyzed. This approach could potentially be an excellent and general tool for protein analysis.
我们开发了一种新的二维（2D）高效液相色谱（HPLC）方法，用于完整蛋白质分析。二维 HPLC 的发展存在一个瓶颈问题 - 第二维（第二-D）分离速度有限。我们通过合并多个第二-D 柱来解决这个问题，允许进行多个第二-D 分离并行进行。为了证明该方法用于全面蛋白质分析的可行性，我们选择离子交换色谱作为第一维，反相色谱作为第二-D。我们以创新的方式合并三个第二-D 柱，以便可以同时进行三种反相分离。我们使用该系统测试了分离标准蛋白质和大肠杆菌裂解物的效果，并实现了十一种标准蛋白质的基线分辨率，并获得了大肠杆菌裂解物的 500 多个峰。这表明样品的复杂性大大降低。当通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析第二-D 流出物的每个馏分时，我们看到的条带少于 10 个，而原始样品分析时则有数百个 SDS-PAGE 条带。该方法可能是蛋白质分析的一种极好且通用的工具。
