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Talanta. 2018 Mar 1;179:588-593. doi: 10.1016/j.talanta.2017.11.060. Epub 2017 Nov 28.

Two-dimensional chromatographic analysis using three second-dimension columns for continuous comprehensive analysis of intact proteins.

Author information

1
Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, United States.
2
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. Electronic address: cxcao@sjtu.edu.cn.
3
Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, United States. Electronic address: shaorong.liu@ou.edu.

Abstract

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.

KEYWORDS:

Comprehensive protein analysis; Liquid chromatography instrumentation; Parallel second-dimension columns; Protein separation; Two-dimensional liquid chromatography

PMID:
29310280
DOI:
10.1016/j.talanta.2017.11.060
[Indexed for MEDLINE]

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