On-line separation of native proteins by two-dimensional liquid chromatography using a single column

• Published in: Journal of Chromatography A Vol. 1216; no. 16; pp. 3553 - 3562
• Main Authors: Geng, Xindu, Ke, Congyu, Chen, Gang, Liu, Peng, Wang, Fei, Zhang, Huiqiang, Sun, Xuan
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 17.04.2009; Amsterdam; New York: Elsevier; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Biotechnology; Buffers; Cations; Chromatography, Ion Exchange; Chromatography, Liquid - instrumentation; Chromatography, Liquid - methods; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Hydrophobic and Hydrophilic Interactions; Intact proteins; Mixed-mode; Muramidase - isolation & purification; On-line buffer exchange; On-line two-dimensional liquid chromatography; Online Systems; Proteins; Proteins - isolation & purification; Proteomics; Reference Standards; Biotechnology; Mixed-mode; On-line buffer exchange; Intact proteins; On-line two-dimensional liquid chromatography; Proteomics; Human; Biological fluid; Peak resolution; Two dimensional chromatography; Hydrophilic Interaction Liquid chromatography; HPLC chromatography; On-line two-dimensional liquid; Protein; Blood; Ion exchange chromatography; Separation method; Plate efficiency; chromatography; Ion exchanger; Blood serum
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2009.01.085

This paper reports the on-line separation of native (N) proteins by two-dimensional liquid chromatography (2D-LC) using a single column with one phase (called 2D column). The 2D column exhibits excellent resolution, selectivity, and retention of proteins in the N state and functions in two retention modes—hydrophobic interaction chromatography (HIC) and weak-cation exchange chromatography (WCX). We describe a new approach to on-line buffer exchange and collection of fractions from the first retention mode and their quantitative re-injection into the same column, followed by re-separation in the second retention mode. Thus, liquid chromatography in a closed system and in an on-line manner could be successfully carried out. This method was termed on-line protein separation by 2D-LC using only a single column (on-line 2D-LC-1C). The applicability of this method was experimentally demonstrated using standard proteins and a human serum sample. The total hypothetical maximum possible peak capacity n c,total and total sample peak capacity n c , total * of the 2D column were 329 and 199, respectively. By comparison against several popular commercially available columns, it was found that the 2D column had not only comparable resolution and better selectivity but also some unique characteristics. This 2D-LC-1C method could be applied to the fast purification of intact proteins in the N state, such protein drugs from natural products, and recombinant proteins and also for the fast pre-fractionation of intact proteins in the “top-down” MS strategy in proteomics.