Two-Dimensional Separations of Intact Proteins Using Ultra High Pressure Liquid Chromatography

        A single cell of a living organism can produce a complement of thousands of proteins which may range in abundance over five or more orders of magnitude.  Comprehensive two-dimensional liquid chromatography (LCxLC) is well suited to analysis of complex mixtures of proteins because of its capability of generating very high resolution separations [1].  Furthermore, LCxLC can be directly interfaced with mass spectrometry, unlike more traditional gel-based 2D separation methods.  My research involves the development of a method that uses anion exchange chromatography coupled with ultra high pressure reversed-phase liquid chromatography and online electrospray-time of flight mass spectrometry for the separation and detection of intact proteins.  The first dimension of the 2D separation is carried out on an anion exchange column using volatile buffers.  Fractions are collected and concentrated prior to further analysis.  For the second dimension of the 2D separation, reversed-phase chromatography is performed using capillary columns packed in-house with very small diameter particles.  These particles provide increased chromatographic efficiency as compared with conventional 3-5 μm particles, but generate much higher backpressures.  Therefore custom built ultra-high pressure pumps are used to perform gradient elution at pressures over 20,000 psi [2].  On-line coupling of the reversed-phase capillary column with ESI-TOF MS provides intact mass information for all detected proteins.  This method has been used to separate an E. coli soluble protein extract and has resolved of hundreds of proteins in a single 2D separation.

1.   Evans, C. R. and Jorgenson, J. W. Anaytical and Bioanalytical Chemistry 2004 378, 1952-1961

2.   MacNair, J. E., Patel, K. D., and Jorgenson, J. W. Analytical Chemistry 1999, 71, 700-708.