Programmable Isoelectric Chromatofocusing and Its Application to Targeted Proteomics and Preparative Purification of Proteins
Derek Y.C. Choy, Chemical and Biological Engineering, Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC V6T 1Z4, Canada and Charles Haynes, Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC V6T 1Z4, Canada.
Chromatofocusing (CF), which primarily separates analytes on the basis of differences in isoelectric point (pI), theoretically achieves peak capacities greater than ion exchange or size exclusion chromatography, making it attractive both as a first separation dimension in multidimensional liquid chromatography platforms for resolving proteomes and as an efficient mode for high-resolution protein purification at the preparative scale. However, reaching this theoretical limit with complex protein mixtures requires improvements in chromatofocusing theory and related methodologies. Toward this goal, we have developed a model and new method for designing CF loading and elution buffers such that the buffering components can be added in a programmed manner to sculpt elution pH gradients over the range of pH 10 to 3.5, and thereby maximize peak capacity on a strong anion exchange column. The method uses small volatile buffering species as opposed to larger polyampholyte molecules, allowing for the facile removal of salts that may interfere with downstream analytical steps such as mass spectrometry. The use of a strong anion exchange column permits application to both bottom-up and top-down proteomics applications. When this chromatofocusing technique is applied with a linear pH gradient to the non-denaturing separation of the blood plasma proteome, superior fractionation performance is observed relative to previously reported chromatofocusing strategies and to traditional anion exchange chromatography. High resolution is also achieved in the non-denaturing fractionation of other proteomes, including cell extracts from E. coli, mouse C2C12, human HEPG2, MMAN and platelet cells; as well as culture media and vesicle extracts for various strains of Cryptococcus, demonstrating the power of optimized CF for protein purification, targeted proteomics or general protein inventory studies. Finally, our programmable CF technology can be applied to individual proteins, providing a means to selectively capture and purify therapeutic products from cell culture on the basis of small differences in pI and gradients in net change with pH near the target protein's pI. Linear velocities as high as 20m/h can be applied to these separations, as demonstrated by our studies on the purification of recombinant human transferrin from cultures of the methylotrophic yeast Pichia pastoris.