The Chromatography Forum

of Delaware Valley

Peak capacity of one-dimensional separations for comprehensive bottom-up proteomics is routinely inadequate for the number of peptides in a sample. Various multidimensional separation methodologies, as well as advanced mass spectrometry instrumentation, have been employed to improve peak capacity. The pressure capabilities of commercial instruments limit the length of the column and dimensions of the stationary phase used to improve the peak capacity of one-dimension reversed-phase separations. We describe a system that operates at constant pressures up to 45,000 psi. The system is comprised of a commercial UHPLC instrument, for gradient generation and sample management, and modified with a gradient storage loop and pneumatic amplifier pump for elevated pressure separations. The system’s performance is assessed with a complex peptide mixture on a range of microcapillary columns packed with sub-2μm C18 particles. For multidimensional separations of the S. cerevisiae proteome, the improved separation efficiency, made possible through this instrument, resulted in a greater number of protein identifications and improved peptide coverage. (This research was completed while working in the Jorgenson Group at the University of North Carolina).