Early studies in ultrahigh pressure liquid chromatography of intact proteins

The analysis of proteomic mixtures is quite complex due to the abundance of species to characterize. Most modern methods utilize a bottom-up approach, but as mass spectrometer methods improve, the need to high-resolution top-down methods have also been identified. The separation of intact proteins for top-down proteomics by reverse-phase liquid chromatography (RPLC) has received renewed interest due to the ease of coupling to ESI-MS. Unfortunately, modern RPLC methods do not have high enough resolving power to analyze complex proteomic mixtures with great success.; It has been well documented that the use of smaller diameter packing material in a chromatographic column can greatly increase the resolving power. These particles, which have a diameter of <2 mum, require a substantially higher backpressure to produce an equivalent flow. Ultrahigh pressure liquid chromatography (UHPLC) can produce pressures up to 100 kpsi and can be used with these smaller particles. Previous work has explored isocratic separations small organics and gradient separation of peptides. This work will investigate gradient UHPLC of intact proteins.; A custom gradient UHPLC system capable of up to 40 kpsi for use with proteomic samples is presented. This system was first used to investigate separations of an E Coli lysate on a column packed with 1.5 um porous particles. The results present some of the highest chromatographic peak capacities seen to date. Next, the carryover behavior of four standard proteins was explored. It is evident from the results that the use of pressures above 15 kpsi greatly improves the carryover and recovery of intact proteins, which is an unanticipated benefit of using ultrahigh pressures.; Reversed-phase methods show the greatest separation power, but also require the use of long gradient times and mobile phases that reduce the activity of the protein in its native form. Hydrodynamic chromatography (HDC) utilizes the parabolic flow profile in the interstitial spaces between particles in a packed column to drive separation of analytes based on their size in reverse order. Since there is no direct interaction with the stationery phase, non-porous silica (NPS) beads can be utilized, making this technique well-suited for ultrahigh pressures.; Based on the hydrodynamic radius of proteins in solution, particles in the size range of 0.2-0.6mum are required for proteins between 25--300 kDa. The preparation of sub-micron NPS packed capillary columns and performance of these columns applied to protein separations by HDC was also investigated as a potential alternative to RPLC methods.