Early Studies in Ultrahigh Pressure Liquid Chromatography of Intact Proteins

Early Studies in Ultrahigh Pressure Liquid Chromatography of Intact Proteins Public Deposited

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Creator

Eschelbach, John W.
- Affiliation: College of Arts and Sciences, Department of Chemistry

Abstract

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 ESIMS. 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 micrometers, 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.

Date of publication