The separation of intact proteins by reverse-phase liquid chromatography (RPLC) has received renewed interest due to its application to top-down proteomics. RPLC is easily coupled to ESI-MS and can be used to separate the complex mixtures typically found when working with proteomic samples. This method, however, has historically exhibited problems with intact protein recovery and run-to-run carryover (ghosting) that has potentially limited it usefulness.

An improvement to traditional 3-5 μm diameter RPLC particles is the use of smaller stationary phase support particles that allow for improved separation of complex mixtures. These particles, which have a diameter of <2 μm, require a substationally higher backpressure to produce an equivalent flow. Modern LC instrumentation is limited to <6 kpsi, which is not sufficient to use with these small-diameter particles. Ultra-high pressure liquid chromatography (UHPLC), however, can produce pressures up to 100 kpsi and can be used with these smaller particles. Previous work has studied the improvements in the separation of small biological molecules, but less work has been done with intact proteins or the effects of using ultra-high pressures on these molecules.

This work focuses on the improvements in protein ghosting and recovery that is seen when using pressures above 20 kpsi to separate intact proteins. To study the effect, a mixture of four proteins was separated by reverse-phase at both traditional (<6 kpsi) and ultra-high (>20 kpsi) pressures. The results show that protein ghosting was eliminated within the LOD of the mass spectrometer, and protein recovery was enhanced at various concentration levels. Work is ongoing to study the nature of this improvement, although it appears to be solely related to the increased backpressure of the system.