Supercritical fluid chromatography reduces back exchange after solution-phase hydrogen/deuterium exchange

ANYL 241

Mark R. Emmett, emmett@magnet.fsu.edu1, Sasa Kazazic1, Alan G. Marshall, marshall@magnet.fsu.edu1, Wei Chen2, Stone. D. Shi, stone.shi@pfizer.com2, Ben Bolaņos, ben.bolanos@pfizer.com2, and Michael J. Greig, michael.greig@pfizer.com2. (1) Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, FL 32310, (2) Structural and Computational Biology & Design, Pfizer Global R&D - La Jolla, 10770 Science Center Dr, San Diego, CA 92121
The biggest problem with H/D exchange as a mass spectrometric probe of surface exposure in a protein/protein complex is back-exchange of H for D. (normally ~30%). HPLC greatly contributes to this back-exchange. Since the mobile phase for SFC is predominately CO2, back exchange is greatly reduced. A fully deuterated penta-peptide (IFVQK) was used in initial tests to mimic peptic fragments seen in an HDX HPLC run. The MS spectrum of the SFC eluted peptide showed ~7.5% back-exchange (as compared to 50% by similar methods). HDX of myoglobin was performed and the peptic fragments were desalted/separated by SFC (91.5% sequence coverage compared to 98.7% coverage with HPLC). The SFC incorporation was 2 to 4 fold higher at all time points as compared to the HPLC. SFC shows increased deuterium incorporation for the early time points of the H/D exchange, thereby recovering information lost in previous conventional solution HDX experiments due to high back-exchange (~90% back-exchange in the early time periods for the fastest-exchanging hydrogens with HPLC separation).
 

Methods Development for Pharmaceutical Analysis
1:30 PM-5:00 PM, Tuesday, 12 September 2006 Moscone Center -- Room 124, Oral

Division of Analytical Chemistry

The 232nd ACS National Meeting, San Francisco, CA, September 10-14, 2006