Speaker: Harry Hines, USAMRIID, Fort Detrick, Maryland

Topic: Mass Spectrometry and Protein Conformation

Place: Building 426, Conference Room, NCI-Frederick, Frederick, MD

Time: Thursday, April 20, 2000, at 2:00 PM

Abstract: Protein activity is strongly influenced by secondary and higher orders of structure, which ultimately depend upon amino acid sequence. While mass spectrometry is well established for assessing amino acid sequences, only recently has it been used to examine protein folding. Some approaches adapted to or developed for mass spectrometry include limited proteolysis, charge state distribution, chemical modifications, and amide hydrogen/deuterium exchange. The principal benefits of using mass spectrometry for these analyses are improved sensitivity, mass assignments, and mass range extension to include larger proteins. Conversely, mass spectrometry is used principally to assess conformational changes, not make structural assignments that are possible with techniques such as X-ray crystallography, circular dichroism, and Fourier transform infrared spectroscopy.

Two different experimental approaches, limited proteolysis and amide hydrogen/deuterium exchange, were applied separately to two substances of interest. One substance was ricin, including deglycosylated ricin (dg-ricin). Deglycosylated ricin may be useful as a vaccine against ricin intoxication. Limited proteolysis was used to evaluate conformational changes that may occur upon ricin deglycosylation. Using limited proteolysis to monitor vaccine stability during storage was also investigated. Amide hydrogen/deuterium exchange was used to investigate secondary structure induced by 2,2,2-trifluoroethanol in VAMP, the second substance of interest. VAMP40 is a polypeptide derived from synaptobrevin, the neural substrate for botulinum neurotoxin B, that may be useful to model synaptobrevin structure. For both of these approaches, mass spectrometry provided relevant data using comparatively small quantities of each substance.