| AVS 65th International Symposium & Exhibition | |
| Plasma Science and Technology Division | Tuesday Sessions |
| Session PS+PB-TuM |
| Session: | Plasma Medicine |
| Presenter: | Faraz Choudhury, University of Wisconsin-Madison |
| Authors: | F.A. Choudhury, University of Wisconsin-Madison D.I. Benjamin, University of Wisconsin-Madison B.B. Minkoff, University of Wisconsin-Madison J. Blatz, University of Wisconsin-Madison M.R. Sussman, University of Wisconsin-Madison J.L. Shohet, University of Wisconsin-Madison |
| Correspondent: | Click to Email |
The protein-therapeutic industry, typified by anti-cancer proteins like Herceptin, has accelerated the need to be able to analyze the 3-dimensional structure of proteins in solution. Mass spectrometry is widely used for this. This has generated a need for new types of probes that covalently label protein solvent-accessible sites. Two common ones rely upon the creation of and covalent modification with highly reactive hydroxyl (OH) radicals. The first, fast photochemical oxidation of proteins (FPOP), generates hydroxyls from H2O2 via laser photolysis, and the second, synchrotron X-ray beam exposure, produces OH radicals via direct radiolysis of water. These techniques suffer from issues such as: a reaction times that are potentially too long to correctly measure protein structure or the necessity to add chemicals. Furthermore, both are cumbersome & expensive, either by accessing a synchrotron or building an instrument to perform FPOP. As a result of a collaboration at the interface of plasma physics and biochemistry, we developed a technique that generates μsecond bursts of OH radicals, using a surface-barrier discharge, for labeling proteins at solvent-accessible amino acid side chains. We call this Plasma-Induced Modification of Biomolecules (PLIMB). PLIMB does not require chemical additives, circumvents the issues associated with reaction timescale, and ultimately costs far less. Using a model protein in solution, cytochrome C, the protein was modified in a dose-dependent fashion in only a lightly buffered water-based solution, demonstrating that the system can generate OH radicals capable of labeling proteins without additional reagents. In addition, only discrete peptides within the protein are modified. Perturbing protein structure via digestion prior to plasma exposure significantly increases the observed covalent modification, suggesting that conformational structure is maintained during exposure. Experiments with myoglobin, a second protein, also revealed distinct regions of modification despite examining in depth the entirety of the protein’s sequence. Mapping the oxidized peptides to myoglobin’s crystal structure reveals that all of these peptides fall within the same face of the protein, suggesting the preservation of a higher-order structure under the solution conditions described. These experiments suggest that PLIMB provides a means of efficaciously generating microsecond bursts of OH radicals while providing a low cost and readily accessible means of probing the conformation of proteins in solution using mass spectrometry. We envision PLIMB being useful in a wide range of biological, medical and pharmaceutical fields.