Nanosecond Laser-Induced Photochemical Oxidation Method for Protein Surface Mapping with Mass Spectrometry Thin Thin Aye, † Teck Yew Low, † and Siu Kwan Sze* ,†,‡ Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, and Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543 We have developed an ultrafast pulse method for protein surface footprinting by laser-induced protein surface oxidations. This method makes use of a pulse UV laser that produces, in nanoseconds, a high concentration of hydroxyl (OH) free radicals by photodissociation of a hydrogen peroxide (H 2 O 2 ) solution. The OH radicals oxidize amino acid residues located on the protein surface to produce stable covalent modifications. The oxidized protein is then analyzed by mass spectrometry to map the oxidized amino acid residues. Ubiquitin and apomyoglo- bin were used as model proteins in this study. Our results show that a single laser pulse can produce extensive protein surface oxidations. We found that monooxidized ubiquitins were more susceptible to further oxidations by subsequent laser irradiation, as compared to nonoxidized ones. This is due to the conformational changes of proteins by oxidation that increases the solvent-accessible surface area. Therefore, it is crucial to perform this experiment with a single pulse of laser so as to avoid oxidation of proteins after conformation of the protein changes. Subsequently, to obtain a high frequency and coverage of the oxidation sites while keeping the number of laser shots to one, we further optimized the laser power and concentration of hydrogen peroxide as well as the concentration of protein. This ultrafast OH radical genera- tion method allows for rapid and accurate detection of surface residues, enabling mapping of the solvent-acces- sible regions of a protein in its native state. Mass spectrometry (MS) is one of the most widely used analytical techniques in proteomics. In addition to being used for protein identification, it is also gaining popularity for deciphering protein structure, particularly for probing the solvent-accessible surfaces of proteins. 1-3 Portions of a protein that are solvent- accessible are available to interact with ligands or other proteins. Interactions at these sites can induce conformational changes that can modify the solvent-accessible surfaces of proteins. Current methods for protein structure determination include hydrogen- deuterium exchange 4-8 and chemical modifications 9 with chemi- cals such as methyl bromide 10 followed by MS analysis. However, hydrogen-deuterium exchange is hampered by complex back- exchange kinetics. As for the latter, the reactivity and universality of such chemical probes to all amino acids are not yet well-defined. Recently, the hydroxyl (OH) free radical has been chosen for labeling the surface residues of proteins due to its numerous advantageous attributes. 11 First, OH radicals are highly reactive 12 and are able to oxidize a variety of amino acid side chains. 2,13 In addition, the rate of oxidation of the side chains of amino acids by OH radicals is much higher than oxidation-induced backbone cleavages. 2,13,14 Therefore, proteins can readily be oxidized without much backbone fragmentation. Currently, OH radicals used for such studies are usually generated by Fenton chemistry, 15 UV irradiation of hydrogen peroxide, 11 or by radiolysis of water with a high-energy X-ray synchrotron beam. 16-19 Each of these three methods has its own shortcomings. Apart from the long incubation time required, iron salts and EDTA used in the Fenton reaction may distort the native conformation of a protein. 20 As for UV irradiation, a time frame of * Corresponding author: Phone: 065-64788111. Fax: 065-64789060. 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