Nanosecond Laser-Induced Photochemical Oxidation Method for Protein Surface Mapping with Mass Spectrometry

Abstract

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 (H2O2) 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 apomyoglobin 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 generation method allows for rapid and accurate detection of surface residues, enabling mapping of the solvent-accessible regions of a protein in its native state.

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

“…Its small size, high reactivity, and water-like properties makes it an excellent reagent for tagging solvent-accessible surface areas. ⋅OH can be produced by Fenton chemistry [20,21], electrochemical methods [22], corona discharge techniques [23], water radiolysis [24][25][26], and photochemical cleavage of H 2 O 2 [27][28][29]. All 20 amino acids can react with ⋅OH; about half of them generate products (often +16 Da modifications) that are readily detectable by MS [25].…”

Laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale

“…We presented a preliminary account of this work at the 2005 American Society for Mass Spectrometry conference. Presented also at the conference was a description of a similar approach using 266 nm irradiation of the protein in a static system but without scavenger to control the reaction duration and a 7-fold higher hydrogen peroxide concentration [9].…”

Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

“…Hydroxyl radical (·OH) is an interesting covalent probe due to its small size and high reactivity. Of the many methods for generating ·OH [13][14][15][16][17][18][19][20], the photolysis of H 2 O 2 by a pulsed UV laser [21][22][23] is particularly convenient. The microsecond ·OH lifetime implies that this photochemical technique is free of oxidation-induced artifacts if the measurements are conducted under single-exposure conditions [21,24,25].…”