Nanosecond laser-induced photochemical oxidation method for protein surface mapping with mass spectrometry.
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.
Aye
Thin Thin
TT
Genome Institute of Singapore, 60 Biopolis Street, Singapore.
Low
Teck Yew
TY
Sze
Siu Kwan
SK
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D007834:Lasers; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010777:Photochemistry; D013499:Surface Properties; D013997:Time Factors; D025801:Ubiquitin / Q000737:chemistry*
D025801:Ubiquitin