title: Enhancing Protein Disulfide Bond Cleavage by UV Excitation and Electron Capture Dissociation for Top-Down Mass Spectrometry.
abstract: The application of ion pre-activation with 266 nm ultraviolet (UV) laser irradiation combined with electron capture dissociation (ECD) is demonstrated to enhance top-down mass spectrometry sequence coverage of disulfide bond containing proteins. UV-based activation can homolytically cleave a disulfide bond to yield two separated thiol radicals. Activated ECD experiments of insulin and ribonuclease A containing three and four disulfide bonds, respectively, were performed. UV-activation in combination with ECD allowed the three disulfide bonds of insulin to be cleaved and the overall sequence coverage to be increased. For the larger sized ribonuclease A with four disulfide bonds, irradiation from an infrared laser (10.6 µm) to disrupt non-covalent interactions was combined with UV-activation to facilitate the cleavage of up to three disulfide bonds. Preferences for disulfide bond cleavage are dependent on protein structure and sequence. Disulfide bonds can reform if the generated radicals remain in close proximity. By varying the time delay between the UV-activation and the ECD events, it was determined that disulfide bonds reform within 10-100 msec after their UV-homolytic cleavage.
authors.affiliation: Department of Chemistry and Biochemistry, University of California-Los Angeles Los Angeles, CA 90095.
authors.forename: Piriya
authors.initials: P
authors.lastname: Wongkongkathep
authors.affiliation: Department of Biological Chemistry, University of California-Los Angeles Los Angeles, CA 90095.
authors.forename: Huilin
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Chemistry, University of California-Riverside, Riverside, CA 92521.
authors.forename: Xing
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Biological Chemistry, University of California-Los Angeles Los Angeles, CA 90095.
authors.forename: Rachel R Ogorzalek
authors.initials: RR
authors.lastname: Loo
authors.affiliation: Department of Chemistry, University of California-Riverside, Riverside, CA 92521.
authors.forename: Ryan R
authors.initials: RR
authors.lastname: Julian
authors.affiliation: Department of Chemistry and Biochemistry, University of California-Los Angeles Los Angeles, CA 90095 ; Department of Biological Chemistry, University of California-Los Angeles Los Angeles, CA 90095 ; UCLA/DOE Institute of Genomics and Proteomics, University of California-Los Angeles Los Angeles, CA 90095.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo