title: Mass tagging approach for mitochondrial thiol proteins.
abstract: A mass tagging approach is described for mitochondrial thiol proteins under nondenaturing conditions. This approach utilizes stable isotope-coded, thiol-reactive (4-iodobutyl)triphenylphosphonium (IBTP) reagents, i.e., the isotopomers IBTP-d(0) and IBTP-d(15). The mass spectrometric properties of IBTP-labeled peptides were evaluated using an ESI-q-TOF and a MALDI-TOF/TOF instrument. High energy collision induced dissociation (CID) in the TOF/TOF instrument caused side-chain fragmentation in the butyltriphenylphosphonium moiety-containing Cys-residue. By contrast, low energy CID in the qTOF instrument yielded sequence tags of IBTP-labeled peptides that were suitable for automated database searching. The IBTP labeling strategy was then applied to the analysis of a protein extract obtained from cardiac mitochondria. The relative abundance measurements for identified IBTP-labeled peptides showed an average variability for peptide quantitation of approximately 10% based on peak area ratios of ion signals for the d(0)/d(15)-tagged peptide pairs. The reactivity of the IBTP reagents was further studied by molecular modeling and visualization. The present study suggests that the IBTP reagent seems to show a bias toward highly surface-exposed protein thiols. Hence, the described mass tagging approach might become potentially useful in redox proteomics studies designed to identify protein thiols that are particularly prone to oxidative modifications.
authors.affiliation: Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Marley
authors.forename: Duane T
authors.initials: DT
authors.lastname: Mooney
authors.forename: Gretchen
authors.initials: G
authors.lastname: Clark-Scannell
authors.forename: Tony T-H
authors.initials: TT
authors.lastname: Tong
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Watson
authors.forename: Tory M
authors.initials: TM
authors.lastname: Hagen
authors.forename: Jan F
authors.initials: JF
authors.lastname: Stevens
authors.forename: Claudia S
authors.initials: CS
authors.lastname: Maier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D007782:Lactoglobulins / Q000737:chemistry / Q000235:genetics; D013058:Mass Spectrometry / Q000379:methods; D008929:Mitochondria, Heart / Q000737:chemistry; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009943:Organophosphorus Compounds / Q000737:chemistry; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D051381:Rats; D015203:Reproducibility of Results; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics
chemical_list: C456341:(4-iodobutyl)triphenylphosphonium; D007782:Lactoglobulins; D024101:Mitochondrial Proteins; D009943:Organophosphorus Compounds; D010455:Peptides; D013438:Sulfhydryl Compounds; D013879:Thioredoxins