Mass tagging approach for mitochondrial thiol proteins.
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.
Marley
Kevin
K
Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA.
Mooney
Duane T
DT
Clark-Scannell
Gretchen
G
Tong
Tony T-H
TT
Watson
Jeffrey
J
Hagen
Tory M
TM
Stevens
Jan F
JF
Maier
Claudia S
CS
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
C456341:(4-iodobutyl)triphenylphosphonium; D007782:Lactoglobulins; D024101:Mitochondrial Proteins; D009943:Organophosphorus Compounds; D010455:Peptides; D013438:Sulfhydryl Compounds; D013879:Thioredoxins