Journal
JOURNAL OF PROTEOME RESEARCH
Volume 4, Issue 4, Pages 1403-1412Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr050078k
Keywords
isotope-coded labeling; mass spectrometry; mitochondria; thiol modifications
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Funding
- NIA NIH HHS [AG 025372] Funding Source: Medline
- NIEHS NIH HHS [P30 ES 00210] Funding Source: Medline
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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.
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