|Abstract or Summary
- Dysfunction of mitochondria has been linked to aging and the pathogenesis of many degenerative diseases. Aside from their primary function in energy production, mitochondria are considered as a major source and target of reactive oxygen and nitrogen species (ROS/RNS) in cells as well. The mitochondrial thiol proteome is a subset of proteins inside mitochondria particularly vulnerable to oxidative modifications often caused by ROS/RNS due to the high reactivity of thiol groups. In this dissertation, we used chemoselective labeling approaches to target mitochondrial thiol proteomes with two distinct purposes. In the first part of our study, a mitochondria-targeted cationic sulfhydryl reagent, (4-iodobutyl)triphenyl- phosphonium (IBTP), was used to label the protein thiols. This study was focused on the investigation of the tandem mass spectrometric behaviors of the thiol peptides modified by IBTP. In the latter part of the dissertation, we tested the hypothesis that the thiol proteomes of cardiac mitochondria undergo chemical changes as a consequence of aging. The thiol-reactive isotope-coded affinity tags (ICATs) were utilized to estimate the possible age-related impacts on the relative abundances of protein thiols in two mitochondrial subpopulations present in rat myocardium, respectively.
Fixed charge chemical modifications on peptides and proteins can have significant effects on the ion fragmentation behaviors in tandem mass spectrometry (MS/MS). The MS/MS fragmentation behavior of butyltriphenylphosphonium (BTP)-modified peptides was evaluated via the comparison to their carbamidomethylated (CAM) analogues using a quadrupole ion trap mass spectrometer (LCQ) and/or a quadrupole time-of-flight (QTOF) instrument under conditions of low energy collision-induced dissociation (CID). Besides the expected higher charge states observed in peptides and fragment ions containing the BTP moiety, the charged BTP group also had a significant effect on the amide bond fragmentation products of modified cysteine-containing peptides. The presence of a phosphonium ion was speculated to reduce the tendency for the protonation of the proximal amide bonds in the peptide backbones, and consequently decrease the product ion abundances at the corresponding cleavage sites when compared to those from the CAM-modified derivatives. This effect was particularly noticeable when an Xxx-Pro bond was in the vicinity of a BTP group. Calculations indicated that proton affinities were generally 20-50 kcal/mol lower for BTP-modified peptides in contrast to the respective unmodified or CAM-modified analogues.
In the latter part of the dissertation, we focused on the evaluation of age-related effects on protein thiol abundances in cardiac subsarcolemmal and interfibrillar mitochondria (SSM/IFM). A total of 243 cysteine residues from 115 proteins in SSM and 149 cysteine sites from 65 proteins in IFM were identified from both young and old rats using ICAT labeling approach, respectively. Significant age-related differences (p-value < 0.05) in protein thiol abundances were only observed for five proteins from IFM. These proteins were associated with the functional pathways of the oxidative phosphorylation (NDUV1 and QCR1), the tricarboxylic acid cycle (MDHM and ODPB) and fatty acid beta-oxidation (ACSL1). Additionally, five other proteins in SSM and two proteins in IFM were detected with potentially significant age-related effects (0.05 < p-value < 0.1) with regard to their respective protein thiol abundances, however, no common protein was identified from both subpopulations. These results demonstrated the differences in age-related effects on protein thiol abundances between cardiac SSM and IFM.
Further, the reactivities of cysteine residues, identified in the current ICAT reaction, were evaluated and sorted into high (78 in SSM vs. 47 in IFM), moderate (30 vs. 32) and low (135 vs. 70) labeling frequency groups. Thirty eight thiol groups were also identified from other cysteine-targeted studies conducted in our group, i.e., Michael addition by 2-alkenals and disulfide exchange via glutathionylation. The comparative results from these three studies revealed eight cysteine residues as the common modification sites targeted by three distinct reaction mechanisms. This resulted in the identification of candidates of vulnerable cysteine residues in the aging heart mitochondria.