Author: Suresh Narayanasamy

Publisher:

Published: 2014

Total Pages: 212

ISBN-13:

The importance of the role of oxidative stress in aging and numerous age-related diseases has become undeniable due to enormous weight of supporting evidence from the field of free radical biology. One of the most prominent oxidative modifications of proteins is the introduction of carbonyl groups. As with oxidative stress, protein carbonylation has been correlated with various disease states and natural aging. While protein carbonyls are relatively stable, reproducible detection and modification site localization are still challenging. This thesis details attempts to improve the analytical methodology used for the identification and quantitation of carbonylated proteins. We have studied the impact of exogenous and endogenous methods for increasing the flux of the progenitor reactive oxygen species, the superoxide radical anion, in Drosophila melanogaster using protein carbonylation measurements. Furthermore, we have applied the same methodology to plasma and serum obtained from trauma patients. Superoxide flux was manipulated first by administering Paraquat to increase generation rates throughout the cell and second through RNA interference knockdown of Mn or mitochondrial superoxide dismutase (Sod2) to decrease destruction rates in mitochondria. Protein carbonylation was measured since carbonyls are not present in the twenty canonical amino acids and are amenable to labeling and enrichment strategies (biotin hydrazide was used for labeling and streptavidin bead-immobilization for enrichment). On-bead digestion was used to release carbonylated protein peptides, which were then subjected to the iTRAQ mass spectrometry-based proteomics approach to obtain Paraquat-exposed and Sod2 knockdown versus control carbonylated protein relative abundance ratios. Western blotting and biotin quantitation assay approaches were also investigated. Considering the whole set of detected carbonylated proteins, lipid droplet and mitochondrial proteins were found to be particularly prevalent. By both western blotting and proteomics, Paraquat exposure resulted in a greater global increase in carbonylation than Sod2 knockdown. However, the Sod2 knockdown dataset included an interesting outlier, cytochrome c oxidase subunit Vb, which could shed light on the mechanism of superoxide generation or the Sod2 knockdown phenotype. An alternative to the iTRAQ quantitation approach, the label-free "Spectral counting" technique was also investigated, using the Paraquat-exposed versus control Drosophila melanogaster samples. Thus, we investigated the correlation between spectral count and iTRAQ-based quantitation. We observed good reproducibility for the iTRAQ approach but not for spectral counting. Comparing mitochondrial relative protein abundances, we found only a weak positive correlation between these two approaches. Discovery proteomics approaches that aim to identify biomarkers or signaling pathways associated with human diseases have gained much attention in recent years. Due to the minimally invasive nature of collecting blood, plasma proteomics has become a common means of studying the entire human proteome, even though cellular proteins leaking into the circulatory system are present at very low concentrations. Severe blunt trauma produces significant changes in leukocyte mRNA levels, corresponding to changes in expression for >80% of human genes. Thus, the early leukocyte genomic response involves simultaneously increasing expression of genes involved in the systemic inflammatory, innate immune, and compensatory anti-inflammatory responses, as well as with suppressing genes involved in adaptive immunity. Inflammation and oxidative stress are often correlated. Thus we analyzed the carbonylated protein proteome at multiple time points in the blood of patients that had endured traumatic injuries. First, temporal changes in carbonylated protein concentrations were measured. Second, the ProteoMiner affinity-ligand strategy was added to the procedure to increase proteome coverage through the depletion of high-abundance plasma proteins. This improved method resulted in the identification of greater numbers of low-abundance proteins. Since the sample size (n=2) was very small, no biological conclusion could be drawn; however, we have shown that measurements of changes in the carbonylated protein proteome over time are possible by this method. The hope is that protein carbonylation biomarkers could someday aid in predicting patient outcomes in clinical settings.