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.

Oxidative stress can result in changes to many biomolecules and also affect their activities. We are interested in protein carbonylation, a type of unnatural oxidation which has been associated with numerous degenerative disease states and is also a consequence of the natural aging process. Protein carbonyls are stable species, but countless analytical barriers exist in terms of their identification. Thus, the main goal of this work was to develop and optimize analytical methods that could be used to help us better understand which, where, and how proteins are being carbonylated. Initial studies involved method validation for carbonylating, tagging, and enriching the model protein human serum albumin (HSA). We have developed a reproducible method of producing carbonylated protein in vitro in which HSA is treated with acrolein to carbonylate cysteines, histidines, and lysines. Protein carbonyls are compatible with various affinity labels and enrichment techniques. We strived to learn more about the efficiencies of various biotin affinity labels and avidin enrichment techniques using quantitative assays and mass spectrometry. Results showed a preference for different affinity labels based on their chemical properties and suggested that monomeric columns are selective for particular peptides. Most recently, method development and validation work was done involving a cleavable biotin tag that enables both enrichment and identification of protein carbonylation modification sites. This affinity tag offered the highest labeling efficiency of all tags tested in the past and greater coverage of modification sites than biotin hydrazide reagents. We applied our analytical methods to two sets of human blood samples. The first sample set was plasma taken from chronic kidney disease (CKD) patients. No carbonylation patterns were elucidated, but this project marked the beginning of blood analyses in which existing protocols were adapted to blood samples. The second sample set was serum/plasma taken from patients with traumatic injuries. We effectively applied our analytical methods to these sample sets and were able to visualize and quantitate temporal protein carbonylation patterns via Western blotting and iTRAQ-based mass spectrometry experiments. ProteoMiner experiments proved successful in that we were able to identify a larger and more diverse amount of carbonylated proteins via mass spectrometry.

Presents a wide variety of mass spectrometry methods used to explore structural mechanisms, protein dynamics and interactions between proteins. Preliminary chapters cover mass spectrometry methods for examining proteins and are then followed by chapters devoted to presenting very practical, how-to methods in a detailed way. Includes footprinting and plistex specifically, setting this book apart from the competition.

Presents Practical Applications of Mass Spectrometry for Protein Analysis and Covers Their Impact on Accelerating Drug Discovery and Development Covers both qualitative and quantitative aspects of Mass Spectrometry protein analysis in drug discovery Principles, Instrumentation, Technologies topics include MS of peptides, proteins, and ADCs , instrumentation in protein analysis, nanospray technology in MS protein analysis, and automation in MS protein analysis Details emerging areas from drug monitoring to patient care such as Identification and validation of biomarkers for cancer, targeted MS approaches for biomarker validation, biomarker discovery, and regulatory perspectives Brings together the most current advances in the mass spectrometry technology and related method in protein analysis

Protein carbonylation has attracted the interest of a great number of laboratories since the pioneering studies at the Earl Stadtman’s lab at NIH started in early 1980s. Since then, detecting protein carbonyls in oxidative stress situations became a highly efficient tool to uncover biomarkers of oxidative damage in normal and altered cell physiology. In this book, research groups from several areas of interest have contributed to update the knowledge regarding detection, analyses and identification of carbonylated proteins and the sites where these modifications occur. The scientific community will benefit from these reviews since they deal with specific, detailed technical approaches to study formation and detection of protein carbonyls. Moreover, the biological impact of such modifications in metabolic, physiologic and structural functions and, how these alterations can help understanding the downstream effects on cell function are discussed. Oxidative stress occurs in all living organisms and affects proteins and other macromolecules: Protein carbonylation is a measure of oxidative stress in biological systems Mass spectrometry, fluorescent labelling, antibody based detection, biotinylated protein selection and other methods for detecting protein carbonyls and modification sites in proteins are described Aging, neurodegenerative diseases, obstructive pulmonary diseases, malaria, cigarette smoke, adipose tissue and its relationship with protein carbonylation Direct oxidation, glycoxidation and modifications by lipid peroxidation products as protein carbonylation pathways Emerging methods for characterizing carbonylated protein networks and affected metabolic pathways

Covers all major modifications, including phosphorylation, glycosylation, acetylation, ubiquitination, sulfonation and and glycation Discussion of the chemistry behind each modification, along with key methods and references Contributions from some of the leading researchers in the field A valuable reference source for all laboratories undertaking proteomics, mass spectrometry and post-translational modification research

This book is designed to be a central text for young graduate students interested in mass spectrometry as it relates to the study of protein structure and function as well as proteomics. It is a definite must-have work for:- libraries at academic institutions with Master and Graduate programs in biochemistry, molecular biology, structural biology and proteomics- individual laboratories with interests covering these areas - libraries and individual laboratories in the pharmaceutical and biotechnology industries.*Serves as an essential reference to those working in the field*Incorporates the contributions of prominent experts *Features comprehensive coverage and a logical structure