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
This volume, along with its companion (volume 474), presents methods and protocols dealing with thiol oxidation-reduction reactions and their implications as they relate to cell signaling. The critically acclaimed laboratory standard for 40 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Over 450 volumes have been published to date, and much of the material is relevant even today--truly an essential publication for researchers in all fields of life sciences. Along with companion volume, provides a full overview of techniques necessary to the study of thiol redox in relation to cell signaling Gathers tried and tested techniques from global labs, offering both new and tried-and-true methods Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines
Oxidative damage appears to play a central role in the development of a wide range of tissue pathology, including neurodegenerative disease, drug side-effects, xenobiotic toxicity, carcinogenesis, and the aging process, to name just a few. Because of the centrality of oxidative processes to normal and abnormal tissue function, it has become imperative to develop appropriate analytical techniques to facilitate the quantitation of significant reactants. Without advances in methodology, corresponding advances in our knowledge of underlying biochemical events will be necessarily limited. Drs. Hensley and Floyd have done an outstanding job of assembling the work of world-class experts into Methods in Biological Oxidative Stress. The contributors have presented concise, yet thorough, descriptions of the state-of-the-art methods that any investigator working in the field needs to access. Mannfred A. Hollinger v Preface Free radicals and reactive oxidizing agents were once ignored as biochemical entities not worth close scrutiny, but are now recognized as causes or contributing factors in dozens, if not hundreds, of disease states. In addition, free radical metabolisms of xenobiotics have become increasingly important to pharmacologists. Accordingly, the need has arisen to accurately quantify reactive oxygen species and their byproducts. Methods in Biological Oxidative Stress is practical in scope, providing the details of up-to-date techniques for measuring oxidative stress and detecting oxidizing agents both in vitro and in vivo. The contributors are recognized experts in the field of oxidative stress who have developed novel strategies for studying biological oxidations.
Oxidants, Antioxidants and Impact of the Oxidative Status in Male Reproduction is an essential reference for fertility practitioners and research and laboratory professionals interested in learning about the role of reactive oxygen species in sperm physiology and pathology. The book focuses on unravelling the pathophysiology of oxidative stress mediated male infertility, recruiting top researchers and clinicians to contribute chapters. This collection of expertise delves into the physico-chemical aspects of oxidative stress, including a new focus on reductive stress. Furthermore, the inclusion of clinical techniques to determine oxidative stress and the OMICS of reductive oxidative stress are also included. This is a must-have reference in the area of oxidative stress and male reproductive function. Offers comprehensive information on oxidative stress and its role in male reproduction, including new therapeutic approaches Deals with current approaches to oxidative stress using OMICS platform“/li> Designed for fertility practitioners, reproductive researchers, and laboratory professionals interested in learning about the role of reactive oxygen species in sperm physiology and pathology
Accumulated evidence indicates oxidative stress plays important roles in disease and aging. Under oxidative stress, lipid peroxidation (LPO) leads to reactive carbonyl species (RCS) that can modify a wide range of biomolecules including protein, DNA and carbohydrate. In this dissertation, we investigate the modification of two model proteins, human serum albumin (HSA) and aconitase (ACO), by the LPO-relevant a, b-unsaturated aldehydes, acrolein (ACR) and 4-hydroxy-2-nonenal (HNE). The investigation is focused on the characterization and quantification ACR and HNE addition to the model proteins. A correlation between HNE modification and ACO activity is also determined. These results provide insights into the impact of oxidative stress at the molecular level and are relevant to aging and disease states. We finally investigate protein carbonylation in ischemic mouse heart mitochondria, and develop a quantitative method for detecting carbonylated protein in this system. The research is based on liquid chromatography/mass spectrometry (LC/M.S.), Western Blots, and enzymatic assay.
"Oxidative stress" is used as the generic term describing the involve ment of reactive oxygen species in various human diseases. The scope of such a topic is becoming increasingly wide. The recent interest in radicals such as nitric oxide and the discovery of new mechanisms such as the effect of free radicals on redox sensitive proteins and genes are enlarging our understanding of the physiological role of free radicals. Oxidative stress is involved in numerous pathological. processes such as ageing, respiratory or cardiovascular diseases, cancer, neurological pathologies such as dementia or Parkinson's disease. It still remains difficult, however, to demonstrate by chemical measurement the in vivo production of free radicals and even more to realise their speciation. Therefore, the development of new tools and indicators is engrossing many researchers working in this field. Reliable indicators are abso lutely necessary not only to monitor the evolution of oxidative stress in patients but also to evaluate the efficiency of new antioxidant treat ments. The French Free radical club of Grenoble, the CERLIB has been involved for many years in the organisation of international training programs on methodology, in order to provide both theoretical and practical help to researchers from various countries. Such training sessions have been highly successful and participants value the oppor tunity to learn reliable techniques. This positive echo explains why the researchers of CERLIB decided, with the help of Prof. Dr. B. Kalyanaraman, to publish selected techniques on free radical re search.
In this, the post-genomic age, our knowledge of biological systems continues to expand and progress. As the research becomes more focused, so too does the data. Genomic research progresses to proteomics and brings us to a deeper understanding of the behavior and function of protein clusters. And now proteomics gives way to neuroproteomics as we beg
Oxidative Stress and Dietary Antioxidants in Neurological Diseases provides an overview of oxidative stress in neurological diseases and associated conditions, including behavioral aspects and the potentially therapeutic usage of natural antioxidants in the diet. The processes within the science of oxidative stress are described in concert with other processes, such as apoptosis, cell signaling, and receptor mediated responses. This approach recognizes that diseases are often multifactorial and oxidative stress is a single component of this. The book examines basic processes of oxidative stress—from molecular biology to whole organs—relative to cellular defense systems, and across a range of neurological diseases. Sections discuss antioxidants in foods, including plants and components of the diet, examining the underlying mechanisms associated with therapeutic potential and clinical applications. Although some of this material is exploratory or preclinical, it can provide the framework for further in-depth analysis or studies via well-designed clinical trials or the analysis of pathways, mechanisms, and components in order to devise new therapeutic strategies. Very often oxidative stress is a feature of neurological disease and associated conditions which either centers on or around molecular and cellular processes. Oxidative stress can also arise due to nutritional imbalance during a spectrum of timeframes before the onset of disease or during its development. Offers an overview of oxidative stress from molecular biology to whole organs Discusses the potentially therapeutic usage of natural antioxidants in the patient diet Provides the framework for further in-depth analysis or studies of potential treatments
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.
