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.

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

Analytical separations in conjunction with powerful detection methods make possible the quantitation of large and small molecules important in environmental science, food science, pharmaceutical, and medical matrices. Many of these separations utilize instrumental methods such as capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) to alleviate the complexity of these various matrices. Fluorescence, laser-induced fluorescence (LIF), evaporative light scattering detection (ELSD), and mass spectrometry (MS) are detection methods commonly employed in conjunction with these separation strategies. Three distinct projects to develop and exploit such analytical technologies are described. First, few biological molecules are natively fluorescent, so they often must be labeled with a fluorescent tag in order to be detected via LIF. Furthermore, protein separations using CE-LIF may be complicated by protein interactions with the negatively charged surface of the inner capillary wall, arising from the ionization of surface silanol groups. Presented here is a method for quantitation of protein-dye complexes using a self assembled, surfactant-coated capillary and on-column protein labeling with a non-covalent squarylium dye, Red-1c. Second, seasonal sugar levels are relevant to the relationship between winter anthocyanin production and drought stress in angiosperm evergreen species, and so the development of a method for rapid analysis of sugars in seasonal tree leaf tissue is needed. An HPLC-ELSD method was developed for the quantitation of three sugars, glucose, fructose, and sucrose. The method was subjected to standard addition validation and was shown to provide precise and accurate measurement of all three analytes in real samples. Third, in addition to their many uses as solvents, ionic liquids (IL) have been utilized as highly effective substrates for complexation and subsequent quantitation of anions by electrospray ionization mass spectrometry (ESI-MS) and high performance liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). The present work provides an alternative method utilizing capillary electrophoresis coupled with electrospray ionization mass spectrometry (CE-ESI-MS). This method offers advantages over LC-MS in that CE, in general, provides shorter separation times, requires less reagent and solvent, and needs less sample volume. Mono- and divalent anionic analytes, were complexed with di- and trivalent cationic liquids. The complexation reaction was undertaken in a variety of modes, including pre-column, on-column and post-column, and the resulting complexes, which were positively charged and of higher mass than the native analytes, were subsequently analyzed by MS. This method allowed for greater sensitivity than could be achieved by direct detection.

The first protocols book, Free Radical and Antioxidant Protocols (1) was published in late 1998. Sections were divided into three parts, covering selected biochemical techniques for measuring oxidative stress, antioxidant (AOX) activity, and combined applications. In choosing the 40 methods to be included in that book, I realized there were considerably more of equal value than that which we could have presented in a single volume. To produce a comprehensive resource, this book and a third are being compiled to expand coverage of the field. A summary of papers (2) published on this important subject emphasizes the continuing rapid growth in oxidative stress investigations relating to our understanding of biochemical reactions, their relevance to pathophysiological mechanisms, how disease may arise, and how therapeutic intervention may be achieved(3). Although there is some overlap between the categories, the ana- sis shown below illustrates where current studies are concentrated and are almost evenly distributed between free radicals and AOX. Over the last 4 yr, there has been a 55% increase in the number of papers published in the area.

Selection of the HPLC Method in Chemical Analysis serves as a practical guide to users of high-performance liquid chromatography and provides criteria for method selection, development, and validation. High-performance liquid chromatography (HPLC) is the most common analytical technique currently practiced in chemistry. However, the process of finding the appropriate information for a particular analytical project requires significant effort and pre-existent knowledge in the field. Further, sorting through the wealth of published data and literature takes both time and effort away from the critical aspects of HPLC method selection. For the first time, a systematic approach for sorting through the available information and reviewing critically the up-to-date progress in HPLC for selecting a specific analysis is available in a single book. Selection of the HPLC Method in Chemical Analysis is an inclusive go-to reference for HPLC method selection, development, and validation. - Addresses the various aspects of practice and instrumentation needed to obtain reliable HPLC analysis results - Leads researchers to the best choice of an HPLC method from the overabundance of information existent in the field - Provides criteria for HPLC method selection, development, and validation - Authored by world-renowned HPLC experts who have more than 60 years of combined experience in the field