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.

As one of the most extensive and important protein post-translational modifications, glycosylation plays a vital role in regulating organisms and is associated with various physiological and pathological processes. Recently, researchers have focused on the need to characterize protein glycosylation sites, structures, and their degree of modification, to better understand their biological functions while also looking for potential biomarkers for diagnosis and treatment of disease. Mass spectrometry (MS) is one of the most powerful tools used to study biomolecules including glycoproteins and glycans. With the continuous development of glycoproteomics and glycomics based on MS analysis, more techniques have evolved and contribute to understanding the structure and function of glycoproteins and glycans. This book reviews advancements achieved in MS-based glycoproteomic analysis, including a wide range of analytical methodologies and strategies involved in selective enrichment; as well as qualitative, quantitative, and data analysis, together with their clinical applications. Significant examples are discussed to illustrate the principles, laboratory protocols, and advice for key implementation to ensure successful results. Mass Spectrometry–Based Glycoproteomics and Its Clinic Application will serve as a valuable resource to elucidate new techniques and their applications for students, postdocs, and researchers working in proteomics, glycoscience, analytical chemistry, biochemistry, and clinical medicine. Editor: Haojie Lu is a professor at Fudan University, specializing in proteomics based on mass spectrometry with particular emphasis on novel technologies for separation and identification of low-abundant proteins and post-translationally modified proteins (including glycosylation), as well as relative and absolute quantification methods for proteomics.

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 book highlights current approaches and future trends in the use of mass spectrometry to characterize protein therapies. As one of the most frequently utilized analytical techniques in pharmaceutical research and development, mass spectrometry has been widely used in the characterization of protein therapeutics due to its analytical sensitivity, selectivity, and specificity. This book begins with an overview of mass spectrometry techniques as related to the analysis of protein therapeutics, structural identification strategies, quantitative approaches, followed by studies involving characterization of process related protein drug impurities/degradants, metabolites, higher order structures of protein therapeutics. Both general practitioners in pharmaceutical research and specialists in analytical sciences will benefit from this book that details step-by-step approaches and new strategies to solve challenging problems related to protein therapeutics research and development.

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