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New Approaches For Quantitative Analysis Of Glycopeptides Using Liquid Chromatography Mass Spectrometry
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Book Synopsis New Approaches for Quantitative Analysis of Glycopeptides Using Liquid Chromatography-mass Spectrometry by : Yining Huang
Download or read book New Approaches for Quantitative Analysis of Glycopeptides Using Liquid Chromatography-mass Spectrometry written by Yining Huang and published by . This book was released on 2016 with total page 250 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Mass Spectrometry–Based Glycoproteomics and Its Clinic Application by : Haojie Lu
Download or read book Mass Spectrometry–Based Glycoproteomics and Its Clinic Application written by Haojie Lu and published by CRC Press. This book was released on 2021-09-20 with total page 216 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Book Synopsis Advancing Mass Spectrometry Methods for Glycosylation Analysis and Their Application to Disease-related Glyco-alteration Study by : Zhengwei (Tony) Chen
Download or read book Advancing Mass Spectrometry Methods for Glycosylation Analysis and Their Application to Disease-related Glyco-alteration Study written by Zhengwei (Tony) Chen and published by . This book was released on 2018 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry (MS) has evolved as a powerful tool for glycosylation analysis benefiting from its high speed, high resolution and sensitivity. It enables detailed structure characterization of glycans, glycosylation site and the peptide sequences of complex biological samples in a high-throughput. This dissertation is dedicated to developing MS-based analytical methods for the structure characterization and quantitative analysis of both released glycans and intact glycopeptides utilizing a bottom-up approach. We started by developing a hydrophilic interaction liquid chromatography (HILIC) coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) platform for improved quantitative analysis of N-glycans compared to MALDI-MS. Faced with the challenges of N-glycan structure complexity, capillary electrophoresis (CE) emerges as a robust separation technique affording very small amount of sample consumption, fast separation speed, and high resolving power. Therefore, we developed a CE-ESI-MS/MS based 6-plex aminoxy tandem mass tag (TMT) labeling method for quantitative analysis of protein N-glycosylation analysis from human serum, providing the ability of N-glycan quantitation across up to six samples. Improved resolution and quantification accuracy of the labeled human milk oligosaccharides (HMOs) isomers was also achieved by coupling CE with traveling wave ion mobility (TWIM)-CID-MS/MS. The advantage of the glycan-focused approach (glycomics) is that the wealthy structure information and glycan isomer separation could be achieved; however, this approach resulted in loss of the glycosylation site-specific information. Therefore, we developed a powerful glycoproteomics workflow for system-wide structure characterization and quantitation of intact glycopeptides. Improvements of this workflow include improved glycoprotein extraction, sequential enrichment strategies for improved N-/O- glycopeptide enrichment, hybrid novel fragmentation technique electron transfer and higher-energy collision dissociation (EThcD), multiplex quantitation enabled by our custom-made isobaric N, N-dimethyl leucine (DiLeu) tags, and automated FDR-based large-scale data analysis by Byonic. We have successfully applied this workflow to analyze complex biological samples for disease-related glyco-alteration study, including the glycosylation alteration in PKM2 knockout breast cancer cells vs. parental cells and glycosylation pattern alteration in AD patients.
Book Synopsis The Plasma Proteins by : Frank W Putnam
Download or read book The Plasma Proteins written by Frank W Putnam and published by Elsevier. This book was released on 2012-12-02 with total page 498 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Plasma Proteins: Structure, Function, and Genetic Control, Second Edition, Volume I is a systematic account of the structure, function, and genetic control of plasma proteins. Clinical relevance is introduced in terms of principles, with emphasis on human proteins. Animal proteins are also used as examples in some cases. Comprised of nine chapters, this volume begins with a historical background on plasma proteins, along with their nomenclature, characterization, and genetic markers. The primary structure and three-dimensional conformation of plasma proteins are also considered. The discussion then turns to the chemical, physical, and biological properties of various plasma proteins such as serum albumin, lipoproteins, and immunoglobulins. Subsequent chapters deal with protease inhibitors in plasma; purification, physical properties, chemical composition, and molecular structure of transferrin; biosynthesis and metabolism of serum lipoproteins; and physical, chemical, and functional properties of the proteins of the complement system. The final chapter is devoted to ?2-microglobulin, with particular reference to its purification and physical properties; chemical composition and structure; physiological function, biosynthesis, and catabolism; and presence and function in cell membranes. This monograph will be of interest to molecular biologists and biochemists.
Book Synopsis Ultra Performance Liquid Chromatography Mass Spectrometry by : Mu Naushad
Download or read book Ultra Performance Liquid Chromatography Mass Spectrometry written by Mu Naushad and published by CRC Press. This book was released on 2014-03-18 with total page 464 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book presents a unique collection of up-to-date UPLC-MS/MS (ultra performance liquid chromatography-tandem mass spectrometric) methods for the separation and quantitative determination of pesticides, capsaicinoids, heterocyclic amines, aflatoxin, perfluorochemicals, acrylamide, procyanidins and alkaloids, lactose content, phenolic compounds, vitamins, and aroma and flavor compounds in a wide variety of foods and food products. With contributions by experts in interdisciplinary fields, this reference offers practical information for readers in research and development, production, and routing analysis of foods and food products.
Book Synopsis Novel HILIC-MS Strategies for Retention Prediction and Quantitative Analysis of Glycomic and Glycoproteomic Analytes by : Emily Mavreen Mize
Download or read book Novel HILIC-MS Strategies for Retention Prediction and Quantitative Analysis of Glycomic and Glycoproteomic Analytes written by Emily Mavreen Mize and published by . This book was released on 2017 with total page 230 pages. Available in PDF, EPUB and Kindle. Book excerpt: Liquid chromatography mass spectrometry (LC-MS) is a powerful method for glycomic and glycoproteomic research. Many recent advances in research have served to raise awareness that the ability to ascertain more refined linkage and position information is of vital importance, as structure is proving to be of critical importance to the function of a glycan. Even the smallest of changes in linkage can produce a significant change in function, therefore it is essential that analytical methods be able to reliably characterize glycans and glycoproteins. Hydrophilic interaction liquid chromatography (HILIC) is a relatively recent addition to LC methods, but has already begun to gain favor for glycan and glycoprotein analysis owing to the improvement in effective separation, particularly for isomeric and isobaric analytes. The purpose of this work is to improve analytical procedures for glycans and glycoproteins, and to that end explores several aspects of LC-MS analysis in order to enhance current methodologies. One project describes the impact degrees of sialylation has on adduct formation and the influence this has on relative and absolute quantitation via HILIC-MS analysis, demonstrating a significant increase in ammonium adduct formation in particular as the number of sialic acids increased. Quantitation via HILIC-MS with and without the use of an 15N isotopically labeled internal standard is another project detailed herein, which showed a significantly improved correlation to expected trends when the internal standard was used. The ability to partially analyze samples by way of an internal standard without compromising results was also explored, as were the consequences of incomplete analysis when an internal standard was not utilized. The development of a HILIC retention prediction model for released and procainamide labeled N-linked glycans is detailed in depth, and is shown to be capable of accurately predicting retention and differentiating between isomeric and isobaric structures. An additional retention prediction model developed for intact glycopeptides is also described, which is capable of accurate retention predictions for isomeric and isobaric glycoforms. These models demonstrated high correlation coefficients between experimental and calculated values, and were proven effective with changes to instruments and experimental parameters.
Book Synopsis Essentials of Glycobiology by : Ajit Varki
Download or read book Essentials of Glycobiology written by Ajit Varki and published by CSHL Press. This book was released on 1999 with total page 694 pages. Available in PDF, EPUB and Kindle. Book excerpt: Sugar chains (glycans) are often attached to proteins and lipids and have multiple roles in the organization and function of all organisms. "Essentials of Glycobiology" describes their biogenesis and function and offers a useful gateway to the understanding of glycans.
Book Synopsis Systems Biology and Computational Proteomics by : Trey Ideker
Download or read book Systems Biology and Computational Proteomics written by Trey Ideker and published by Springer. This book was released on 2007-09-19 with total page 138 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book constitutes the thoroughly refereed post-proceedings of two joint RECOMB 2006 satellite events: the Second Annual Workshop on Systems Biology, RSB 2006, and the First Biennial Workshop on Computational Proteomics, RCP 2006, held in San Diego, CA, USA in December 2006. The papers cover various aspects of systems biology and explore the use of computational mass spectrometry in various proteomic applications.
Book Synopsis Advancing Qualitative and Quantitative Proteomics/peptidomics Via Development of Novel Mass Spectrometry-based Approaches by : Qing Yu
Download or read book Advancing Qualitative and Quantitative Proteomics/peptidomics Via Development of Novel Mass Spectrometry-based Approaches written by Qing Yu and published by . This book was released on 2017 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Liquid chromatography mass spectrometry (LC-MS) has evolved as a powerful tool in protein and peptide identification and quantitation. It has the capability to monitor thousands of proteins and peptides simultaneously and therefore is a critical component in the study of underlying biological mechanisms and the discovery of novel therapeutic targets. This dissertation is devoted to the development and application of novel LC-MS based techniques to characterize proteomics and peptidomics qualitatively and quantitatively by incorporating various sample preparation strategies, novel MS methods and isobaric labeling technology. A portion of this dissertation describes the design and evaluation of several dimethylated amino acid tags and their potentials to be used as isobaric labeling reagents for protein and peptide quantitation. Their utilities are further demonstrated through their application in the study of global proteomic changes in models of restenosis. Although isobaric tags are originally designed for higher-energy collisional dissociation (HCD), we expand their use by enabling them to be coupled with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation. The benefits of using such strategy in quantitative proteomics, especially phosphorylation studies, are recounted in this dissertation. Quantitative strategies presented in this dissertation are complemented by advances in MS-based protein and endogenous peptide sequencing methodologies. We established a workflow to sequence intact glycopeptides, enabling detailed investigation of complex protein glycosylation and its microheterogeneity. The application of this improved workflow revealed a hyperglycosylation trend from day 0 to day 7 post angioplasty along the progression of restenosis. Similarly, by employing a further modified strategy, this dissertation documented the discovery of O-linked glycosylation on endogenous signaling peptides, specifically insulin and its related peptide hormones. The significance of such discovery can be critical toward diabetes research and treatment. Meanwhile, this dissertation also describes the strategy to characterize large neuropeptides in a species with no genome information, which can be greatly useful for future neuropeptide research. Furthermore, we employ ion mobility MS (IM-MS) to study peptide structures and conformations, leading to discovery of D-amino acid containing peptides based on subtle changes in gas-phase conformations despite identical mass-to-charge ratio. The IM-MS strategy enables the study of this unique isobaric post-translational modification (PTM) in neuropeptides occurring via amino acid isomerization. Overall, this dissertation research not only improves upon isobaric labeling technique by exploring novel chemical tags and new fragmentation methods, but also presents a useful platform to enable in-depth investigation on complex PTMs and subtle conformational differences. Collectively, we expect that the technology advancements presented in this work will enable broad applications in various biological systems and lead to improved understanding of underlying molecular mechanisms.
Book Synopsis Mass Spectrometry-Based Chemical Proteomics by : W. Andy Tao
Download or read book Mass Spectrometry-Based Chemical Proteomics written by W. Andy Tao and published by John Wiley & Sons. This book was released on 2019-07-10 with total page 448 pages. Available in PDF, EPUB and Kindle. Book excerpt: PROVIDES STRATEGIES AND CONCEPTS FOR UNDERSTANDING CHEMICAL PROTEOMICS, AND ANALYZING PROTEIN FUNCTIONS, MODIFICATIONS, AND INTERACTIONS—EMPHASIZING MASS SPECTROMETRY THROUGHOUT Covering mass spectrometry for chemical proteomics, this book helps readers understand analytical strategies behind protein functions, their modifications and interactions, and applications in drug discovery. It provides a basic overview and presents concepts in chemical proteomics through three angles: Strategies, Technical Advances, and Applications. Chapters cover those many technical advances and applications in drug discovery, from target identification to validation and potential treatments. The first section of Mass Spectrometry-Based Chemical Proteomics starts by reviewing basic methods and recent advances in mass spectrometry for proteomics, including shotgun proteomics, quantitative proteomics, and data analyses. The next section covers a variety of techniques and strategies coupling chemical probes to MS-based proteomics to provide functional insights into the proteome. In the last section, it focuses on using chemical strategies to study protein post-translational modifications and high-order structures. Summarizes chemical proteomics, up-to-date concepts, analysis, and target validation Covers fundamentals and strategies, including the profiling of enzyme activities and protein-drug interactions Explains technical advances in the field and describes on shotgun proteomics, quantitative proteomics, and corresponding methods of software and database usage for proteomics Includes a wide variety of applications in drug discovery, from kinase inhibitors and intracellular drug targets to the chemoproteomics analysis of natural products Addresses an important tool in small molecule drug discovery, appealing to both academia and the pharmaceutical industry Mass Spectrometry-Based Chemical Proteomics is an excellent source of information for readers in both academia and industry in a variety of fields, including pharmaceutical sciences, drug discovery, molecular biology, bioinformatics, and analytical sciences.
Book Synopsis The Determination of Glycopeptides and Glycoproteins by Liquid Chromatography/mass Spectrometry and Tandem Mass Spectrometry by : James Joseph Conboy
Download or read book The Determination of Glycopeptides and Glycoproteins by Liquid Chromatography/mass Spectrometry and Tandem Mass Spectrometry written by James Joseph Conboy and published by . This book was released on 1992 with total page 350 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Standing in the Way of Profiling Depth by : Daniel Graham Delafield
Download or read book Standing in the Way of Profiling Depth written by Daniel Graham Delafield and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: This thesis outlines research focusing on the development and application of chromatography separation and mass spectrometry data acquisition methods aimed at improving profiling depth in proteomic analyses. This work is briefly outlined and explained in Chapter 1, highlighting how reliance on common analytical methodologies inherently limits our view of the proteome. Chapter 2 details the initial reports of utilizing porous graphitic carbon (PGC) chromatography as an alternative to reversed-phase liquid chromatography (RPLC). This report describes demonstrable improvements in peptide, glycopeptide, and protein identifications while highlighting the various analytical advantages seen over traditional separations. In this preliminary work, we observed elevated column temperatures imparted potentially detrimental effects of various glycopeptides, a phenomenon explored in Chapter 3. This following report provides heuristic guidance in PGC-based glycoproteomic analyses and details how column temperature must be optimized to suit the given analytical need. Chapter 4 provides a topical overview of quantitative approaches in glycan and glycopeptide analyses, providing meaningful consideration and comparison of methodologies most appropriate for use in future glycoproteomic analyses. In Chapter 5, we return to PGC separations with a specific focus on further detailing the benefits found in proteomic analyses. This report highlights substantial improvements in the number of peptide and protein identifications compared to what was seen previously and highlights the breadth of information lost during routine RPLC analyses. Chapter 6 departs from chromatographic separation and turns attention towards quantitative mass spectrometry (MS) methods useful for biological discovery. The knowledge and information garnered from this report informed the experimental design used in Chapter 7, which describes the employment of data-independent acquisition (DIA)-MS to study a novel, progressive prostate cancer cell model. Here we quantified 6,614 proteins across 9 biological samples, finding 1,242 to be significantly dysregulated in malignant cancer phenotypes. Thes proteins demonstrate potential for disease diagnosis, phenotypic stratification, and therapeutic targeting. In Chapter 8 we expand on the utility of DIA-MS, detailing the ability to reuse and repurpose prior proteomic measurements for enhanced biomolecular identification. We apply this workflow to the analysis of neurological disorder patient cohorts, revealing 1,642 dysregulated proteins that speak to the biomolecular organization related to Alzheimer's Disease. Chapter 9 describes new and emerging ion mobility (IM)-based analytical modalities and discuss their capacity for biomolecular interrogation and structural analysis. Given instrumentation of this kind is not utilized in the works preceding, this report explains potential advantages and necessary considerations, should this technology become of interest. Finally, we conclude with Chapter 10, briefly discussing the various investigations that may follow this body of work.
Download or read book The Glycome written by Adeel Malik and published by CRC Press. This book was released on 2021-07-07 with total page 305 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume provides a comprehensive understanding of the enigmatic identity of the glycome, a complex but important area of research that has been largely ignored due to its complexity. The authors thoroughly deal with almost all aspects of the glycome, i.e., elucidation of the glycan identity enigma and its role in regulation of the cellular process, and in disease etiology. The book bridges the knowledge gap in understanding the glycome, from being a cell signature to its applications in disease etiology. In addition, it details many of the major insights regarding the possible role of the glycome in various diseases as a therapeutic marker. The book systematically covers the major aspects of the glycome, including the significance of substituting the diverse monosaccharide units to glycoproteins, the role of glycans in disease pathologies, and the challenges and advances in glycobiology. The authors stress the significance and huge encoding power of carbohydrates as well as provide helpful insights in framing the bigger picture. The Glycome: Understanding the Diversity and Complexity of Glycobiology details state-of-the-art developments and emerging challenges of glycome biology, which are going to be key areas of future research, not only in the glycobiology field but also in pharmaceutics.
Book Synopsis Advancing Mass Spectrometry Methods for Quantification and Characterization of Peptides and Proteins in Complex Biological Samples by :
Download or read book Advancing Mass Spectrometry Methods for Quantification and Characterization of Peptides and Proteins in Complex Biological Samples written by and published by . This book was released on 2015 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Protein quantification using liquid chromatography mass spectrometry (LC-MS) is a critical component in the discovery and verification of protein candidate biomarkers. Label-free and label-based quantitative methods are both commonly employed, and both have advantages and disadvantages. Label-based methods are capable of analyzing relative protein abundances from multiple samples in one LC-MS experiment, thus enabling high throughput analysis. Unfortunately, the high cost of commercial labels prevents many groups from including them in their proteomics workflows. Our group previously developed a cost-effective isobaric label set, DiLeu, through dimethylation of L-leucine isotopes. The DiLeu reagent performed comparably to commercial options, but multiple improvements and variations to this reagent were needed. For example, the multiplexing capacity of the original DiLeu reagent only permits four quantitative channels, necessitating development of a new label with greater throughput. A portion of this thesis is dedicated to the design, synthesis, and characterization of a new 8-plex DiLeu reagent. This dissertation also recounts the development of isotopic DiLeu (iDiLeu), a 5-plex mass difference variant of DiLeu that allows for researchers to quantify analyte peptide concentrations from a four-point calibration curve in one LC-MS run. One glaring omission in the development of noncommercial isobaric labels is that no protein candidate biomarkers have been discovered by them. Our work rectifies this oversight by identifying several potential biomarkers using custom-synthesized 4-plex DiLeu isobaric tags from the urine of men diagnosed with lower urinary tract symptoms (LUTS). Quantitative strategies presented in this dissertation are complemented by advances in our neuropeptidomics methodologies. In one project, we study the role of protein degradation in crustacean neuropeptidomics and apply a heat stabilization strategy to mitigate the number of protein fragments that interfere with neuropeptide identification. The final project investigates an alternative method to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) in crab neuropeptide and lipid imaging. This technique, nanostructure initiator mass spectrometry (NIMS), was unable to ionize neuropeptides in C. borealis brain tissue despite its capability to ionize lipids, showing that sometimes traditional methods like MALDI MSI remain advantageous over more recent but less vetted techniques. The future outlook for our LUTS collaboration and quantitative tag projects is positive because of the new directions still being explored. Overall, the new quantitative techniques developed in this dissertation and the advances in neuropeptide identification will benefit research groups interested in cost-effective label-based quantification methods and neuropeptide characterization in LC-MS and MSI studies.
Book Synopsis Antibody Glycosylation by : Marija Pezer
Download or read book Antibody Glycosylation written by Marija Pezer and published by Springer Nature. This book was released on 2021-10-22 with total page 588 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book summarizes recent advances in antibody glycosylation research. Covering major topics relevant for immunoglobulin glycosylation - analytical methods, biosynthesis and regulation, modulation of effector functions - it provides new perspectives for research and development in the field of therapeutic antibodies, biomarkers, vaccinations, and immunotherapy. Glycans attached to both variable and constant regions of antibodies are known to affect the antibody conformation, stability, and effector functions. Although it focuses on immunoglobulin G (IgG), the most explored antibody in this context, and unravels the natural phenomena resulting from the mixture of IgG glycovariants present in the human body, the book also discusses other classes of human immunoglobulins, as well as immunoglobulins produced in other species and production systems. Further, it reviews the glycoanalytical methods applied to antibodies and addresses a range of less commonly explored topics, such as automatization and bioinformatics aspects of high-throughput antibody glycosylation analysis. Lastly, the book highlights application areas ranging from the ones already benefitting from antibody glycoengineering (such as monoclonal antibody production), to those still in the research stages (such as exploration of antibody glycosylation as a clinical or biological age biomarker), and the potential use of antibody glycosylation in the optimization of vaccine production and immunization protocols. Summarizing the current knowledge on the broad topic of antibody glycosylation and its therapeutic and biomarker potential, this book will appeal to a wide biomedical readership in academia and industry alike. Chapter 4 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.
Book Synopsis Determination and Quantitation of Protein Site-specific Glycosylation Using Mass Spectrometry by : Jincui Huang
Download or read book Determination and Quantitation of Protein Site-specific Glycosylation Using Mass Spectrometry written by Jincui Huang and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Glycosylation is one of the most common but complicated post-translational modifications (PTMs) of proteins. Estimates vary widely, but a common assessment is that about 70% of eukaryotic proteins are modified by some type of glycan. It is well known that glycosylation plays important roles in protein folding, protein stability, cell-cell signaling, and cell-cell adhesion. Over the past 30 years, insight into the biological roles of glycan modification has grown dramatically, yet the field of glycosylation has often struggled due to the inadequacies of accessible analytical methods as well as the inherent complexity of protein glycosylation. This dissertation addresses the current techniques to study comprehensive protein glycosylation including conventional glycomics method, site-specific glycosylation via non-specific protease and specific protease digestion. The effectiveness of the different methodologies for specific analysis such as the profiling of glycan-heterogeneity or quantification of site-specific glycosylation is described. The first chapter of this dissertation provides an overview of the analysis of protein glycosylation using liquid chromatography - mass spectrometry (LC-MS). This includes an overview of the technology and methodology in glycomics and glycoproteomics analyses. In addition, a brief overview is provided of commonly used stationary phases in LC separation as well as various mass analyzers used for glycomics and glycoproteomics analysis. Particular emphasis is placed on methods and technologies that provide site-specific glycan heterogeneity and quantitation.The second chapter describes the glycan site mapping of human milk secretory immunoglobulin A (sIgA) based on a combination of non-specific protease digestion and LC-MS. sIgA, a protein complex composed of four polypeptides with potentially more than 20 glycosites, is digested via non-specific protease to create glycopeptides with small peptide tags, thereby enabling the porous graphitized carbon (PGC) based LC separation and MS profiling of all the glycopeptides to reveal site-specific information. With this method, site-specific heterogeneity and relative quantitation of each glycan composition and the occupation at each site of sIgA was determined. The third chapter describes the development of an analytical method using multiple reaction monitoring (MRM) for the quantitation of milk proteins and their glycoforms in a rapid throughput manner. The goal was accomplished via UPLC separation of peptides and glycopeptides from trypsin digestion and MRM QqQ quantitation of these peptides and glycopeptides. The absolute concentration of proteins was calculated from the linear fit equation of the corresponding standard protein response. Site-specific quantitation of glycoforms from each protein is achieved by the normalization to the corresponding protein abundance to remove the contribution of protein concentration. This method was verified with three human milk samples to provide quantitation of proteins and their glycoforms, which yielded a low limit of quantitation, high reproducibility, and high sensitivity. The fourth chapter presents a comprehensive glycosylation analysis with a conventional glycomics, site-specific glycoproteomics, and proteomics methods. The combination of different approaches was used for glycosylation characterization of high density lipoproteins (HDL), which provided a simultaneous and detailed analysis of glycoproteins (protein ID, both N- and O-glycan composition, as well as site-specific glycopeptide composition) and gangliosides (both the glycan and ceramide portion).
Book Synopsis Capillary Gel Electrophoresis by : Andras Guttman
Download or read book Capillary Gel Electrophoresis written by Andras Guttman and published by Newnes. This book was released on 2021-12-04 with total page 391 pages. Available in PDF, EPUB and Kindle. Book excerpt: Capillary Gel Electrophoresis and Related Microseparation Techniques covers all theoretical and practical aspects of capillary gel electrophoresis. It also provides an excellent overview of the key application areas of nucleic acid, protein and complex carbohydrate analysis, affinity-based methodologies, micropreparative aspects and related microseparation methods. It not only gives readers a better understanding of how to utilize this technology, but also provides insights into how to determine which method will provide the best technical solutions to particular problems. This book can also serve as a textbook for undergraduate and graduate courses in analytical chemistry, analytical biochemistry, molecular biology and biotechnology courses. Covers all theoretical and practical aspects of capillary gel electrophoresis Excellent overview of the key applications of nucleic acid, protein and complex carbohydrate analysis, affinity-based methodologies, micropreparative aspects and related microseparation methods Teaches readers how to use the technology and select methods that are ideal for fundamental problems Can serve as a textbook for undergraduate and graduate courses in analytical chemistry, analytical biochemistry, molecular biology and biotechnology courses
