Highly Sensitive Qualitative and Quantitative Top-down Proteomics Using Capillary-zone Electrophoresis-electrospray Ionization-tandem Mass Spectrometry PDF Download
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Author: Rachele Anne Lubeckyj
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 152
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Book Description
Author: Rachele Anne Lubeckyj
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 152
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Book Description
Author: Christian Neusüß
Publisher: Springer Nature
ISBN: 1071624938
Category : Science
Languages : en
Pages : 264
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Book Description
This volume details aspects and applications of interfacing capillary electrophoresis (CE) with mass spectrometry (MS). Chapters guide readers through approaches based on different types of CE-MS interfaces such as (nano)sheath liquid, porous tip, and liquid junction, as well as various capillary coatings, and a broad range of applications including several top-down and bottom-up proteomic approaches. Additionally, a list of analyte targets was provided consisting of amphetamines, antibiotics, carbohydrates (including glycosaminoglycans and glycopeptides), enantiomers, extracellular matrix metabolites, monoclonal antibodies, and nanoparticles, and therefore covers numerous fields of applications such as pharmaceutical, biomedical, food, agrochemical, and environmental analysis. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. Authoritative and cutting-edge, Capillary Electrophoresis-Mass Spectrometry: Methods and Protocols aims to provide highly valuable information for both beginners and experts in the field be it students, technical staff, and scientists.
Author: Elijah Neal McCool
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 182
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Book Description
Proteomes are very complex with a large number of unique proteoforms spread across a wide concentration dynamic range. This means that an MS-based platform with highly efficient separation and highly sensitive detection of proteoforms is required. Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has been suggested as one such platform. When coupled to offline liquid chromatography-based fractionation, CZE-MS/MS has proven to be invaluable to the TDP community.In Chapter 2, the first optimization of dynamic pH junction-based sample stacking for TDP is provided along with one of the first comparisons of reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS) and CZE-MS/MS. Optimization of dynamic pH junction is performed with a standard protein mixture, and this platform was ultimately applied to an Eschericia coli (E. coli) whole cell lysate. This resulted in the largest TDP dataset for single-shot CZE-MS/MS. The comparison of RPLC-MS/MS and CZE-MS/MS also included analysis of an E. coli cell lysate and resulted in high numbers of identifications and highlighted the various pros and cons of each method.In Chapter 3, two dimensional LC fractionation (size exclusion chromatography (SEC) and RPLC) was coupled to CZE-MS/MS for deep TDP of E. coli cells. This study resulted in the largest TDP dataset, at the time, for E. coli, identifying 5700 proteoforms and 850 proteins. We were also able to identify and localize various interesting PTMs and estimate protein abundances using a spectral counting method. From this study it was clear thatour platform was comparable to other RPLC-MS/MS methods for deep TDP in terms of number of proteoform identifications and total instrument time.In Chapter 4, we applied our TDP platform to two isogenic colorectal cancer (CRC) cell lines, SW480 and SW620, from primary and metastatic tumors. Genetic changes have been known for a long time to affect CRC progression but this was the first proteoform-level deep TDP study of CRC metastasis. In total, we identified over 23000 proteoforms and over 2000 proteins, for the largest TDP dataset of any cell type and was a 400% increase in terms of identifications over previous deep TDP studies. We used a special database searching tool to identify single amino acid variants (SAAVs) for the largest dataset of proteoforms containing SAAVs. Quantitative analysis identified 460 proteoforms with significant differences in abundance between SW480 and SW620. Several of these proteoforms were also phosphorylated which could further impact disease progression and outcome for a specific patient phenotype and could serve as biomarkers for deciding how to treat a patient or for drug development.In Chapter 5, both activated ion electron transfer dissociation (AI-ETD) and ultraviolet photodissociation (UVPD) at 213 nm were coupled to CZE for deep TDP of E. coli and zebrafish brain samples, respectively. Optimized CZE-AI-ETD and CZE-UVPD resulted in large numbers of proteoform identifications, and many important modifications were identified and localized using these effective fragmentation techniques. This included N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation.In Chapter 6, a variety of insights into the future of TDP are provided. This includes important applications for TDP, such as personalized medicine, drug development, embryonic development, and pathogen identification. Also, a few advancements to the TDP workflow that may have increased focus on in the future are mentioned.
Author: Xiaojing Shen
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 194
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Book Description
Mass spectrometry (MS) coupled with online liquid-phase separation is the major tool for large-scale bottom-up proteomics (peptide-centric), top-down proteomics (proteoform-centric), and native proteomics (protein complex-centric). While liquid chromatography (LC)-MS is the dominant method for proteomics at different levels, capillary zone electrophoresis (CZE)-MS has emerged as a valuable and complementary technique, which provides high-capacity separation and highly sensitive detection of peptides, proteoforms and even protein complexes under native conditions. This work focuses on developing novel CZE-MS/MS methods for multi-level proteomics (bottom-up, top-down, and native).In Chapter 2, a high-throughput bottom-up proteomics workflow was developed by coupling immobilized trypsin-based speedy protein digestion with fast CZE-MS/MS. Immobilized trypsin produced almost the same digestion performance as free trypsin for complex proteomes with about 50-times higher speed (15 min vs. 12 h). Integration of immobilized trypsin (IM)-based rapid protein cleavage and fast CZE-MS/MS enables the identification of thousands of proteins from the mouse brain proteome in only 3 h, which is significantly faster than the typical LC-MS-based bottom-up proteomics workflow (3 h vs. >12 h). The high-throughput workflow was expected to be useful for bottom-up proteomics of human clinical samples (e.g., serum and urine).Chapter 3 presents the first example of CZE-MS/MS with activated ion-electron capture dissociation (AI-ECD) on a high-end quadrupole-time-of-flight (Q-TOF) mass spectrometer for top-down proteomics, enabling high-resolution separation, highly sensitive detection, and extensive gas-phase backbone cleavages of proteoforms. The CZE-AI-ECD method will be useful to the top-down proteomics community for the comprehensive characterization of proteoforms in complex proteomes. Chapter 4 and 5 focus on the development of novel CZE-MS methods for native proteomics, delineating proteins and protein complexes under native conditions. In Chapter 4, a native CZE-MS/MS platform with an Orbitrap mass spectrometer was established for native proteomics of a complex proteome (E. coli), leading to the identification of 23 protein complexes in discovery mode. The work represents the first example of native proteomics via coupling online liquid-phase separation to native MS and MS/MS. The characterization of large protein complexes (up to 200 kDa) was also achieved with a new CZE-MS system on a high-end Q-TOF mass spectrometer.In Chapter 5, a novel native capillary isoelectric focusing (cIEF)-assisted CZE-MS method is presented for the characterization of monoclonal antibodies (mAbs) with large sample loading capacity and high separation resolution. Using the method, the potential separations of different conformations of the SigmaMAb and the detection of its various glyco-proteoforms and homodimer were documented. The method separated the NISTmAb into three peaks with a microliter sample loading volume, corresponding to its different proteoforms. In addition, eight glyco-proteoforms of the NISTmAb and its homodimer were detected. The results demonstrate the potential of the native cIEF-assisted CZE-MS method for advancing the characterization of large proteins (i.e., mAbs) and protein complexes under native conditions.
Author: Gerhardus de Jong
Publisher: John Wiley & Sons
ISBN: 3527339248
Category : Science
Languages : en
Pages : 364
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Book Description
Diese Monographie bietet einen vollständigen Überblick über die Prinzipien und Anwendungen der Kapillarelektrophorese (CE) und der Massenspektrometrie (MS) und legt den Nachdruck insbesondere auf Kopplungsschnittstellen. Ausführlich erläutert werden auch alle relevanten Substanzklassen. Ein einzigartiges Wissenskompendium für alle, die sich CE-MS beschäftigen!
Author: Tian Xu
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 0
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Book Description
Top-down proteomics (TDP) enables the proteome profiling of biological subjects at the proteoform level and understanding of differential functions associated with proteoform heterogeneity, such as sequence variation, post-translational modifications (PTMs), etc. Drastic advances on TDP technologies (e.g. sample preparation, separation/fractionation, fragmentation, bioinformatics, etc.) have been achieved in the past decades. Further improvements in separation remain desired for better analysis throughput and deeper proteome coverage. Capillary electrophoresis (CE), including capillary zone electrophoresis (CZE) and capillary isoelectric focusing (cIEF), provide superior separation performance for proteoforms. This dissertation focuses on the advancement of CE-MS-based tools on throughput, separation resolution, and capacity for TDP and utility of these tools for biological applications.In Chapter 2, we developed high-throughput and high-capacity cIEF-MS/MS platforms. The high-throughput platform enables efficient identification and quantification of proteoforms (less than one hour per run), whereas the high-capacity cIEF-MS/MS provides large number of proteoform identifications (IDs, more than 700 proteoforms in a single shot analysis) which is valuable for deep TDP. In Chapter 3, we further improved the stability and robustness of cIEF-MS platform using optimized linear polyacrylamide (LPA) capillary coating and catholyte with lower pH (pH~10). The work achieved high-resolution characterization and accurate isoelectric point (pI) determination of charge variants (~0.1 pI difference) of monoclonal antibodies (mAbs). In Chapter 4, we developed a nondenaturing cIEF-MS platform for ultrahigh resolution characterization of microheterogeneity of a variety of protein complexes. Typically, pI determinations of variants in protein complexes allow us to decipher how sequence or PTM variations modulate the pIs of the protein complexes. In Chapter 5, while CZE-MS/MS is a well-developed approach, for the first time, we coupled FAIMS to CZE-MS/MS to facilitate online gas-phase fractionation of proteoforms. The FAIMS greatly enhanced the sensitivity of the system and expanded the number of proteoform IDs, especially large proteoform IDs. The work renders CZE-FAIMS-MS/MS as a new powerful multidimensional platform for deep TDP.In Chapters 6 and 7, we applied cIEF-MS/MS and CZE-MS/MS for studying the sexual dimorphism of zebrafish brains and proteoform-level differences between metastatic and nonmetastatic colorectal cancer (CRC) cells, respectively. In Chapter 6, quantitative TDP of thousands of proteoforms from male and female zebrafish brains by cIEF-MS/MS based approach discovered various overexpressed proteoforms in male or female brains that are closely associated with hormone activity. In Chapter 7, We performed deep TDP study of non-metastatic and metastatic CRC cells (SW480 and SW620) using CZE-MS/MS based multidimensional platform and identified more than 20,000 proteoforms of over 2,000 proteins from the two cell lines, which presents around 5-folds higher number of proteoform IDs in comparison with previous TDP studies of human cancer cells. The work revealed significant discrepancies between the two isogenic cell lines regarding proteoform and single amino acid variant (SAAV) profiles. Quantitative data disclosed differentially expressed proteoforms between the two cell lines and their corresponding genes were connected to cancer pathways and networks.
Author: Geoffrey S. Ginsburg
Publisher: Academic Press
ISBN: 0123822270
Category : Science
Languages : en
Pages : 1343
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Book Description
Genomic and Personalized Medicine, Second Edition - winner of a 2013 Highly Commended BMA Medical Book Award for Medicine - is a major discussion of the structure, history, and applications of the field, as it emerges from the campus and lab into clinical action. As with the first edition, leading experts review the development of the new science, the current opportunities for genome-based analysis in healthcare, and the potential of genomic medicine in future healthcare. The inclusion of the latest information on diagnostic testing, population screening, disease susceptability, and pharmacogenomics makes this work an ideal companion for the many stakeholders of genomic and personalized medicine. With advancing knowledge of the genome across and outside protein-coding regions of DNA, new comprehension of genomic variation and frequencies across populations, the elucidation of advanced strategic approaches to genomic study, and above all in the elaboration of next-generation sequencing, genomic medicine has begun to achieve the much-vaunted transformative health outcomes of the Human Genome Project, almost a decade after its official completion in April 2003. Highly Commended 2013 BMA Medical Book Award for Medicine More than 100 chapters, from leading researchers, review the many impacts of genomic discoveries in clinical action, including 63 chapters new to this edition Discusses state-of-the-art genome technologies, including population screening, novel diagnostics, and gene-based therapeutics Wide and inclusive discussion encompasses the formidable ethical, legal, regulatory and social challenges related to the evolving practice of genomic medicine Clearly and beautifully illustrated with 280 color figures, and many thousands of references for further reading and deeper analysis
Author: Xiaojing Yan
Publisher:
ISBN:
Category :
Languages : en
Pages : 104
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Book Description
Author: Timothy D. Veenstra
Publisher: Academic Press
ISBN: 0080569153
Category : Science
Languages : en
Pages : 445
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Book Description
The content of this volume is designed to reach a wide audience, including those involved with relevant technologies such as electrophoresis and mass spectrometry, to those interested in how proteomics can benefit research. A wide range of techniques are discussed, each specifically designed to address different needs in proteomic analysis. The concluding chapter discusses the important issue related to handling large amounts of data accumulated in proteomic studies. - Discusses proteomics in the postgenomic age - Includes various strategies for quantitative proteomics - Covers the role of MS in structural functional proteomics and proteomics in drug discovery and bioinformatics
Author: Gyorgy Marko-Varga
Publisher: Elsevier
ISBN: 044451810X
Category : Language Arts & Disciplines
Languages : en
Pages : 663
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Book Description
Proteomics and peptidomics is the detailed understanding of the role that proteins and peptides play in health and disease and is a necessary compliment to genetic analysis. The functional expression analysis of both proteins and peptides plays a central role in modern drug discovery as well as drug development, and is also a key research area in systems biology. Proteomics and Peptidomics captures the width as well as the depth within the area and exemplifies the variety as well as the traditional basis of analytical chemistry that is needed in order to move forward in expression analysis studies. As a fast emerging field, it gives and overview of parts within the field combined with highly specialized and dedicated topics that are intended to compliment each other.
