Fast Detection and Quantification of Proteoforms in Therapeutic Proteins Using Intact Protein Mass Spectrometry PDF Download

Author: Manasi Gaikwad
Publisher:
ISBN:
Category :
Languages : en
Pages : 0

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Author: Laura Heikaus
Publisher:
ISBN:
Category :
Languages : en
Pages :

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Book Description
A characteristic of many proteoforms, derived from a single gene, is their similarity regarding the composition of atoms, making their analysis very challenging. Many overexpressed recombinant proteins are strongly associated with this problem, especially recombinant therapeutic glycoproteins from large-scale productions. In contrast to small molecule drugs, which consist of a single defined molecule, therapeutic protein preparations are heterogenous mixtures of dozens or even hundreds of very similar species. With mass spectrometry, currently high-quality spectra of intact proteoforms can be obtained only, if the complexity of the mixture of individual proteoform-ions, entering the gas phase at the same time is low. Thus, prior to mass spectrometric analysis, an effective separation is required for getting fractions with a low number of individual proteoforms. This is especially true not only for recombinant therapeutic proteins, because of their huge heterogeneity, but also relevant for top-down proteomics. Purification of proteoforms is the bottleneck in analyzing intact proteoforms with mass spectrometry. This review is focusing on the current state of the art, especially of liquid chromatography for preparing proteoforms for mass spectrometric top-down analysis. The topic of therapeutic proteins has been chosen, because this group of proteins is most challenging regarding their proteoform analysis.

Author: Mike S. Lee
Publisher: John Wiley & Sons
ISBN: 1119359368
Category : Science
Languages : en
Pages : 282

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Book Description
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

Author: Hartmut Schl√oter
Publisher:
ISBN:
Category : Science
Languages : en
Pages : 0

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Book Description
A characteristic of many proteoforms, derived from a single gene, is their similarity regarding the composition of atoms, making their analysis very challenging. Many overexpressed recombinant proteins are strongly associated with this problem, especially recombinant therapeutic glycoproteins from large-scale productions. In contrast to small molecule drugs, which consist of a single defined molecule, therapeutic protein preparations are heterogenous mixtures of dozens or even hundreds of very similar species. With mass spectrometry, currently high-quality spectra of intact proteoforms can be obtained only, if the complexity of the mixture of individual proteoform-ions, entering the gas phase at the same time is low. Thus, prior to mass spectrometric analysis, an effective separation is required for getting fractions with a low number of individual proteoforms. This is especially true not only for recombinant therapeutic proteins, because of their huge heterogeneity, but also relevant for top-down proteomics. Purification of proteoforms is the bottleneck in analyzing intact proteoforms with mass spectrometry. This review is focusing on the current state of the art, especially of liquid chromatography for preparing proteoforms for mass spectrometric top-down analysis. The topic of therapeutic proteins has been chosen, because this group of proteins is most challenging regarding their proteoform analysis.

Author: Bifan Chen
Publisher:
ISBN:
Category :
Languages : en
Pages : 215

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Book Description
The study of proteins is critical for understanding cellular functions at the molecular level. Top-down mass spectrometry (MS) has emerged as a premier tool for global and comprehensive analysis of proteoforms. The top-down approach retains intact mass information, providing a "bird's-eye" view of the proteome and allowing for identification of novel proteoforms, in-depth sequence characterization, and quantification of disease associated post-translational modifications (PTMs). However, many technical challenges still exist. The research described here involves analytical development in top-down MS, particularly in the areas of enrichment, separation, and characterization of samples ranging from standard proteins and complex lysates, to large therapeutic biomolecules. Chapter 1 provides an introduction and review of recent advances in different aspects of top-down proteomics. Chapters 2 and 3 are related to the study of intact phosphoproteins. Specifically, chapter 2 describes the use of functionalized nanoparticles for enrichment and the subsequent coupling of online liquid chromatography (LC)-MS for characterizing endogenous phosphoproteins from complex cell lysates. Chapter 3 investigates how phosphorylation moieties might influence the efficiency of electron capture dissociation (ECD). Chapters 4 and 5 focus on the development of hydrophobic interaction chromatography (HIC) that could be coupled online directly with MS and its applications to therapeutic molecules (monoclonal antibodies). Chapter 6 describes a middle-down approach to obtain multi-attribute of both cysteine and lysine conjugated antibody-drug conjugates, which overcomes some current challenges using HIC-MS and the top-down approach. Overall, these analytical developments expand the toolbox of the top-down approach and generally facilitate the analysis of intact proteins.

Author: Guodong Chen
Publisher: Springer Science & Business Media
ISBN: 1441978623
Category : Science
Languages : en
Pages : 408

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Book Description
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.

Author: Liangliang Sun
Publisher: Humana
ISBN: 9781071623244
Category : Science
Languages : en
Pages : 240

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Book Description
This volume discusses the latest mass spectrometry (MS)-based technologies for proteoform identification, characterization, and quantification. Some of the topics covered in this book include sample preparation, proteoform separation, proteoform gas-phase fragmentation, and bioinformatics tools for MS data analysis. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, Proteoform Identification: Methods and Protocols is a valuable resource for researchers in both academia and the biopharmaceutical industry who are interested in proteoform analysis using MS.

Author: Michael Nshanian
Publisher:
ISBN:
Category :
Languages : en
Pages : 175

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Book Description
The advent of top-down protein mass spectrometry (MS), or direct analysis of intact proteins forgoing proteolysis, has transformed the field of protein mass spectrometry, ushering in a new era of protein identification and characterization together with a new set of challenges. The analysis of intact proteins and their direct fragmentation in tandem (MS/MS) mode helps overcome the "inference" problem associated with peptide-based bottom-up proteomics; that is, correctly assigning given peptide fragments and their modifications to the intact protein from which they originated. Despite its many advantages, however, the top-down approach requires extensive sample fractionation and suffers from low sensitivity but much progress has been made. From recently-developed cross-linked polyacrylamide gels, from which intact proteins can be more easily recovered, to the discovery of reagents that enhance protein charging in electrospray ionization (ESI), there have been considerable gains in detection and sensitivity, offering the potential for a more complete and accurate characterization of a "proteoform": the full complement of the combinatorial possibilities that could arise from a given gene product. Top-down MS also includes the study of proteins in their native or native-like states. This is especially important in characterizing disease-related proteins, particularly in the context of protein aggregation. Native MS, using electron-capture dissociation (ECD) and ion mobility spectrometry (IMS), enables the study of protein-inhibitor complexes in the gas phase, offering structural insight into stoichiometry, site of inhibitor binding and mechanism of inhibition. In addition, intact analysis and electron-based fragmentation enable the detection of thermally-labile post-translational modifications like phosphorylation, known to play key regulatory roles in shifting proteins towards cytotoxic states. Top-down method developments in protein recovery, separation and supercharging have led to improvements in detection and sensitivity, while top-down MS applications to structural characterization of disease-related proteins have shed more light on the mechanisms of cytotoxic aggregation, offering greater promise of therapeutic development.

Author: Robert James Milliken
Publisher:
ISBN:
Category :
Languages : en
Pages :

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Book Description
The identification of peptides by automated database searching of fragmentation (MS2)spectra began with the SEQUEST algorithm in 1994. Since then, the rapidly increasing size and complexity of proteomic data sets have made algorithms for the identification and quantification of peptides, proteins, and protein forms (proteoforms) increasingly important. This thesis describes new algorithms for the interpretation of mass spectra in proteomics, along with details of their implementation. Chapter 1 contains an overview of the past, present, and possible future of proteomics. Chapter 2 describes FlashLFQ, an algorithm for high-speed label-free quantification of peptides and proteins following a search of bottom-up mass spectrometry data. FlashLFQ is an order of magnitude faster than previous label-free quantification methods. The algorithm is incorporated into the MetaMorpheus search software program, allowing the quantification of post-translationally modified peptides discovered by MetaMorpheus's global post-translational modification discovery (G-PTM-D) engine. Chapter 3 describes a Bayesian statistical test to estimate the probability that a protein is differentially expressed between two experimental conditions. Compared to Student's t-test, this novel statistical analysis increases the number of truly changing proteins detected in two benchmark data sets, and has been incorporated into FlashLFQ. Finally, Chapter 4 describes Proteoform Explorer, a tool for displaying chromatograms and isotopic envelopes for intact-mass (MS1) proteoform data to aid proteomics researchers in validation and data exploration. Two metrics are also introduced: the Deconvoluted Summed Ion Current (D-SIC), meaning the summed ion current of all deconvoluted proteoform ions in a chromatographic run, and the Identified Summed Ion current (I-SIC), meaning the summed ion current of all identified proteoform ions in a chromatographic run. These metrics can be used to measure progress towards identifying all proteoform ions observed in a chromatographic run. From another perspective, they can also be used to quantify the "dark matter" of the proteome in a given sample (i.e., the proteoforms that are observable in the mass spectra but were not identified).

Author: Ingvar Eidhammer
Publisher: John Wiley & Sons
ISBN: 111849377X
Category : Mathematics
Languages : en
Pages : 290

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Book Description
The definitive introduction to data analysis in quantitative proteomics This book provides all the necessary knowledge about mass spectrometry based proteomics methods and computational and statistical approaches to pursue the planning, design and analysis of quantitative proteomics experiments. The author’s carefully constructed approach allows readers to easily make the transition into the field of quantitative proteomics. Through detailed descriptions of wet-lab methods, computational approaches and statistical tools, this book covers the full scope of a quantitative experiment, allowing readers to acquire new knowledge as well as acting as a useful reference work for more advanced readers. Computational and Statistical Methods for Protein Quantification by Mass Spectrometry: Introduces the use of mass spectrometry in protein quantification and how the bioinformatics challenges in this field can be solved using statistical methods and various software programs. Is illustrated by a large number of figures and examples as well as numerous exercises. Provides both clear and rigorous descriptions of methods and approaches. Is thoroughly indexed and cross-referenced, combining the strengths of a text book with the utility of a reference work. Features detailed discussions of both wet-lab approaches and statistical and computational methods. With clear and thorough descriptions of the various methods and approaches, this book is accessible to biologists, informaticians, and statisticians alike and is aimed at readers across the academic spectrum, from advanced undergraduate students to post doctorates entering the field.