Top Down Characterization of Proteins by Electron Capture Dissociation and Blackbody Infrared Radiative Dissociation Mass Spectrometry PDF Download

Author: Ying Ge
Publisher:
ISBN:
Category :
Languages : en
Pages : 354

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Author: Haroun N. Shah
Publisher: John Wiley & Sons
ISBN: 1119991927
Category : Science
Languages : en
Pages : 545

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New advances in proteomics, driven largely by developments in mass spectrometry, continue to reveal the complexity and diversity of pathogenic mechanisms among microbes that underpin infectious diseases. Therefore a new era in medical microbiology is demanding a rapid transition from current procedures to high throughput analytical systems for the diagnosis of microbial pathogens. This book covers the broad microbiological applications of proteomics and mass spectrometry. It is divided into six sections that follow the general progression in which most microbiology laboratories are approaching the subject –Transition, Tools, Preparation, Profiling by Patterns, Target Proteins, and Data Analysis.

Author: Piriya Wongkongkathep
Publisher:
ISBN:
Category :
Languages : en
Pages : 170

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Mass spectrometry (MS) has made significant contributions to protein and proteomics analysis during the past decades from its advantages of speed, sensitivity, specificity, and low sample consumption. While the proteomics field grows rapidly to identify thousands of proteins in a single analysis, "native" mass spectrometry, exploiting the unique features of electrospray ionization (ESI) for delivering large macromolecules to the mass spectrometer, has provided many potential exciting capabilities and applications to structural biology and biochemistry. It can analyze proteins in their native states, i.e., structures present in their native configurations from physiological pH solutions, with minimal sample preparation. In this thesis, I describe the application of native ESI combined with top-down MS using electron capture dissociation (ECD) and ion mobility (IM) to characterize the molecular features of protein-ligand complexes. Binding and structural information can be comprehensively obtained from this experimental platform. Native ESI-MS alone provides molecular mass, stoichiometry, and binding affinity, all from a single analysis. We demonstrate that top-down MS, the fragmentation of intact proteins and protein complexes using MS, offers a powerful capability to elucidate the location of ligand binding on a protein's structure and for probing the surface topology of proteins. Ion mobility mass spectrometry, a recently developed technique that yields information on the structural conformation of molecules, was used to reveal structural changes of proteins upon ligand binding. My thesis focuses on several proteins, including -synuclein (AS), which is a small protein related to Parkinson's disease. AS is natively unfolded at physiological pH, which makes it difficult to study by standard methods such as X-ray crystallography or NMR. Using our mass spectrometry techniques, transition metal binding (copper, cobalt, and manganese) to AS that is associated with accelerating fibril formation was monitored. The binding of a small molecule amyloid inhibitor called molecular tweezer (MT or CLR01) on two model proteins important in neurodegenerative diseases, AS and superoxide dismutase (SOD1), was studied. Tandem mass spectrometry (MS/MS) techniques such as collisionally activated dissociation (CAD) along with ECD were used to characterize the sites of binding of small molecule ligands to proteins. Ion mobility mass spectrometry was implemented to reveal the conformational changes of AS upon metal binding. It was demonstrated that copper can induce the AS protein to collapse into a more compact state, which may provide a hint of the mechanisms behind amyloid fibrillation. Additionally, two new methods to extend the application of top-down MS for protein structure characterization were developed. First, the same molecular tweezer molecule, which has a specificity to bind lysine residues, was used to probe surface residues of proteins. The lysines found to bind to the molecular tweezers identified by top-down MS correlates well with solvent accessibility values, suggesting that the MT compound can be applied as a molecular probe to pinpoint surface active lysine residues. Lastly, supplemental activation methods by ultraviolet and infrared laser irradiation prior to ECD was applied to assist disulfide bond cleavage of complex multiple intermolecular and intramolecular disulfide bond-containing proteins. Backbone bond cleavage from top-down MS was significantly increased when the disulfide bonds were cleaved, allowing more sequence information to be obtained. The new methods described in this thesis extend the applicability of mass spectrometry to provide a more complete picture of a protein's structure.

Author: Huili Zhai
Publisher:
ISBN:
Category :
Languages : en
Pages : 332

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Author: Richard B. Cole
Publisher: John Wiley & Sons
ISBN: 1118211553
Category : Science
Languages : en
Pages : 900

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Discover how advances in mass spectrometry are fueling new discoveries across a broad range of research areas Electrospray and MALDI Mass Spectrometry brings both veteran practitioners and beginning scientists up to date with the most recent trends and findings in electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In particular, this Second Edition highlights how advances in electrospray and MALDI mass spectrometry are supporting important discoveries in new and emerging fields such as proteomics and metabolomics as well as in traditional areas of chemistry and physics research. Electrospray AND MALDI Mass Spectrometry, SECOND EDITION is divided into five parts: Part A, Fundamentals of ES, explains the fundamental phenomena underlying the electrospray process, including selectivity in ionization and inherent electrochemistry, and concludes with a chapter offering a comparative inventory of source hardware Part B, Fundamentals of MALDI, confronts ionization mechanisms, instrument development, and matrix selection, and includes a final chapter that explores the special application of MALDI to obtain two-dimensional images of spatial distributions of compounds on surfaces Part C, ES and MALDI Coupling to Mass Spectrometry Instrumentation, examines the coupling of these ionization techniques to various mass analyzers, including quadrupole ion trap, time-of-flight, Fourier transform ion cyclotron resonance, and ion mobility mass spectrometers Part D, Practical Aspects of ES and MALDI, investigates analytical issues including quantification, charge-state distributions, noncovalent interactions in solution that are preserved as gas-phase ions, and various means of ion excitation in preparation for tandem mass spectrometry, and offers a guide to the interpretation of even-electron mass spectra Part E, Biological Applications of ES and MALDI, examines the role of mass spectrometry in such areas as peptide and protein characterization, carbohydrate analysis, lipid analysis, and drug discovery Written by a team of leading experts, the book not only provides a critical review of the literature, but also presents key concepts in tutorial fashion to help readers take full advantage of the latest technological breakthroughs and applications. As a result, Electrospray and MALDI Mass Spectrometry will help researchers fully leverage the power of electrospray and MALDI mass spectrometry. The judicious compartmentalization of chapters, and the pedagogic presentation style throughout, render the book highly suitable for use as a text for graduate-level courses in advanced mass spectrometry.

Author: Raymond E. March
Publisher: CRC Press
ISBN: 1420083740
Category : Science
Languages : en
Pages : 568

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Widely used in medical research, pharmaceutical and fine chemicals industries, biological and physical sciences, and security and environmental agencies, mass spectrometry techniques are continually under development. In Practical Aspects of Trapped Ion Mass Spectrometry: Volume V, Applications of Ion Trapping Devices, an international panel of aut

Author: Lisa Anne Vasicek
Publisher:
ISBN:
Category :
Languages : en
Pages : 338

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There continue to be great strides in the field of proteomics but as samples become more complex, the ability to increase sequence coverage and confidence in the identification becomes more important. Several methods of derivatization have been developed that can be used in combination with tandem mass spectrometry to identify and characterize proteins. Three types of activation, including infrared multiphoton dissociation, ultraviolet photodissociation, and electron transfer dissociation, are enhanced in this dissertation and compared to the conventional method of collisional induced dissociation (CID) to demonstrate the improved characterization of proteins. A free amine reactive phosphate group was synthesized and used to modify the N-terminus of digested peptides. This phosphate group absorbs at the IR wavelength of 10.6 [mu]m as well as the Vacuum-ultraviolet (VUV) due to an aromatic group allowing modified peptides to be dissociated by infrared multi-photon dissociation (IRMPD) or ultraviolet photodissociation (UVPD) whereas peptides without this chromophore are less responsive to IR or UV irradiation. The PD spectra for these modified peptides yield simplified MS/MS spectra due to the neutralization of all N-terminal product ions from the incorporation the negatively charged phosphate moiety. This is especially advantageous for UVPD due to the great number of product ions produced due to the higher energy deposition of the UV photons. The MS/MS spectra also produce higher sequence coverage in comparison to CID of the modified or unmodified peptides due to more informative fragmentation pathways generated upon PD from secondary dissociation and an increased ion trapping mass range. IRMPD is also implemented for the first time on an orbitrap mass spectrometer to achieve high resolution analysis of IR chromophore-derivatized samples as well as top-down analysis of unmodified proteins. High resolution/high mass accuracy analysis is extremely beneficial for characterization of complex samples due to the likelihood of false positives at lower resolutions/accuracies. For electron transfer dissociation, precursor ions in higher charge states undergo more exothermic electron transfer and thus minimize non-dissociative charge reduction. In this dissertation, cysteine side chains are alkylated with a fixed charge to deliberately increase the charge states of peptides and improve electron transfer dissociation. ETD can also be used to study protein structure by derivatizing the intact structure with a hydrazone reagent. A hydrazone bond will be preferentially cleaved during ETD facilitating the recognition of any modified residues through a distinguishing ETD fragmentation spectrum.

Author: Bifan Chen
Publisher:
ISBN:
Category :
Languages : en
Pages : 0

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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: Agnieszka Kraj
Publisher: John Wiley & Sons
ISBN: 047039580X
Category : Science
Languages : en
Pages : 358

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With contributions from noted experts from Europe and North America, Mass Spectrometry Instrumentation, Interpretation, and Applications serves as a forum to introduce students to the whole world of mass spectrometry and to the many different perspectives that each scientific field brings to its use. The book emphasizes the use of this important analytical technique in many different fields, including applications for organic and inorganic chemistry, forensic science, biotechnology, and many other areas. After describing the history of mass spectrometry, the book moves on to discuss instrumentation, theory, and basic applications.

Author: Michael L. Gross
Publisher:
ISBN: 9780080438474
Category : Science
Languages : en
Pages : 1098

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Book Description
Presents information on the biographies of recognized pioneers and innovators in the field of mass spectrometry. - Highlights over 120 innovators in mass spectrometry, including several Nobel Prize winners. Discusses instrumentation and their uses, also providing interesting information on the careers, characters, and life stories of the people who did the work. Offers unique insight into the careers and personalities of luminaries in the field.