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Enhancing Detection Of Large Noncovalent Protein Complexes And Characterization Of Vault Protein Complexes By Ion Mobility And Mass Spectrometry
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Book Synopsis Enhancing Detection of Large Noncovalent Protein Complexes and Characterization of Vault Protein Complexes by Ion Mobility and Mass Spectrometry by : Shirley Hom Lomeli
Download or read book Enhancing Detection of Large Noncovalent Protein Complexes and Characterization of Vault Protein Complexes by Ion Mobility and Mass Spectrometry written by Shirley Hom Lomeli and published by . This book was released on 2011 with total page 258 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Integration of Tandem Mass Spectrometry and Ion Mobility Spectrometry for Protein Characterization and Structural Analysis by : Deepali Rathore
Download or read book Integration of Tandem Mass Spectrometry and Ion Mobility Spectrometry for Protein Characterization and Structural Analysis written by Deepali Rathore and published by . This book was released on 2016 with total page 213 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry (MS) based proteomics and intact protein analyses are important tools for the structural study of proteins and provide powerful methods for solving biochemical puzzles involving proteins. The work described in this dissertation is aimed at the development of novel, efficient, and information rich strategies for protein structure and sequence analysis. The approaches developed have been applied to analytes ranging from proteolytic peptides to large non-covalent protein complexes.
Book Synopsis Characterization of Peptides, Proteins, and Protein Complexes Using Infrared Multiphoton Dissociation Spectroscopy, Ion Mobility Spectrometry, and Surface-induced Dissociation Mass Spectrometry by : Erin M. Panczyk
Download or read book Characterization of Peptides, Proteins, and Protein Complexes Using Infrared Multiphoton Dissociation Spectroscopy, Ion Mobility Spectrometry, and Surface-induced Dissociation Mass Spectrometry written by Erin M. Panczyk and published by . This book was released on 2021 with total page 210 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry-based techniques have emerged as powerful analytical tools to investigate the structure of proteins from the primary to quaternary levels. The advancement of mass spectrometry instrumentation and methods has allowed researchers to go beyond just measuring an analyte’s mass-to-charge ratio, but to also probe gas-phase dissociation behaviors and conformations of peptides, proteins, and protein complexes. The primary structure of a protein refers to the linear sequence of amino acids linked together via peptide bonds. The presence, and the order, of specific amino acids in a peptide can strongly influence how a peptide fragments in the gas-phase. Particular amino acids can direct where along the peptide backbone fragmentation is favored and the structure of the fragment ions formed. One method for probing the structure of peptide fragment ions is infrared multiphoton dissociation (IRMPD) mass spectrometry coupled with theoretical quantum chemical calculations. This approach is used to investigate the role of peptide bond conformation on the structure of b2+ fragment ions formed from proline and dimethylproline-containing peptides (Chapter 3). Additionally, IRMPD is used to study the fragmentation patterns of proline containing pentapeptides into b3+ ions (Chapter 4). Native mass spectrometry (nMS) analyzes the intact structures of proteins and protein complexes and offers complementary information to traditional biophysical methods, such as NMR or cryo-EM. Tandem mass spectrometry, specifically surface-induced dissociation (SID), provides information on protein complex connectivity, stoichiometry, and gas-phase structural rearrangement. SID is utilized to monitor deviation from native structure for protein complexes generated from submicrometer nanoelectrospray capillaries (Chapter 5), as well as to provide insight into connectivity of protein complexes selected by trapped ion mobility spectrometry (Chapter 6). In addition to SID, ion mobility spectrometry provides information on the gas-phase shape or conformation of biomolecules. Here, ion mobility spectrometry is utilized to separate multiple conformers of proline-containing peptides (Chapter 3), compare the collision cross sections of protein complexes generated from submicrometer and micrometer sized nanoelectrospray capillaries (Chapter 5), and select protein complexes and isomeric peptides prior to dissociation on an ultrahigh resolution mass spectrometry platform (Chapter 6). Finally, the development and optimization of Trapped Ion Mobility Spectrometry (TIMS) for native mass spectrometry applications is applied to the widely available timsTOF Pro mass spectrometry platform to promote the dissemination of native ion mobility technology.
Book Synopsis Protein and Peptide Mass Spectrometry in Drug Discovery by : Michael L. Gross
Download or read book Protein and Peptide Mass Spectrometry in Drug Discovery written by Michael L. Gross and published by John Wiley & Sons. This book was released on 2011-09-26 with total page 484 pages. Available in PDF, EPUB and Kindle. Book excerpt: The book that highlights mass spectrometry and its application in characterizing proteins and peptides in drug discovery An instrumental analytical method for quantifying the mass and characterization of various samples from small molecules to large proteins, mass spectrometry (MS) has become one of the most widely used techniques for studying proteins and peptides over the last decade. Bringing together the work of experts in academia and industry, Protein and Peptide Mass Spectrometry in Drug Discovery highlights current analytical approaches, industry practices, and modern strategies for the characterization of both peptides and proteins in drug discovery. Illustrating the critical role MS technology plays in characterizing target proteins and protein products, the methods used, ion mobility, and the use of microwave radiation to speed proteolysis, the book also covers important emerging applications for neuroproteomics and antigenic peptides. Placing an emphasis on the pharmaceutical industry, the book stresses practice and applications, presenting real-world examples covering the most recent advances in mass spectrometry, and providing an invaluable resource for pharmaceutical scientists in industry and academia, analytical and bioanalytical chemists, and researchers in protein science and proteomics.
Book Synopsis Mass Spectrometry of Protein Interactions by : Kevin Downard
Download or read book Mass Spectrometry of Protein Interactions written by Kevin Downard and published by John Wiley & Sons. This book was released on 2007-08-24 with total page 153 pages. Available in PDF, EPUB and Kindle. Book excerpt: The authoritative guide to analyzing protein interactions by mass spectrometry Mass spectrometry (MS) is playing an increasingly important role in the study of protein interactions. Mass Spectrometry of Protein Interactionspresents timely and definitive discussions of the diverse range of approaches for studying protein interactions by mass spectrometry with an extensive set of references to the primary literature. Each chapter is written by authors or teams of authors who are international authorities in their fields. This leading reference text: * Discusses the direct detection of protein interactions through electrospray ionization (ESI-MS); ion mobility analysis; and matrix-assisted laser desorption/ionization (MALDI-MS) * Covers the indirect analysis of protein interactions through hydrogen-deuterium exchange (HX-MS); limited proteolysis; cross-linking; and radial probe (RP-MS) * Guides researchers in the use of mass spectrometry in structural biology, biochemistry, and protein science to map and define the huge number and diversity of protein interactions * Reviews the latest discoveries and applications and addresses new and ongoing challenges This is a comprehensive reference for researchers in academia and industry engaged in studies of protein interactions and an excellent text for graduate and postgraduate students.
Book Synopsis Top-Down Mass Spectrometry Characterization of Protein-Ligand Complexes Important to Neurodegenerative Diseases by : Piriya Wongkongkathep
Download or read book Top-Down Mass Spectrometry Characterization of Protein-Ligand Complexes Important to Neurodegenerative Diseases written by Piriya Wongkongkathep and published by . This book was released on 2015 with total page 170 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Book Synopsis Methodologies and Applications for the Analysis of Intact Proteins and Protein-ligand Interactions by Top-down Mass Spectrometry by : Michael Nshanian
Download or read book Methodologies and Applications for the Analysis of Intact Proteins and Protein-ligand Interactions by Top-down Mass Spectrometry written by Michael Nshanian and published by . This book was released on 2018 with total page 175 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Book Synopsis Incorporation of Surface Induced Dissociation Into a Commercial Ion Mobility by : Mowei Zhou
Download or read book Incorporation of Surface Induced Dissociation Into a Commercial Ion Mobility written by Mowei Zhou and published by . This book was released on 2013 with total page 242 pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract: There is a growing interest in application of mass spectrometry as a high throughput technique for quaternary structure studies of protein complexes. One way to study protein complexes by mass spectrometry is to specifically label peptides segments that carry critical structural information, and after protein digestion subsequently identify the labeled peptides using liquid chromatography - mass spectrometry. A chemical crosslinker forms covalent bonds at specific amino acid sidechains that are in proximity in the protein structure. This approach is used to probe the binding interface of LexA/RecA proteins in Escherichia coli (Chapter 3). In contrast, intact noncovalent protein complexes can be directly transferred into the gas phase, while retaining memory of their solution structures. Accurate molecular weight measurement by mass spectrometry can be used for stoichiometry determination of protein-protein and protein-ligand systems, as manifested by the two examples of stoichiometry determination of differently treated adiponectin oligomers (Chapter 4), and the silver binding properties of the N-terminal region of a bacterial protein CusB (Chapter 5).
Book Synopsis Structural Studies on Noncovalent Protein Complexes by Mass Spectrometry and Travelling Wave Ion Mobility Spectrometry by : José Pedro Correia Afonso
Download or read book Structural Studies on Noncovalent Protein Complexes by Mass Spectrometry and Travelling Wave Ion Mobility Spectrometry written by José Pedro Correia Afonso and published by . This book was released on 2012 with total page 444 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis New Analytical Tools for Native Mass Spectrometry and Ion Mobility Mass Spectrometry Analysis of Intact Proteins by : Seoyeon Hong
Download or read book New Analytical Tools for Native Mass Spectrometry and Ion Mobility Mass Spectrometry Analysis of Intact Proteins written by Seoyeon Hong and published by . This book was released on 2020 with total page 146 pages. Available in PDF, EPUB and Kindle. Book excerpt: Native mass spectrometry (MS) is a structural biology tool that probes proteins and protein complexes in the gas phase. In native MS, electrospray ionization (ESI) of protein samples are prepared in nondenaturing conditions and generate "native-like" protein ions, which retain noncovalent interactions observed in solution. Therefore, native MS is useful to provide information about the stoichiometry, topology, and ligand binding of protein complexes. Native MS coupled with ion mobility (IM) provides the momentum transfer collision cross section (omega), which is indicative of the size and shape of ions. Collision-induced unfolding (CIU) is an energy-dependent IM-MS technique that probes the unfolding of protein structures monitored by omega as a function of energy. This dissertation explores the utility of native MS and IM-MS analysis to study protein complexes. First, native MS analysis is used to investigate the stoichiometry of iron and sulfur in the endogenous Fe-S clusters binding to the F-box and leucine-rich protein 5 (FBXL5) and Skp1 from the Skp1-Cul1-Fbox (SCF) ubiquitin ligase in Chapter 2. The thermal activation in solution prior to ESI in combination with instrumental activation effectively elucidates the binding of [2Fe-2S] to FBXL5-Skp1 by reducing the presence of nonspecifically binding adducts (NSA). The effects of polarity on native-like avidin tetramers are characterized using native MS and IM-MS analysis in Chapter 3. The native MS of native-like avidin tetramer shows that the average charge state distribution is different between the cations and the anions. The native IM-MS results show that the omega of native-like avidin tetramer is similar regardless of the charge state and polarity. However, the CIU results display differences due to the charge state and polarity. In particular, the differences between the CIU results of 14+ and 14- avidin ions indicate that CIU analysis of the avidin ions is sensitive to solely polarity. In order to quantitatively compare the CIU results of 14+ and 14- avidin ions, a similarity score is developed. Similarity score analysis comparing the 14+ and 14- ions indicates that the largest difference in omega is observed at near 800 eV, while the greatest similarity is observed at low energy range (56 to ~400 eV). The utility of native MS and IM-MS analysis is explored to characterize antibodies (Abs) using two IgG2 samples (SIgG2 and AIgG2) in Chapter 4. These two samples are from the same subclass (IgG2), have the kappa light chain and were each purified from human myeloma plasma, but were from different manufacturing origins. Native MS results of the two samples indicate that the two Abs display vastly different apparent mass (SIgG2: ~154 kDa and AIgG2: 157, 159 kDa respectively) and the relative mass heterogeneity based on the peak width and shape. The IM-MS analysis demonstrates that the omega of the native-like Abs depends on the z, which contrasts from the omega of most proteins. The strong dependence of omega on z may be due to large differences in structural populations and/or the presence of flexible hinge region. The CIU analysis of SIgG2 and AIgG2 demonstrates that the greatest difference in omega between the two Abs is present at low energy with greater difference for anions than for cations. Overall, these results indicate that anions and low energy may preferentially provide significant differences when comparing similar proteins using native IM-MS and CIU analysis. Collision-induced unfolding (CIU) is increasingly used to study the effects of ligand binding to proteins and protein complexes. In chapter 5, a workflow is developed to more accurately assess the effects of ligand binding on the CIU stability. Mass spectra of the quadrupole-selected precursor ions at varying collision-energy display signals indicate that the precursor ions for CIU analysis is interfered by the presence of NSA despite extensive buffer exchange. Therefore, Srelative method is developed to determine the minimum collision-energy threshold at which all of the apparent NSA are removed from the initial m/z window of the precursor ions. More generally, Srelative may be used for quality control of CIU analysis.
Book Synopsis Applying Tandem Mass Spectrometry Coupled with Ion Mobility to Probe the Structure of Non-covalent Protein Complexes and Their Interactions with Ligands, Peptides and Other Proteins by : Royston Samuel Quintyn
Download or read book Applying Tandem Mass Spectrometry Coupled with Ion Mobility to Probe the Structure of Non-covalent Protein Complexes and Their Interactions with Ligands, Peptides and Other Proteins written by Royston Samuel Quintyn and published by . This book was released on 2015 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: By monitoring the surface induced dissociation (SID) products as a function of increasing collision energy or from sub-complexes initially produced from SID of the native complexes (in SID-IM-SID experiments), we were able to gain a fundamental mechanistic insight into the assembly of several tetrameric protein complexes. In addition, the SID collision energies associated with appearance of the SID fragments may be used as a means of characterizing the relative strengths of the inter-subunit contacts. The coupling of IM with collision-induced dissociation (CID) and SID allowed us to identify and characterize the structures of the conformational intermediates present during the unfolding of several complexes, and thus illustrates its capability to probe the folding mechanisms of protein complexes.
Book Synopsis Development of Ultraviolet Photodissociation Based Tandem Mass Spectrometry Methods for the Characterization of Protein Macromolecular Structures and Glycolipids by : John Patrick O'Brien
Download or read book Development of Ultraviolet Photodissociation Based Tandem Mass Spectrometry Methods for the Characterization of Protein Macromolecular Structures and Glycolipids written by John Patrick O'Brien and published by . This book was released on 2014 with total page 616 pages. Available in PDF, EPUB and Kindle. Book excerpt: Photon-based tandem mass spectrometry provides a versatile ion activation strategy for the analysis of polypeptides, proteins, and lipids. 351-nm ultraviolet photodissociation mass spectrometry (UVPD-MS) is a facile and selective tandem dissociation technique used to elucidate chromophore-modified peptides within large mixtures. A bis-aryl chromogenic chemical probe was utilized to target solvent exposed primary amine residues within native protein states. Collision-induced dissociation (CID) was employed to indiscriminatly characterize the complete proteolytic digest while chromophore containing peptides were selectively dissociated with 351-nm UVPD; thus streamlining the identification of targeted peptides with structurally informative residues. Protein amine residue reactivities were then compared with predicted solvent exposures to elucidate protein tertiary structures, their mechanistic properties, and ligand-binding interactions. High-energy 193-nm UVPD is a fast, high-energy tandem mass spectrometry method and frequently generates fragment ions typically inaccessible to CID-based methods. Native mass spectrometry was coupled to top-down 193-nm UVPD for the gas phase characterization of non-covalent protein-ligand and protein-protein complexes. This method yielded a unique array of fragment ions for a comprehensive analysis of protein structures. UVPD of non-covalent complexes generated many polypeptide backbone fragments to characterize the primary sequence of proteins. Furthermore, top-down UVPD engendered cleavages with intact electrostatic interactions; this provided insight into the binding interfaces within protein-ligand complexes and the higher order structural architectures of oligomeric complexes. High-resolution 193-nm UVPD was paired with high performance liquid chromatography (LC) for the streamlined structural analysis of amphiphilic glycolipids within complex mixtures. For all glycolipids, UVPD provided the most comprehensive structural analysis tool by affording a diverse array of fragment ions to characterize both hydrophobic and hydrophilic moieties. UVPD based LC-MS separations of gangliosides shed light on the ceramide lipid bases, glycan moieties, and their isobaric structural variants. UVPD activation of lipid A and lipooligosaccharides (LOS) compounds generated a mixture of C-C, C-O, and C-N fragment ions to illustrate the hydrophobic acyl structures, while cleavages within the glycosidic, and cross-ring cleavages allowed the determination of acylation patterns. Novel LC-MS separation strategies were developed to elucidate and structurally characterize complex mixtures of lipopolysaccharide containing compounds.
Book Synopsis New Methods for the Analysis of Noncovalent Biological Complexes and Protein Posttranslational Modifications by : Yonghao Yu
Download or read book New Methods for the Analysis of Noncovalent Biological Complexes and Protein Posttranslational Modifications written by Yonghao Yu and published by . This book was released on 2006 with total page 480 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Leveraging Capillary Zone Electrophoresis-mass Spectrometry for Multi-level Proteomics by : Xiaojing Shen
Download or read book Leveraging Capillary Zone Electrophoresis-mass Spectrometry for Multi-level Proteomics written by Xiaojing Shen and published by . This book was released on 2020 with total page 194 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Book Synopsis Identification and Analysis of Proteins Using Matrix Assisted Laser Desorption Ionization and Electrospray Ionization Mass Spectrometry by : Neil Robert Quebbemann
Download or read book Identification and Analysis of Proteins Using Matrix Assisted Laser Desorption Ionization and Electrospray Ionization Mass Spectrometry written by Neil Robert Quebbemann and published by . This book was released on 2020 with total page 143 pages. Available in PDF, EPUB and Kindle. Book excerpt: Sample complexity continues to hinder the effectiveness of Top-Down mass spectrometry, which aims to become a high-throughput platform for proteomics. One possible solution to this issue is the separation and measurement of protein mixtures using virtual 2D gel electrophoresis/mass spectrometry (virtual 2D gel/MS), where intact proteins are initially separated by isoelectric focusing on immobilized pH gradient (IPG) gels followed by mass analysis using matrix-assisted laser desorption/ionization (MALDI) MS. Here, we report on improvements made to the virtual 2D gel/MS platform. With increased automation, we have reduced the time required to acquire and visualize proteins separated on a 180 mm IPG gel from several days to under 1 hour. This automation includes the implementation of a high-speed MALDI time-of-flight mass spectrometer operating with specialized MS imaging software to acquire data. Analysis of the MS data was also automated through the development of a custom program written in MATLAB. Mass spectrometry signal intensity, signal-to-noise ratio, and sensitivity were all improved with a novel MALDI matrix application method where gels are immersed in matrix solution overnight, improving matrix crystallization. We also demonstrate for the first time the use of a 15 Tesla Fourier transform-ion cyclotron resonance mass spectrometer equipped with a MALDI source to acquire virtual 2D gel/MS data offering both an increase in resolution and accuracy of mass measurement results. Using the improved virtual 2D gel/MS technique, we identify changes to the E. coli proteome caused by both cold shock and antibiotic induced stress. The aggregation and accumulation of -synuclein in the brain have been linked to numerous neurodegenerative disorders including Parkinson's disease. To prevent these synucleinopathies much effort has been made to understand the cause of this protein aggregation and to find ways to prevent it. It has been shown that various ligands affect -synuclein's propensity towards aggregation with the small molecule compound, CLR01, a lysine molecular tweezer, decreasing aggregation and divalent heavy metals increasing aggregation. Here, we use electrospray ionization-MS and collision induced unfolding (CIU) coupled with ion mobility spectrometry to probe the effects that CLR01, Mn(II), Co(II), and Cu(II) have on the structural stability of -synuclein in the gas phase. Our results indicate that the binding of CLR01, Mn(II), 1x Cu(II), and 3x Cu(II) all have a stabilizing effect on the structure of the protein while Co(II) destabilizes the protein. The work presented in this thesis demonstrate new mass spectrometry-based experimental platforms to qualitatively and quantitatively profile complex protein mixtures rapidly and accurately, and to probe the structural stability of protein/ligand complexes that are complementary to other biophysical methods.
Book Synopsis Leveraging Native Mass Spectrometry and 193 Nm Ultraviolet Photodissociation as Structural Biology Tools by : Megan Rachel Mehaffey
Download or read book Leveraging Native Mass Spectrometry and 193 Nm Ultraviolet Photodissociation as Structural Biology Tools written by Megan Rachel Mehaffey and published by . This book was released on 2020 with total page 726 pages. Available in PDF, EPUB and Kindle. Book excerpt: Structural biology studies aimed at the elucidation of protein-dependent disease mechanisms have traditionally relied on high-resolution techniques, including X-ray crystallography, nuclear magnetic resonance, and cryogenic electron microscopy. While such methodologies remain standard for gaining information on the core structure of proteins, specific drawbacks including time or large sample quantities associated with these approaches have spawned the development of other pipelines. Mass spectrometry (MS) is one such tool that has gained traction as a rapid and sensitive low-resolution structural biology technique. Routinely protein complexes of interest are reacted in solution with covalent chemical probes to preserve structural information prior to enzymatic digestion and mass spectrometric read-out. However, with the advent of native MS, protein complexes can now be efficiently transferred intact into the gas phase using high ionic strength solutions while retaining structures reminiscent of their solution conformations, and directly interrogated using MS/MS methods. Ultraviolet photodissociation (UVPD) is one such ion activation method that has been extensively developed to break apart protein complexes in a manner that allows conclusions about structure to be drawn based on the fragmentation behavior. The work presented here leverages native mass spectrometry in conjunction with 193 nm UVPD to probe a variety of biologically important protein-ligand and protein-protein complexes. The utility in a native UVPD-MS approach for structural examination of protein-ligand complexes is demonstrated through characterization of conformational changes associated with the catalytic cycle of a phosphotransferase enzyme as well as elucidation of structural changes resulting from mutation or inhibition of an enzyme responsible for conferring antibiotic resistance to bacteria. An oncogenic protein and several clinical variants bound to a downstream effector protein provides an example of the capabilities of native MS and UVPD to characterize the structure of a protein-protein complex. Native UVPD-MS is also used for epitope mapping of the main antigenic determinant of the influenza virus. Aimed at improving analysis of larger complexes, multistage native UVPD-MS is developed to probe the structure of a protein implicated in chemotherapeutic resistance in glioblastoma tumors. Lastly, uniting on-line capillary electrophoresis (CE) with multistage native UVPD-MS offers a high-throughput workflow for structural characterization of ribosomal protein complexes
Book Synopsis Enhancing the Characterization of Intact Proteins by Ultraviolet Photodissociation Mass Spectrometry by : Sean Duncan Dunham
Download or read book Enhancing the Characterization of Intact Proteins by Ultraviolet Photodissociation Mass Spectrometry written by Sean Duncan Dunham and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Access to high resolution mass spectrometers and high energy modes of activation such as electron- and photon-based modalities have enabled wider adoption of top-down methodologies, or strategies that allow the study of intact proteins. However, interpretation of MS/MS spectra of large proteins remains difficult owing to spectral congestion, charge capacity limitations, and other challenges. In particular, for ultraviolet photodissociation (UVPD) of intact proteins, a single laser pulse is typically used to avoid secondary dissociation of fragment ions that occurs when multiple pulses are employed. Consequently, a large amount of the precursor ion population remains undissociated, meaning a large portion of the potential signal is not effectively utilized. Secondary dissociation results in the generation of less informative small terminal and internal fragment ions. Internal fragments are typically ignored due to the computational challenges associated with accounting for them. The following research focuses on the use of fragment ion protection (FIP) during 193 nm UVPD to counter secondary dissociation when utilizing multiple laser pulses and the exploration of the benefits and pitfalls when considering internal fragment ions generated by 193 nm UVPD. In, summary, FIP increased the center sequence coverage of large proteins, but there is room for improvement. The inclusion of internal fragment ions can aid in enhancing the sequence coverage of intact proteins. However, the majority of internal fragment ions are not reliably identified across multiple replicates, reflecting a high risk of false positive identifications when they are considered. These findings are described in this thesis
