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Book Supercharging Methods for Improving Analysis and Detection of Proteins by Electrospray Ionization Mass Spectrometry

Download or read book Supercharging Methods for Improving Analysis and Detection of Proteins by Electrospray Ionization Mass Spectrometry written by Catherine Cassou Going and published by . This book was released on 2015 with total page 155 pages. Available in PDF, EPUB and Kindle. Book excerpt: The characterization of mechanisms, analytical benefits, and applications of two different methods for producing high charge state protein ions in electrospray ionization (ESI) mass spectrometry (MS), or "supercharging", are presented in this dissertation. High charge state protein ions are desirable in tandem MS due to their higher fragmentation efficiency and thus greater amount of sequence information that can be obtained from them. The first supercharging method, supercharging with reagents (typically non-volatile organic molecules), is shown in this work to be able to produce such highly charged protein ions from denaturing solutions that about one in every three residues carries a charge. The high Coulomb repulsion in these ions results in these ions adopting near-linear gas-phase structures with little to no non-covalent interactions, making them ideal for efficient fragmentation in tandem MS experiments and for the minimization of gas phase HD scrambling during tandem MS. Supercharging with reagents from aqueous solutions typically produces much lower charge states as compared to that observed from a denaturing solution. However, two new reagents are presented in this work that increase protein ion charge past that from denaturing solutions when added to aqueous solutions at just 2% by volume. Increases in charge of up to 168% are reported in the presence of these reagents. The mechanism of the increases in protein ion charging with these reagents from aqueous solutions was investigated with fluorescence experiments and correlated to a destabilization of the protein structure by these reagents toward denaturation. The actual protein denaturation event likely occurs in the ESI droplet itself, consistent with previous studies of the mechanism of supercharging with reagents. Thus, efficient tandem MS of high charge states is possible from ESI of aqueous solutions in which a protein maintains its native or native-like structure and activity, enabling tandem MS analysis of protein modifications, ligand binding, or structural changes in real time. Interestingly, another application for supercharging reagents is protein desalting in the ESI droplet. Supercharging reagents bind to sodium ions, resulting in less non-specific sodium ion adduction to proteins, which can improve signal-to-noise ratios of protein ions, lower limits of detection, and enable the detection of bound ligands or specific binding of salts that might otherwise be obscured by sodium adduction. The second supercharging method, electrothermal supercharging (ETS), requires the presence of particular buffer salts rather than organic reagents to increase protein ion charge in the ESI droplet. An investigation of the effect of several different buffer salts on ETS is presented in this work, revealing that the choice of buffer salt is very important to obtaining effective ETS and that buffer salts likely stabilize or destabilize protein structure in the ESI droplet via Hofmeister effects. The application of ETS to tandem MS of proteins produced by ESI and its utility on proteins ranging in size over an order of magnitude (8.6 kDa to 83.0 kDa) is demonstrated. Hydrogen-deuterium exchange experiments can be performed in aqueous solutions and measured continuously with ETS coupled to tandem MS for protein structure analysis in real time with a spatial resolution of 1.3 residues and without gas phase hydrogen-deuterium scrambling. This work demonstrates the wide applicability of ETS for the study of primary and higher order protein structure for small and large proteins alike.

Book Fundamental Studies of Protein Ionization for Improved Analysis by Electrospray Ionization Mass Spectrometry and Related Methods

Download or read book Fundamental Studies of Protein Ionization for Improved Analysis by Electrospray Ionization Mass Spectrometry and Related Methods written by Kevin A. Douglass and published by . This book was released on 2014 with total page 190 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry is an analytical technique in which a sample is converted to gas phase ions that are subsequently separated and detected. It offers great speed, selectivity, and sensitivity during analysis, characteristics which have enabled it to become a leading method for the study of proteins. The applications of MS for these biologically significant macromolecules range from accurately determining identity and sequence to shedding light on post-translational modifications and protein molecule interactions. As a first step towards analysis by MS, gas-phase protein ions must be formed. A common method for ionization is electrospray ionization, where a liquid sample including the protein is charged, nebulized, and evaporated, resulting in bare protein ions. Although ESI has been used in this way for over two decades, many aspects of the protein charging mechanism remain unclear. To address this problem, my research has focused on identifying the factors that determine the extent of protein multiple charging during ESI and improving the ionization of proteins by desorption electrospray ionization. DESI is a method similar to ESI, except that the sample is desorbed from a surface by the spray instead of being present in it from the onset. A simple model was developed that enables the accurate prediction of protein multiple charging observed during ESI-MS if the protein sequence is known. Furthermore, the enhancement of multiple charging that is observed upon the addition of certain organic reagents, a phenomenon known as supercharging, was investigated and a novel mechanism of protein supercharging was proposed. The difficulty in analyzing large proteins by DESI-MS was studied using an innovative approach where DESI was separated into its individual sub-processes and their individual contributions to the DESI process were evaluated. As a result, core limitations to the DESI-MS of large proteins were identified. The results of my cumulative research efforts should lead to the improved MS analysis of proteins by spray ionization methods, including ESI and DESI.

Book Methodologies and Applications for the Analysis of Intact Proteins and Protein ligand Interactions by Top down Mass Spectrometry

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 Protein Analysis using Mass Spectrometry

Download or read book Protein Analysis using Mass Spectrometry written by Mike S. Lee and published by John Wiley & Sons. This book was released on 2017-05-26 with total page 282 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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

Book Mechanisms and Applications of Improved Protein Analysis by Desorption Electrospray Ionization Mass Spectrometry  DESI MS

Download or read book Mechanisms and Applications of Improved Protein Analysis by Desorption Electrospray Ionization Mass Spectrometry DESI MS written by Roshan Javanshad and published by . This book was released on 2021 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Electrospray ionization mass spectrometry (ESI-MS) is a soft ionization technique that allows detection of macromolecules, such as intact proteins, by the formation of multiply charged ions from solutions. Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient ionization technique that directly samples analyte from a surface during ESI-MS analysis. Although DESI-MS is highly accomplished at the analyses of metabolites, lipids, and other small molecules, it is far more limited when it comes to protein analysis. While most of the field in ambient ionization MS has moved towards primarily applications, our approach has been to explore the use of DESI-MS and direct ESI-MS to answer fundamental scientific questions. Understanding the mechanisms by which proteins are analyzed with these techniques provides essential insight into protein behavior and enables improving these techniques even further. The presented work focuses on improving DESI-based protein analysis via solution- phase and gas-phase additives and understanding the underlying mechanisms by which these additives improve protein signal. DESI-MS and complementary direct ESI-MS experiments were used to (1) investigate the effect of amino acid additives on protein signal, (2) understand the mechanism by which amino acid additives improve protein signal during DESI-MS, (3) investigate the effect of organic solvent vapors on protein signal, and (4) incorporate these techniques and findings into developing a novel method for rapid analysis of immobilized His- tagged proteins. As a result, we were able to successfully improve protein analysis by DESI-MS through the addition of L-serine to the desorption solvent. Serine was shown to act as a solubility enhancing additive through improving dissolution of unfolding proteins during the extraction/desorption step of DESI-MS, potentially by inhibiting aggregation. Exposing the DESI-MS sampling region to ethyl acetate vapors also improved the signal intensity of proteins similar to previously reported ESI-MS observations. Finally, the potential application of direct ESI-MS and DESI-MS for rapid analysis of immobilized recombinant His-tagged proteins from Ni-NTA and Cu-NTA coated surfaces was evaluated. We successfully demonstrated the capture and release of recombinant His-tagged human ubiquitin from Ni-NTA and Cu-NTA surfaces by DESI-MS. Furthermore, we show the detection of His-tagged recombinant protein directly out of complex solutions containing the total protein fraction of the E. coli expression system and the lysis buffer, after purifying on Ni- and Cu-NTA plates. This work demonstrated the potential of direct ESI-MS and DESI-MS for rapid analysis of recombinant His-tagged proteins from crude bacterial cell lysate.

Book Improving Protein Analysis by Desorption Electrospray Ionization  DESI MS

Download or read book Improving Protein Analysis by Desorption Electrospray Ionization DESI MS written by Elahe Honarvar and published by . This book was released on 2019 with total page 155 pages. Available in PDF, EPUB and Kindle. Book excerpt: Electrospray ionization mass spectrometry (ESI-MS) is one of the most well-known and versatile techniques for analyzing a broad range of molecules and it has become one of the leading techniques to study biomolecules, such as proteins. ESI-MS can accurately determine the molecular weight of proteins and provide information about their peptide sequence, post-translational modifications as well as their interaction with other molecules. During ESI-MS analysis, by spraying a sample of proteins, prepared in form of a solution, charged droplets are produced using an electric field. As the solvent molecules gradually evaporate from these droplets, freely hovering bare protein ions remain. The ions are then sampled into the mass spectrometer where they are separated and detected based on their mass to charge (m/z) ratios. In the recent years, a new extension of ESI-MS has been developed that allows analysis of molecules from their immediate surroundings. The technique is called desorption electrospray ionization (DESI-MS). In DESI-MS the sample preparation steps take place in close proximity to ionization step. Such features also provide the advantage of surface analysis and imaging to study spatial distribution of molecules. While DESI shares the ionization mechanism of ESI-MS, it lacks its ability to analyze large biopolymers, and struggles to analyze proteins larger than 25kDa. Previously our research group suggested that the loss in protein signal intensity was not due to problems with physical desorption or ionization, but rather due to incomplete protein dissolution during the desorption step. The studies conducted in this dissertation address this shortcoming by improving protein dissolution during DESI-MS. Effect of addition of volatile ammonium salts during DESI is studied, among which ammonium bicarbonate shows significant improvement in signal to noise (S/N) ratio of proteins, specifically those with higher isoelectric points (pI). The improved S/N ratio seems to be caused by extensive removal of potassium from the protein ions. While these additives lead to improvements in the performance of DESI, their addition does not cause the same effect in ESI. The different effects of these additives in DESI and ESI are studied in terms of proteins signal intensity, S/N ratio as well as charge state distribution. The effect of addition of the amino acid of serine to the electrospray solvent of DESI is investigated. For proteins with different molecular weights and pI values, serine shows promising improvements in the signal intensity. Application of vaporized organic reagents in the nebulizing gas flow of the electrospray solvent of DESI is described. To add these vapors, DESI sprayer is enclosed and the vapor is delivered to the inner environment of the enclosure. By adding the vapor of ethyl acetate during DESI analysis of proteins, the attained signal intensity is increased. Such improvements can potentially be combined during a single analysis to further better the outcome of protein analysis by DESI-MS.

Book Biophysical Mass Spectrometry Techniques for Probing the Higher Order Structure of Proteins and Complexes

Download or read book Biophysical Mass Spectrometry Techniques for Probing the Higher Order Structure of Proteins and Complexes written by Sterling, Jr. (Harry John) and published by . This book was released on 2012 with total page 366 pages. Available in PDF, EPUB and Kindle. Book excerpt: Electrospray ionization mass spectrometry (ESI-MS) is a powerful analytical platform for answering a wide variety of questions about the identity, quantity, structure, function, dynamics and energetics of biological molecules. Key advantages of ESI-MS include unrivaled specificity, attomole sensitivity, and the capacity for simultaneous analysis of complex mixtures with analyte masses that differ by less than 1 ppm. The low flow rates and sub-micron sized droplets formed with "nano" ESI allows biomolecular ions to be readily formed from purely aqueous or buffered aqueous solutions, and these ions have been shown to retain a "memory" of their solution-phase structures so that higher-order structural information can be obtained directly from a gas-phase measurement. All of the work described in this dissertation was undertaken in an effort to develop new nanoESI-based techniques that augment the existing array of biophysical mass spectrometry techniques for probing the structure/function relationships of biological molecules in their native environments. In part one, a hypothesis for the origin of nanoESI "supercharging" is developed and exhaustively tested utilizing a variety of solution- and gas-phase techniques with a range of different proteins and protein complexes. The results of all of these studies support the hypothesis that the origin of aqueous solution supercharging is the rapid chemical and/or thermal denaturation of a protein or protein complex analyte in an evaporating ESI droplet due to enrichment of the reagent caused by its high boiling point relative to that of water. Aqueous solution supercharging has recently been used in a variety of new applications and an understanding of its underlying mechanism is therefore essential. In part two, two new biophysical mass spectrometry applications are described. The first is a tandem-MS application of aqueous solution supercharging for obtaining hydrogen/deuterium exchange (HDX) rate constants in real-time with nearly single amino acid spatial resolution, and the second describes an MS method to obtain the quaternary structure of protein complexes that require high concentrations of essential salts. Finally, two ideas for new HDX-MS methods that capitalize on the mechanism of aqueous solution supercharging are outlined.

Book Applied Surface Thermodynamics

Download or read book Applied Surface Thermodynamics written by Jan Spelt and published by CRC Press. This book was released on 1996-06-27 with total page 670 pages. Available in PDF, EPUB and Kindle. Book excerpt: Offers a treatment of applied surface dynamics in relation to contact angles and surface tensions, providing a foundation for the subject and detailed presentations of recent techniques. The work supplies a theoretical framework for the study and measurement of surface tensions and contact angles, and acts as a day-to-day guide for laboratory pract

Book Computational and Statistical Methods for Protein Quantification by Mass Spectrometry

Download or read book Computational and Statistical Methods for Protein Quantification by Mass Spectrometry written by Ingvar Eidhammer and published by John Wiley & Sons. This book was released on 2012-12-10 with total page 290 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Book Analysis of Protein Post Translational Modifications by Mass Spectrometry

Download or read book Analysis of Protein Post Translational Modifications by Mass Spectrometry written by John R. Griffiths and published by John Wiley & Sons. This book was released on 2016-11-07 with total page 414 pages. Available in PDF, EPUB and Kindle. Book excerpt: Covers all major modifications, including phosphorylation, glycosylation, acetylation, ubiquitination, sulfonation and and glycation Discussion of the chemistry behind each modification, along with key methods and references Contributions from some of the leading researchers in the field A valuable reference source for all laboratories undertaking proteomics, mass spectrometry and post-translational modification research

Book Development of Electrospray Ionization   Mass Spectrometry for Analysis of Water Soluble and Membrane Proteins and Educational Protocols for an Analytical Chemistry Class

Download or read book Development of Electrospray Ionization Mass Spectrometry for Analysis of Water Soluble and Membrane Proteins and Educational Protocols for an Analytical Chemistry Class written by Wonhyeuk Jung and published by . This book was released on 2021 with total page 160 pages. Available in PDF, EPUB and Kindle. Book excerpt: Native mass spectrometry (MS) is a branch of MS analysis in which the structure of the target analytes of interest are kept intact and remaining in their "native" functional structure (as much as possible). This approach was made possible by the development of electrospray ionization (ESI), a soft ionization technique that does not fragment the target analyte during the ionization process while inducing multiple charging. The multiply charged biomolecules, in turn, can be subjected to fragmentation via collisional activation with a non-reactive gas such as nitrogen. This approach of combining native MS with fragmentation-based analysis, termed native topdown MS analysis, can be applied to large biomolecules such as membrane proteins to gain structural insights. Membrane proteins present unique challenges to conventional high-resolution structural techniques due to their hydrophobic nature. However, they are responsible for various physiological phenomena and account for 60% of known druggable targets in the cell. Thus, there is a need for an approach that can overcome issues with membrane protein analysis while complementing other biophysical techniques used to probe protein structure. Here, how native top-down MS can play this role is presented. The effects of non-ionic saccharide-based detergents, a commonly used class of detergents for membrane protein solubilization, on the resulting charge states of soluble proteins is investigated to gain insights into the mechanism of ESI. The MS-fragmentation patterns from collisionally activated dissociation of membrane proteins and membrane protein-lipid complexes are compared. How new insights into the lipid binding sites can be gained by detecting lipid-bound MS-fragments is presented. The result of the study indicates that native top-down MS analysis can provide unique structural insights for membrane proteins and their non-covalent interactions. When the analytical goal is to investigate the atomic composition of the target analyte, an ionization approach in which the sample is fully atomized before MS analysis is preferred instead. Inductively coupled plasma ionization, which atomizes and ionizes the sample via a plasma, can be coupled with MS analysis (ICP-MS) to quantify heavy metal contamination in complex samples. A protocol for ICP-MS analysis of commercial fish products for mercury contamination detection developed to aid an analytical chemistry class for instruction of undergraduate chemistry students is presented.

Book Protein and Peptide Mass Spectrometry in Drug Discovery

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 Probing Effects on Protein Charge State Distributions by Electrospray Ionization Mass Spectrometry

Download or read book Probing Effects on Protein Charge State Distributions by Electrospray Ionization Mass Spectrometry written by Ping Xu and published by . This book was released on 2006 with total page 169 pages. Available in PDF, EPUB and Kindle. Book excerpt: All mass spectrometers combine ion formation, mass analysis and ion detection. The first chapter of this thesis introduces the fundamentals of some common ionization techniques, especially electrospray ionization source (ESI), and some common mass analyzers, along with their advantages and disadvantages.

Book Characterization of Protein Therapeutics using Mass Spectrometry

Download or read book Characterization of Protein Therapeutics using Mass Spectrometry written by Guodong Chen and published by Springer Science & Business Media. This book was released on 2014-07-08 with total page 408 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Book Novel Analytical Methods for Examining Biomolecular Complexes Using Electrospray Ionization Mass Spectrometry

Download or read book Novel Analytical Methods for Examining Biomolecular Complexes Using Electrospray Ionization Mass Spectrometry written by Tawnya Grace Flick and published by . This book was released on 2012 with total page 344 pages. Available in PDF, EPUB and Kindle. Book excerpt: Several analytical strategies and investigations are presented in this dissertation to improve the quantification, sensitivity, and structural information that can be obtained for gaseous biomolecular ions in electrospray ionization (ESI) mass spectrometry (MS) experiments. Internal or external standards are commonly employed to quantify molecules in complex mixtures because molecular ion abundances cannot be directly related to the concentration of the molecules in solution. A new standard-free quantitation method is used to obtain the relative concentrations of components in a mixture using the abundances of large, nonspecific clusters formed by ESI. Large non-covalent clusters overcome differences in ionization efficiencies between molecules, and are representative of the solution-phase mixture. The sensitivity in MS experiments can be significantly lowered by the presence of high concentrations of salts in the ESI solution because nonspecific ion adduction to biomolecules distributes ion signal into different forms with various numbers of adducts. Studies here demonstrate the extent of both sodium ion and acid molecule adduction to proteins are inversely related, and both depend significantly on the proton affinity of the anion in the ESI solution. Several solution-phase additives that contain anions with low proton affinity values are shown to effectively desalt protein ions generated by ESI, which should result in improved detection limits, more accurate mass measurements, and improved tandem MS sensitivity. Additionally, a solution-phase additive (HClO4) is discovered that can be used to count the number of basic sites accurately in peptides and proteins based on the number of HClO4 adducts to low charge states. High charge states of peptides and proteins can be readily formed by ESI of aqueous solutions that contain trivalent metal ions, and fragmentation of these trivalent metal ion-peptide or protein complexes by electron capture dissociation can be used to increase the structural information obtained from these experiments. Metal ion-biomolecule interactions are ubiquitous in nature where they play a role in many biological processes. Here, nonspecific metal ion adduction to protein cation and anions is shown to result in more compact conformations compared to the bare protein ion, likely a result of salt-bridge interactions between the metal ion and the biomolecule.

Book Developing Top down Mass Spectrometry for Intact Protein Identification in the Chromatographic Timescale

Download or read book Developing Top down Mass Spectrometry for Intact Protein Identification in the Chromatographic Timescale written by Rajeswari Lakshmanan and published by . This book was released on 2012 with total page 225 pages. Available in PDF, EPUB and Kindle. Book excerpt: Protein identification by top-down mass spectrometry based methods yield intact mass of the proteins and indicate the presence of post-translational modifications (PTMs) and/or isoforms. Currently, the methods employed for top-down protein identification are performed using instruments with dual mass analyzers and are based on fragmenting isolated charge states, which greatly reduces the duty cycle of the instrument. High throughput top-down methods are required for protein identification in complex sample mixtures. We demonstrate the capability to perform intact protein identifications in a single-stage time-of-flight mass spectrometer during protein elution from a liquid chromatography (LC) column. In addition, we have developed a new data-independent fragmentation method known as `Continuous Accumulation of Selected Ions-Collisionally Activated Dissociation' (CASI-CAD) to fragment multiple charge states of the protein simultaneously for the purpose of identification in the LC timescale. CASI-CAD is performed without any precursor selection and thus, the duty-cycle of the instrument is not lowered. Both these methods unambiguously identified all the proteins in the human proteasome complex used for method development. The presence of PTMs and N-terminal modifications were also characterized for the proteins in this complex. Supercharging reagents are known for their ability to enhance the multiple charging of proteins during electrospray ionization (ESI). This improves the mass measurement accuracy and fragmentation efficiency of proteins during ESI-MS. Currently, the mechanism behind supercharging is unknown. We have analyzed different supercharging reagents under a variety of solvent conditions to probe the mechanisms behind supercharging. In addition, the supercharging ability of sulfolane was utilized for proteins eluting from a column by adding the reagent to the LC solvents. Furthermore, reagent introduction in the vapor phase increased the signal intensity for intact proteins eluting from a column when compared to experiments performed without the reagent. These methods presented here are efficient top-down means to address complex samples in the chromatographic timescale.

Book Charging of Proteins and Protein Complexes in Native Mass Spectrometry

Download or read book Charging of Proteins and Protein Complexes in Native Mass Spectrometry written by Anna Christine Susa and published by . This book was released on 2017 with total page 112 pages. Available in PDF, EPUB and Kindle. Book excerpt: Electrospray ionization (ESI) mass spectrometry is a powerful analytical tool for investigating the identities, structures, functions, and energetics of biomolecules. ESI transfers intact molecules from buffered aqueous solutions in which they are folded or in native conformations into the gas phase for mass spectral analysis. ESI is widely used for the analysis of proteins in mass spectrometry, but the factors that influence charging of protein ions formed by ESI are not well understood. Higher charge states of protein ions are desirable because they fragment more easily in tandem mass spectrometry methods, leading to more sequence coverage than lower charge protein ions. Lower charge protein ions are advantageous in native mass spectrometry when preservation of native protein conformation is desirable. The work described in this dissertation explores factors that control charging of macromolecular ions in native mass spectrometry. This work provides evidence that the charging of macromolecular ions is not significantly limited by ion evaporation of cations, such as alkylammonium or alkali metal ions, or proton transfer to salts that are commonly added to aqueous buffered ESI solutions. In addition, a novel and simple method for ESI of proteins directly from buffers commonly used in biochemistry laboratories is demonstrated. In native mass spectrometry, protein solutions are typically desalted and buffer exchanged into volatile ammonium salt buffers to prevent salt adduction to the protein ions because salt adduction significantly hinders detection and sensitivity of mass spectral analysis. However, some salts are essential for protein structure and function, and this new method of ion desalting allows formation of protein and protein complex ions directly from buffers that contain high ionic strengths of nonvolatile salts to mimic the intracellular and extracellular environments. This technique greatly impacts the way native mass spectrometry is performed because it eliminates the need to reinvestigate properties of the proteins and protein complexes in traditional ammonium salt buffers used in mass spectrometry. Therefore, biochemists will no longer need to adapt their protein solutions to make them suitable for mass spectrometry.