Development Of Top Down Mass Spectrometry Methods For Structural Characterization Of Protein Macromolecules Utilizing 193nm Ultraviolet Photodissociation

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Development of Top-down Mass Spectrometry Methods for Structural Characterization of Protein Macromolecules Utilizing 193nm Ultraviolet Photodissociation

Author : Michael B. Cammarata
Publisher :
ISBN 13 :
Total Pages : 322 pages
Book Rating : 4.:/5 (15 download)

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Book Synopsis Development of Top-down Mass Spectrometry Methods for Structural Characterization of Protein Macromolecules Utilizing 193nm Ultraviolet Photodissociation by : Michael B. Cammarata

Download or read book Development of Top-down Mass Spectrometry Methods for Structural Characterization of Protein Macromolecules Utilizing 193nm Ultraviolet Photodissociation written by Michael B. Cammarata and published by . This book was released on 2016 with total page 322 pages. Available in PDF, EPUB and Kindle. Book excerpt: The dissertation will discuss the advancement of informative structural biology techniques utilizing a top-down centric workflow with 193nm ultraviolet photodissociation (UVPD) mass spectrometry. Native electrospray ionization is used to transport proteins to the gas phase in a native-like state, then UVPD is used for structural characterization to reveal ligand binding sites within a protein-ligand complex as well as detect conformational changes based upon the suppression or enhancement of backbone cleavages. Conformational changes induced by ligand exchange or removal and single amino acid mutations as well as combinations of the two (ligands and mutations) are investigated. The rich fragmentation patterns of UVPD are also used for structural characterization of crosslinked proteins. Typically these crosslinking experiments are performed by bottom-up mass spectrometry with has significant shortcomings. The main drawback is the need for proteolysis which cuts proteins into small peptides, thus increasing the complexity of the samples and its subsequent analysis. Additionally this proteolysis step loses the post-translation modification information or amino acid mutations that may be driving a specific protein-protein interaction. Top-down methods avoid protein digestion and thus are used to directly evaluate the protein interactions or protein complexes. These two methodologies will bring the mass spectrometry and structural biology community a step closer to the realization of high-throughput structural biology for proteins and their interactions with other proteins and small molecules.

Development of Ultraviolet Photodissociation Based Tandem Mass Spectrometry Methods for the Characterization of Protein Macromolecular Structures and Glycolipids

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Author : John Patrick O'Brien
Publisher :
ISBN 13 :
Total Pages : 616 pages
Book Rating : 4.:/5 (919 download)

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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.

Development of Top-down Methods for Evaluating Protein Structure and Protein Unfolding Utilizing 193 Nm Ultraviolet Photodissociation Mass Spectrometry

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Author : Michael B. Cammarata
Publisher :
ISBN 13 :
Total Pages : 134 pages
Book Rating : 4.:/5 (953 download)

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Book Synopsis Development of Top-down Methods for Evaluating Protein Structure and Protein Unfolding Utilizing 193 Nm Ultraviolet Photodissociation Mass Spectrometry by : Michael B. Cammarata

Download or read book Development of Top-down Methods for Evaluating Protein Structure and Protein Unfolding Utilizing 193 Nm Ultraviolet Photodissociation Mass Spectrometry written by Michael B. Cammarata and published by . This book was released on 2014 with total page 134 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ultraviolet photodissocation (UVPD) mass spectrometry was used for high mass accuracy top down characterization of two proteins labeled by the chemical probe, S-ethylacetimidate (SETA), in order to evaluate conformational changes as a function of denaturation. The SETA labeling/UVPD-MS methodology was used to monitor the mild denaturation of horse heart myoglobin by acetonitrile, and the results showed good agreement with known acetonitrile and acid unfolding pathways of myoglobin. UVPD outperformed another ion activation method, electron transfer dissociation (ETD), in terms of sequence coverage, allowing the SETA reactivity of greater number of lysine amines to be monitored and thus providing a more detailed map of myoglobin. This strategy was applied to the third zinc-finger binding domain, domain C, of PARP-1 (PARP-C), to evaluate the discrepancies between the NMR and crystal structures which reported monomer and dimer forms of the protein, respectively. The trends reflected from the reactivity of each lysine as a function of acetonitrile denaturation supported that PARP-C exists as a monomer in solution with a close-packed C-terminal alpha helix. Additionally, those lysines for which the SETA reactivity increased under denaturing conditions were found to engage in tertiary polar contacts such as salt bridging and hydrogen bonding, providing evidence that the SETA/UVPD-MS approach offers a versatile means to probe the interactions responsible for conformational changes in proteins. UVPD mass spectrometry was also employed to investigate the structure of holo-myoglobin as well as its apo form transferred to the gas phase by native electrospray. The fragmentation yields from UVPD showed the greatest overall correlation with B-factors generated from the crystal structure of apo-myoglobin, particularly for the more disordered loop regions. Comparison of UVPD of holo- and apo- myoglobin revealed similarities in fragmentation yields, particularly for the lower charge states (8 and 9+), but those regions involved in harboring the heme group (for the holo form) exhibited significantly lower fragmentation than the apo-myoglobin state. Both holo- and apo-myoglobin exhibited low fragmentation yields for the AGH helical core (reflecting its highest stability).

Leveraging Native Mass Spectrometry and 193 Nm Ultraviolet Photodissociation as Structural Biology Tools

Author : Megan Rachel Mehaffey
Publisher :
ISBN 13 :
Total Pages : 726 pages
Book Rating : 4.:/5 (124 download)

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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

Development of Tandem Mass Spectrometric Methods for Proteome Analysis Utilizing Photodissociation and Ion/ion Reactions

Author : Jared Bryan Shaw
Publisher :
ISBN 13 :
Total Pages : 366 pages
Book Rating : 4.:/5 (858 download)

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Book Synopsis Development of Tandem Mass Spectrometric Methods for Proteome Analysis Utilizing Photodissociation and Ion/ion Reactions by : Jared Bryan Shaw

Download or read book Development of Tandem Mass Spectrometric Methods for Proteome Analysis Utilizing Photodissociation and Ion/ion Reactions written by Jared Bryan Shaw and published by . This book was released on 2013 with total page 366 pages. Available in PDF, EPUB and Kindle. Book excerpt: The utility of 193 nm ultraviolet photodissociation (UVPD) and negative electron transfer dissociation (NETD) for the characterization of peptide anions was systematically evaluated. UVPD outperformed NETD in nearly all metrics; however, both methods provided complementary information to traditional collision induced dissociation (CID) of peptide cations in high throughput analyses. In order to enhance the performance of NETD, activated ion negative electron transfer dissociation (AI-NETD) methods were developed and characterized. The use of low-level infrared photoactivation or collisional activation during the NETD reaction period significantly improved peptide anion sequencing capabilities compared to NETD alone. Tyrosine deprotonation was shown to yield preferential electron detachment upon NETD or UVPD, resulting in N - C[alpha] bond cleavage N-terminal to the tyrosine residue. LC-MS/MS analysis of a tryptic digest of BSA demonstrated that these cleavages were regularly observed under high pH conditions. Transmission mode desorption electrospray ionization (TM-DESI) was coupled with 193 nm UVPD and CID for the rapid analysis and identification of protein digests. Comparative results are presented for TM-DESI-MS/CID and TM-DESI-MS/UVPD analyses of five proteolyzed model proteins. In some cases TM-DESI/UVPD outperformed TM-DESI-MS/CID due to the production of an extensive array of sequence ions and the ability to detect low m/z product ions. 193 nm UVPD was implemented in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa was achieved. The high-energy activation afforded by UVPD exhibited far less precursor ion charge state dependence than conventional methods, and the viability of 193 nm UVPD for high throughput top-down proteomics analyses was demonstrated for the less 30 kDa protein from a fractionated yeast cell lysate. The use of helium instead of nitrogen as the C-trap and HCD cell bath gas and trapping ions in the HCD cell prior to high resolution mass analysis significantly reduced the signal decay rate for large protein ions. As a result, monoclonal IgG1 antibody was isotopically resolved and mass accurately determined. A new high mass record for which accurate mass and isotopic resolution has been achieved (148,706.3391 Da ± 3.1 ppm) was established.

Enhancing the Characterization of Intact Proteins by Ultraviolet Photodissociation Mass Spectrometry

Author : Sean Duncan Dunham
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (136 download)

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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

Advancement of Photodissociation Mass Spectrometry Methods for the Analysis of Protein Post-translational Modifications

Author : Michelle Renee Robinson
Publisher :
ISBN 13 :
Total Pages : 410 pages
Book Rating : 4.:/5 (15 download)

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Book Synopsis Advancement of Photodissociation Mass Spectrometry Methods for the Analysis of Protein Post-translational Modifications by : Michelle Renee Robinson

Download or read book Advancement of Photodissociation Mass Spectrometry Methods for the Analysis of Protein Post-translational Modifications written by Michelle Renee Robinson and published by . This book was released on 2016 with total page 410 pages. Available in PDF, EPUB and Kindle. Book excerpt: Post-translational modifications (PTMs) are important for regulating protein structure and function. Despite significant progress for PTM analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS), opportunities for new method development remain. The research presented in this dissertation promotes 193 nm ultraviolet photodissociation (UVPD) as an alternative activation technique for PTM analysis with specific utility for phosphorylated and sulfated peptides. A novel de novo sequencing method with applications for unbiased PTM discovery was developed utilizing Lys-N proteolysis, N-terminal imidazolinylation, and UVPD to direct fragmentation for the formation of N-terminal ions. The N-terminal a, b, and c ions generated by UVPD were differentiated from one another by characteristic mass shifts. Sets of triplet peaks were used to distinguish N-terminal ions from confounding C-terminal ions and improve the accuracy of de novo sequencing. UVPD was evaluated for the analysis of phosphopeptide cations and anions. Negative mode analysis was advantageous for the detection of casein peptides in high phosphorylation states, while positive mode proved more robust for global phosphoproteomic analysis of HeLa and HCC70 cell lysates. Compared to collisional activation, the depth of coverage was lower using UVPD yet more extensive fragmentation and improved phosphate retention on products ions was achieved. Phosphorylation mapping by LC-UVPD-MS was carried out in the C-terminal domain (CTD) of RNA polymerase II as a function of kinase treatment, ERK2 or TFIIH, and organism, yeast or fruit fly. Single phosphorylations on Ser2 or Ser5 in the consensus heptad, YSPTSPS, were observed across all experimental conditions. Analysis of the non-consensus fruit fly CTD revealed the significance of Tyr1 and Pro residues in the +1 position relative to Ser for phosphorylation to occur. For sulfated peptides, negative mode UVPD yielded a and x ions that largely retained the labile sulfate modification, facilitating peptide sequencing and PTM localization. With appropriate MS/MS tools established, the next step towards global sulfoproteomics was the development of enrichment methods. Weak anion exchange (WAX) was applied for this purpose. Following carbamylation to neutralize primary amines which otherwise repel the anion exchanger; improved WAX retention was observed for sulfopeptides relative to a complex mixture of unmodified bovine serum albumin peptides.

Ultraviolet Spectroscopy of Proteins

Author : Alexander P. Demchenko
Publisher : Springer Science & Business Media
ISBN 13 : 3642708471
Total Pages : 323 pages
Book Rating : 4.6/5 (427 download)

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Book Synopsis Ultraviolet Spectroscopy of Proteins by : Alexander P. Demchenko

Download or read book Ultraviolet Spectroscopy of Proteins written by Alexander P. Demchenko and published by Springer Science & Business Media. This book was released on 2013-11-11 with total page 323 pages. Available in PDF, EPUB and Kindle. Book excerpt: The aim of this book is to give a comprehensive description of the basic methods used in the ultraviolet spectroscopy of proteins, to discuss new trends and development of these methods, and to analyze their different applications in the study of various aspects of protein structure and dynamics. Ultraviolet spectroscopy is one of the oldest and most popular methods in the field of biochemistry and molecular biophysics. At present, it is difficult to imagine the biochemical laboratory without a recording spectrophotometer or spectrofluorimeter. There are several hundreds of publications directly devoted to protein ultraviolet spectroscopy and in a great number of studies UV spectroscopic methods are used for the structural analysis of different proteins. Meanwhile a unified description of the theoretical basis of the methods, experimental techniques, data analysis, and generalization of results obtained in solving the specific problems of protein structure are lacking. There are three reasons for which a monograph on ultraviolet spectroscopy is needed today. Firstly, there has been significant growth in facilities of experimental technique, its precision, and versatility associated with computer data analysts. This new technique is available to a wide circle of scientists engaged in the field of protein research. Most of them are not spectroscopists and, thus, there is a need for a conceivable and precise source of information on how to use this method and what kind of data it should provide.

Characterization of Proteins and Peptides Via Enhanced 266 Nm Ultraviolet Photodissociation Mass Spectrometry Utilizing a Selenium Based Chromophore

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Author : William Ryan Parker
Publisher :
ISBN 13 :
Total Pages : 210 pages
Book Rating : 4.:/5 (14 download)

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Book Synopsis Characterization of Proteins and Peptides Via Enhanced 266 Nm Ultraviolet Photodissociation Mass Spectrometry Utilizing a Selenium Based Chromophore by : William Ryan Parker

Download or read book Characterization of Proteins and Peptides Via Enhanced 266 Nm Ultraviolet Photodissociation Mass Spectrometry Utilizing a Selenium Based Chromophore written by William Ryan Parker and published by . This book was released on 2016 with total page 210 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry and chemical derivatization have been used as tools for the identification of proteins in both top-down and bottom-up studies. Cysteine is the rarest and most nucleophilic amino acid thus making it a popular target for chemical tagging strategies. Ultraviolet photodissociation (UVPD) is a versatile activation technique for fragmentation of peptides and proteins. For successful photodissociation, ions of interest must contain a suitable chromophore that matches the wavelength of irradiation. N-(Phenylseleno)phthalimide (NPSP) is a fast reacting reagent which attaches a selenium based chromophore that absorbs at 266 nm light to free thiols. In the studies presented in this thesis, NPSP was used to derivatize free cysteine residues in both intact proteins and tryptic peptides. Activation with 266 nm photons causes a dominant neutral loss of the benzeneselenol groups on the tagged protein or peptide ions. This diagnostic neutral loss allows the determination of the number of free versus bound cysteine residues in intact proteins. Additionally, tagging peptides with benzeneselenol provides a means to target only the cysteine-containing peptides in bottom-up proteomics experiments. Both of these methods provide a significantly reduced search space for identification of cysteine-containing proteins. Counting the number of cysteine residues also provides an effective way to restrict the number of protein candidates for database searches. Moreover, cysteine peptides are inherently more unique than other peptides created upon enzymatic digestion of proteins due to the low frequency of cysteine in the proteome, thus allowing these peptides to be used as surrogates for protein identification.

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

Author : Michael Nshanian
Publisher :
ISBN 13 :
Total Pages : 175 pages
Book Rating : 4.:/5 (15 download)

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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.

Advancement of Photodissociation and Electron-based Tandem Mass Spectrometry Methods for Proteome Analysis

Author : James Andrew Madsen
Publisher :
ISBN 13 :
Total Pages : 378 pages
Book Rating : 4.:/5 (756 download)

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Book Synopsis Advancement of Photodissociation and Electron-based Tandem Mass Spectrometry Methods for Proteome Analysis by : James Andrew Madsen

Download or read book Advancement of Photodissociation and Electron-based Tandem Mass Spectrometry Methods for Proteome Analysis written by James Andrew Madsen and published by . This book was released on 2011 with total page 378 pages. Available in PDF, EPUB and Kindle. Book excerpt: The number and types of diagnostic ions obtained by infrared multiphoton dissociation (IRMPD) and collision induced dissociation (CID) were evaluated for supercharged peptide ions created by electrospray ionization of solutions spiked with mnitrobenzyl alcohol. IRMPD of supercharged peptide ions increased the sequence coverage compared to that obtained by CID for all charge states investigated. Multiply charged, N-terminally derivatized peptides were subjected to electron transfer reactions to produce singly charged, radical species. Upon subsequent "soft" CID, highly abundant z-type ions were formed nearly exclusively, which yielded simplified fragmentation patterns amenable to de novo sequencing methods. Furthermore, the simplified series of z ions were shown to retain labile phosphoric acid moieties. Infrared multiphoton dissociation (IRMPD) was implemented in a novel dual pressure linear ion trap for rapid "top-down" proteomics. Due to secondary dissociation, IRMPD yielded product ions in significantly lower charge states as compared to CID, thus facilitating more accurate mass identification and streamlining product ion assignment. This outcome was especially useful for database searching of larger proteins (~29 kDa) as IRMPD substantially improved protein identification and scoring confidence. Also, IRMPD showed an increased selectivity towards backbone cleavages N-terminal to proline and C-terminal to acidic residues (especially for the lowest precursor charge states). Ultraviolet photodissociation (UVPD) at 193 nm was implemented on a linear ion trap mass spectrometer for high-throughput proteomic workflows. Upon irradiation by a single 5 ns laser pulse, efficient photodissociation of tryptic peptides was achieved with production of a, b, c, x, y, and z sequence ions, in addition to immonium ions and v and w side-chain loss ions. The factors that influence the UVPD mass spectra and subsequent in silico database searching via SEQUEST were evaluated. 193 nm ultraviolet photodissociation (UVPD) was employed to sequence singly and multiply charged peptide anions. Upon dissociation by this method, a-/x-type, followed by d and w side-chain loss ions, were the most prolific and abundant sequence ions, often yielding 100% sequence coverage. LC-MS/UVPD analysis using high pH mobile phases yielded efficient characterization of acidic peptides from mitogen-activated protein kinases.

Mass Spectrometry Analysis for Protein-Protein Interactions and Dynamics

Author : M. Chance
Publisher : John Wiley & Sons
ISBN 13 : 0470258861
Total Pages : 325 pages
Book Rating : 4.4/5 (72 download)

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Book Synopsis Mass Spectrometry Analysis for Protein-Protein Interactions and Dynamics by : M. Chance

Download or read book Mass Spectrometry Analysis for Protein-Protein Interactions and Dynamics written by M. Chance and published by John Wiley & Sons. This book was released on 2008-09-22 with total page 325 pages. Available in PDF, EPUB and Kindle. Book excerpt: Presents a wide variety of mass spectrometry methods used to explore structural mechanisms, protein dynamics and interactions between proteins. Preliminary chapters cover mass spectrometry methods for examining proteins and are then followed by chapters devoted to presenting very practical, how-to methods in a detailed way. Includes footprinting and plistex specifically, setting this book apart from the competition.

Mass Spectrometry-based Structural Proteomics

Author : Hao Zhang
Publisher :
ISBN 13 :
Total Pages : 189 pages
Book Rating : 4.:/5 (758 download)

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Book Synopsis Mass Spectrometry-based Structural Proteomics by : Hao Zhang

Download or read book Mass Spectrometry-based Structural Proteomics written by Hao Zhang and published by . This book was released on 2011 with total page 189 pages. Available in PDF, EPUB and Kindle. Book excerpt: Converting gene-sequence information into functional information about a protein is a major challenge of post-genomic biology. Proteins have a variety of functions from serving as catalysts to acting as structural components; all these functions are closely related to protein structure. The first step to understand protein function is often a structural study of that protein. Two major approaches, NMR spectroscopy and X-ray crystallography, can provide an atomic-level, 3D structural model of a protein. The applications of these high resolution approaches, however, are limited by protein size, conformational flexibility, and aggregation propensity. To obtain complementary structural information about proteins, a variety of approaches from traditional structural biology (e.g., circular dichroism, fluorescence spectroscopy) to new advances (e.g., computational prediction, protein footprinting) are required. Mass spectrometry (MS) has become an important tool for studying protein structure, dynamics, interactions, and function. In particular, detailed characterization of protein-ligand interactions is now possible, a critical step toward understanding biological function. Mass spectrometric analysis of protein structure can take two approaches. First, protein-ligand interactions can be probed by chemical labeling followed by MS analysis to determine the resulting mass shift (extent of labeling) and the location of the labeling. This approach in a titration format gives protein-ligand affinities. The labeling takes place in solution, where biochemistry occurs, and can be under physiological conditions, whereas the mass spectrometer is used for analysis typically by bottom-up proteomic strategies. In the other approach, protein assemblies can also be transferred directly into the gas phase and interrogated by MS to afford structural insights. One can view this is a top-down approach. The measurements refer to a gas-phase species, and that raises the question of whether the outcomes of the measurements have relevance to the structure and properties of proteins in solution or in a living system. Although there are differences in experimental format, results, and sensitivity between the two approaches of MS-based protein structural analysis, the similarity of those approaches must not be overlooked. All MS-based structural analyses rely heavily on the identification of peptides, purified protein species, or protein complexes. This analysis has been accelerated by the developments of MS instrumentation and methodology in protein analysis; the structural information provided by MS-based analysis is greatly facilitated by having a structural model of the protein. The integrated results from MS approaches, traditional structural biology approaches (e.g., NMR and X-ray), and computational modeling give more complete structural information of proteins than that from any one of the approaches alone. In the first part of thesis, we focus on the development and application of chemical-labeling methods (protein footprinting) in studies of protein conformation. In the second part, a combined top-down approach of native ESI and electron-capture dissociation (ECD) in FTICR MSis presented for structural studies of protein assemblies in the gas phase.

Development of Mass Spectrometric Methods for Proteomics Analysis Utilizing Gas-phase Chemistry and Ultraviolet Photodissociation

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Author : Dustin Donald Holden
Publisher :
ISBN 13 :
Total Pages : 342 pages
Book Rating : 4.:/5 (18 download)

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Book Synopsis Development of Mass Spectrometric Methods for Proteomics Analysis Utilizing Gas-phase Chemistry and Ultraviolet Photodissociation by : Dustin Donald Holden

Download or read book Development of Mass Spectrometric Methods for Proteomics Analysis Utilizing Gas-phase Chemistry and Ultraviolet Photodissociation written by Dustin Donald Holden and published by . This book was released on 2016 with total page 342 pages. Available in PDF, EPUB and Kindle. Book excerpt: The utility of ultraviolet photodissociation (UVPD) in comparison to higher-energy collisional dissociation (HCD) to provide sequence coverage was assessed for various protein cation charge states and sizes. UVPD provided consistently higher sequence coverages through more uniform fragment ion distribution along the protein sequence. HCD provided lower sequence coverage values as well as more preference towards cleavage at the most labile bonds. Assessment of coverage dependence at lower charge states was also performed through proton transfer reactions (PTR) with ion parking. Overall, HCD provided preferential cleavage C-terminal to amino acids with acidic sidechains and N-terminal to proline, while UVPD provided more evenly distributed cleavage sites with enhancement near proline and phenylalanine. Using UVPD as a structural analysis tool, PTR was assessed for perturbations to native-like structure of various protein complexes. Through comparison of UVPD fragment intensities, spectra of protein complexes generated through PTR showed little difference to spectra obtained from native-like protein spectra. Following this, PTR-UVPD was applied to elucidate fragment origins of an ambiguous homodimeric protein complex, otherwise displaying complex a complex mass spectrum of overlapping species. A novel approach to performing UVPD using light emitting diodes (LEDs) was explored involving the engineering of a new planar ion trap. Commercially available ultraviolet LEDs emitting photons with wavelengths ranging from 255 to 275 nm were obtained and interfaced with the new ion trap. Sequestration of sample ions in a small spot allowed optimization of overlap with LED photons and resulting fragmentation efficiencies were assessed. Once optimized, LED-UVPD was successfully performed for electron photodetachment (EPD) of single stand DNA and tyrosine sidechain cleavage of a peptide. Custom instrument function was enabled to automatically resonantly eject un-dissociated precursor ions following UVPD and was applied during liquid chromatography (LC) bottom-up proteomics experiments. Through ejection of uninformative, un-dissociated precursor ions, detrimental mass shifting effects caused by increasing the number of charges during spectrum acquisition were relieved. It was observed that performing PE-UVPD resulted in higher protein identification confidence values than for UVPD alone for an E. coli whole cell lysate digest.

Mass Spectrometry-based Structural Analysis of Photosynthetic Protein Assemblies

Author : Yue Lu (Biochemist)
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (11 download)

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Book Synopsis Mass Spectrometry-based Structural Analysis of Photosynthetic Protein Assemblies by : Yue Lu (Biochemist)

Download or read book Mass Spectrometry-based Structural Analysis of Photosynthetic Protein Assemblies written by Yue Lu (Biochemist) and published by . This book was released on 2017 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: This dissertation focuses on using mass spectrometry-based techniques to study photosynthetic protein assemblies. Photosynthesis is a process that converts light energy into chemical energy, the basis of most life on Earth. The two most crucial protein machineries involved in this process are reaction center and light harvesting complexes. They are usually giant protein complexes with different numbers of co-factors. In a more expanded sense, photosynthesis is not just about the utilization of solar energy, the regulation of light energy is also essential as excess light energy is detrimental to photosynthesis organisms. Again, protein assemblies play an indispensable role in this process. The knowledge of the structure and function as well as the molecular mechanism of those protein complexes are desired. Today, mass spectrometry is being widely used in proteomics studies. Its capabilities include but are not limited to the protein primary structure investigation. The development of MS-based footprinting, native MS and membrane protein MS detection platforms largely benefit the study of photosynthetic proteins. The MS-based footprinting technique can investigate protein conformational change upon its binding to other molecules or under the stimulus of pH change or other factors. Native MS can investigate the conformation and topology of protein complexes in a near-native environment where the non-covalent interactions are preserved. Membrane proteins are notoriously difficult to study. The development of MS-based membrane protein detection platforms largely benefits the study of photosynthesis, as reaction center and light-harvesting complexes are usually membrane proteins. In this dissertation, a variety of MS-based techniques were utilized to study reaction center proteins, light harvesting proteins and the proteins involved in the photoprotection process. We utilized top-down MS to study the components as well the primary structure of LH2 from a purple bacterium (Rb. sphaeroides), which reveals a new post-translational modification and mutation information. In addition, we developed a MS-based platform to footprint this LH2, investigating its topology in a lipid bilayer. The reaction center from another purple bacterium (B. viridis) was studied by both bottom-up and top-down MS and lots of unexpected mutations were identified. We also conducted a native MS study on this reaction center, and the capabilities of retaining the co-factors as well as its collisional cross section in the gas phase are discussed. Lastly, we study the orange carotenoid protein (OCP) and the fluorescence recovery protein, two major players in the non-photochemical quenching process in cyanobacteria. We utilized MS-based techniques to probe the conformation and structure of these two proteins and finally proposed a mechanism for non-photochemical quenching in cyanobacteria.

Advancing Intact Protein Analysis by Top-down Mass Spectrometry

Author : Bifan Chen
Publisher :
ISBN 13 :
Total Pages : 215 pages
Book Rating : 4.:/5 (123 download)

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Book Synopsis Advancing Intact Protein Analysis by Top-down Mass Spectrometry by : Bifan Chen

Download or read book Advancing Intact Protein Analysis by Top-down Mass Spectrometry written by Bifan Chen and published by . This book was released on 2019 with total page 215 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Protein Identification and Characterization by Mass Spectrometry

Author : Joy M. Ginter
Publisher :
ISBN 13 : 9780549754404
Total Pages : pages
Book Rating : 4.7/5 (544 download)

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Book Synopsis Protein Identification and Characterization by Mass Spectrometry by : Joy M. Ginter

Download or read book Protein Identification and Characterization by Mass Spectrometry written by Joy M. Ginter and published by . This book was released on 2008 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: This work examines the use of mass spectrometry for protein identification and characterization. Although techniques from the three current proteomic approaches, bottom-up, shotgun, and top-down, were studied, the main research goal was to develop improved top-down methodology in light of the shortcomings found within other techniques. Mass spectrometry instrumentation not previously suited for top-down analysis of relatively large, intact proteins was used in a modified format. A pseudo MS3 approach was developed using a commercially available quadrupole-time-of-flight mass spectrometer (Q-TOF) through the use of in-source collision induced dissociation (CID) coupled to a more traditional CID experiment utilizing the collision cell of the mass spectrometer. With this approach, sequence tags originating from the termini of the proteins were sequenced that led to unique protein identifications. Experiments showed that for the method to be successful proteins first needed to be in a reduced state as disulfide bonds within the protein's primary structure inhibited fragmentation. This technique was demonstrated with simple protein standard mixtures and then applied to a more complex protein mixture, the proteome of E. Coli . Complex protein mixtures require significant separation prior to introduction into the mass spectrometer, and methods for introducing intact proteins are not as prevalent. Solution isoelectric focusing (sIEF) of whole proteins was studied using a modified commercial isoelectric focusing (IEF) device, and the resulting fractions electrosprayed directly into the mass spectrometer after separation by liquid chromatography (LC). The focus of this work was to develop a method that would be analogous to the traditional 2D gel, but amenable to use with the developed top-down MS approach. The proteome of E. Coli was separated using the sIEF-LC approach, and the resulting fractions were subjected to two different MS based approaches. In the first approach, the intact masses of soluble proteins within each sIEF fraction were determined through the deconvolution of each protein's MS spectrum. The pseudo MS3 approach was then applied to generate sequence tags for the proteins to aid in the identification of the proteins. In this first approach a researcher has a more accurate mass, a pI range, and a sequence tag all available for use when determining a protein's identification. In the second approach used the sIEF fractions were globally digested with the enzyme trypsin, and the resulting tryptic digest solutions were analyzed using a shotgun proteomic method. In this approach, proteins were identified through the sequencing of their tryptic fragments and the pI range from the sIEF. This approach was used to determine if any mass limitations existed within the LC separation of the intact proteins. In the first approach, the highest molecular mass observed was approximately 40kDa whereas in the second approach tryptic fragments were found to match to proteins up to 52kDa. Top-down proteomic approaches have gained a lot of attention as researchers shift their focus from simply identifying proteins to characterizing them in light of a specific question. The pseudo-MS3 approach described herein is advantageous since it allows for characterizing relatively large intact proteins with instrumentation that is already common in most proteomic laboratories. Previous top-down approaches have mainly relied on the use of more advanced MS instrumentation such as a Fourier Transform mass spectrometer (FT-MS). Additionally, the sIEF-LC approach described allows for fractionated proteins that can be used for any of a number of proteomic approaches including a top-down approach where solution-based separations are necessary.