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Characterization Of Proteins And Peptides Via Enhanced 266 Nm Ultraviolet Photodissociation Mass Spectrometry Utilizing A Selenium Based Chromophore
Download Characterization Of Proteins And Peptides Via Enhanced 266 Nm Ultraviolet Photodissociation Mass Spectrometry Utilizing A Selenium Based Chromophore full books in PDF, epub, and Kindle. Read online Characterization Of Proteins And Peptides Via Enhanced 266 Nm Ultraviolet Photodissociation Mass Spectrometry Utilizing A Selenium Based Chromophore ebook anywhere anytime directly on your device. Fast Download speed and no annoying ads. We cannot guarantee that every ebooks is available!
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.
Book Synopsis De Novo Sequencing and Peptide Characterization Via Ultraviolet Photodissociation Mass Spectrometry by : Scott Allen Robotham
Download or read book De Novo Sequencing and Peptide Characterization Via Ultraviolet Photodissociation Mass Spectrometry written by Scott Allen Robotham and published by . This book was released on 2015 with total page 470 pages. Available in PDF, EPUB and Kindle. Book excerpt: Although in silico database search methods remain more popular for shotgun proteomics methods, de novo sequencing offers the ability to identify proteins lacking sequenced genomes and ones with subtle splice variants or truncations. Ultraviolet photodissociation (UVPD) of peptides derivatized by selective attachment of a chromophore at the N-terminus generated characteristic series of y ions. The UVPD spectra of the chromophore-labelled peptides were simplified and thus amenable to de novo sequencing. E.coli lysates were modified by the use of carbamylation and the attachment of a UV chromophore to the N-terminus of digested peptides. UVPD of the resulting peptides generated clean sets of y ions. A novel de novo algorithm, UVnovo, afforded high confidence identification of thousands of peptides from an E. coli lysate and allowed UVPD/CID paired spectra to be searched. E.coli lysate peptides were analyzed in alternating scans by UVPD and CID on the same precursors to generate paired UVPD/CID spectra. UVnovo enabled sequence tag de novo sequencing of peptides in order to find matching sequences from a database. Ultimately the UVnovo functioned as a standalone de novo sequencing program or a hybrid de novo/database search program. In an effort to better characterize the fragmentation pathways promoted by ultraviolet photoexcitation in comparison to CID, six adrenocorticotropic hormone (ACTH) peptides in a range of charge states were subjected to 266 nm ultraviolet photodissociation (UVPD), 193 nm UVPD, and CID. While both UVPD and CID led to preferential cleavage of the Y-S bond for all ACTH peptides (except ACTH (1-39)), UVPD was far less dependent on charge state and location of basic sites for the production of C-terminal and N-terminal ions.
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
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 Enhanced Protein Characterization Through Selective Derivatization and Electrospray Ionization Tandem Mass Spectrometry by : Lisa Anne Vasicek
Download or read book Enhanced Protein Characterization Through Selective Derivatization and Electrospray Ionization Tandem Mass Spectrometry written by Lisa Anne Vasicek and published by . This book was released on 2011 with total page 338 pages. Available in PDF, EPUB and Kindle. Book excerpt: There continue to be great strides in the field of proteomics but as samples become more complex, the ability to increase sequence coverage and confidence in the identification becomes more important. Several methods of derivatization have been developed that can be used in combination with tandem mass spectrometry to identify and characterize proteins. Three types of activation, including infrared multiphoton dissociation, ultraviolet photodissociation, and electron transfer dissociation, are enhanced in this dissertation and compared to the conventional method of collisional induced dissociation (CID) to demonstrate the improved characterization of proteins. A free amine reactive phosphate group was synthesized and used to modify the N-terminus of digested peptides. This phosphate group absorbs at the IR wavelength of 10.6 [mu]m as well as the Vacuum-ultraviolet (VUV) due to an aromatic group allowing modified peptides to be dissociated by infrared multi-photon dissociation (IRMPD) or ultraviolet photodissociation (UVPD) whereas peptides without this chromophore are less responsive to IR or UV irradiation. The PD spectra for these modified peptides yield simplified MS/MS spectra due to the neutralization of all N-terminal product ions from the incorporation the negatively charged phosphate moiety. This is especially advantageous for UVPD due to the great number of product ions produced due to the higher energy deposition of the UV photons. The MS/MS spectra also produce higher sequence coverage in comparison to CID of the modified or unmodified peptides due to more informative fragmentation pathways generated upon PD from secondary dissociation and an increased ion trapping mass range. IRMPD is also implemented for the first time on an orbitrap mass spectrometer to achieve high resolution analysis of IR chromophore-derivatized samples as well as top-down analysis of unmodified proteins. High resolution/high mass accuracy analysis is extremely beneficial for characterization of complex samples due to the likelihood of false positives at lower resolutions/accuracies. For electron transfer dissociation, precursor ions in higher charge states undergo more exothermic electron transfer and thus minimize non-dissociative charge reduction. In this dissertation, cysteine side chains are alkylated with a fixed charge to deliberately increase the charge states of peptides and improve electron transfer dissociation. ETD can also be used to study protein structure by derivatizing the intact structure with a hydrazone reagent. A hydrazone bond will be preferentially cleaved during ETD facilitating the recognition of any modified residues through a distinguishing ETD fragmentation spectrum.
Book Synopsis Structural Characterization of Complex Biological Systems Via Ultraviolet Photodissociation Mass Spectrometry by : Christopher Martin Crittenden
Download or read book Structural Characterization of Complex Biological Systems Via Ultraviolet Photodissociation Mass Spectrometry written by Christopher Martin Crittenden and published by . This book was released on 2019 with total page 552 pages. Available in PDF, EPUB and Kindle. Book excerpt: The work detailed in this dissertation describes the advantages that 193 nm ultraviolet photodissociation (UVPD) affords for characterization of structurally complex biological molecules as compared to traditional ion activation techniques, such as collisional or electron-based dissociation, for mass spectrometry. UVPD, either alone or in tandem with collisional activation such as collision induced dissociation (CID), consistently provides more extensive structural information about biomolecules. One such system where the utility of both UVPD and CID was employed was in the structural characterization of lipid A species. Lipid A, the innermost structural component of lipopolysaccharides (LPS) which decorate the surface of Gram-negative bacteria, may undergo covalent modifications in order to provide resistance to antibiotics. By utilizing a combinatorial approach, CID is able to characterize the covalent modifications that are present while UVPD is able to elucidate which side of the molecule (reducing or nonreducing end) undergoes the modification through selective fragmentation of the diglucosamine backbone. This approach confirmed the presence of aminoarabinose modification present on the LPS of A. baumannii after exposure to the antibiotic polymyxin B. Another instance of utilizing the power of both photodissociation and collisional activation was in the characterization of oligosaccharide molecules from LPS of E. coli. These biomolecules are typically heavily phosphorylated near the reducing end of the saccharide backbone, and as such, collisional activation produces fragment ions originated from cleavages localized near the phosphate sites. UVPD of the oligosaccharides produces a plethora of diagnostic fragment ions throughout the molecule, but this often leads to spectral congestion and ambiguous fragment assignment. UVPD generates charge-reduced precursor ions that can be subjected to subsequent collisional activation in a MS3 event, allowing complete characterization significantly fewer confounding product ions as compared to UVPD alone. Another hallmark of UVPD is its fast, high energy deposition that causes cleavage of covalent bonds while allowing survival of non-covalent interactions. This characteristic allows electrostatic interactions to be mapped in non-covalent complexes, unlike the collisional activation which preferentially cleaves weak non-covalent interactions owing to the stepwise nature of collisional activation. In this work, it is demonstrated that UVPD of the electrostatic complex between a cationic antimicrobial peptides (CAMP) and Kdo2-lipid A illuminates, through the production of diagnostic holo peptide fragment ions retaining the intact mass of the lipid A species, which amino acids in the peptide sequence are responsible for mediating the interaction between the two molecules in the gas phase. In contrast, collisional activation of the electrostatic complex between the two species simply results in the disruption of the network of non-covalent interactions, only yielding apo peptide product ions. In the same vein, this notion of retention of electrostatic interactions post-photodissociation was employed to interrogate where metal ions were sequestered in proteins. UVPD has previously been touted as a means to determine the binding location of ligands (such as drug molecules) to proteins after transporting the protein-ligand complexes to the gas-phase by native ESI. This methodology was extended to determine the binding location of metal ions (such as calcium, copper, silver, and praseodymium, to name a few) to proteins. The binding sites of calcium (II) and a series of lanthanide (III) ions were successfully determined for staphylococcal nuclease, the binding sites of silver (I) and copper (II) were determined for azurin, and multiple binding sites for calcium (II) and select lanthanides (III) were determined for calmodulin, all agreeing with reported crystal structure data. These are but only a few examples of the utility of UVPD as an alternative to ion activation in the gas phase. The unprecedented characterization of ions by UVPD, regardless of polarity, number of charges, size of the molecule, or molecular interactions present, suggests that there are many other potential applications of UVPD in the future
Book Synopsis Ultraviolet Photodissociation Techniques for Selective Activation of Derivatized Peptides and Characterization of Disulfide Bonds in Proteins by : Matthew Montana Quick
Download or read book Ultraviolet Photodissociation Techniques for Selective Activation of Derivatized Peptides and Characterization of Disulfide Bonds in Proteins written by Matthew Montana Quick and published by . This book was released on 2018 with total page 170 pages. Available in PDF, EPUB and Kindle. Book excerpt: Bottom-up strategies have been the most widely utilized workflow for proteomics and have typically relied on collisional activation methods to generate highly diagnostic fragmentation patterns for thousands of peptides. Ultraviolet photodissociation (UVPD) is a versatile alternative to collisional activation that has been adapted for both bottom-up and top-down proteomics methods. The mechanism and outcome of UVPD varies widely depending on photon wavelength, largely owing to the energy deposited per photon and the ability of peptides to efficiently absorb at a specific wavelength. For example, activation of proteins and peptides by 193 nm UVPD, a wavelength of light absorbed by the protein backbone produces a, b, c, x, y, and z sequence ions, as well as immonium ions and side-chain loss ions. Since the protein backbone does not absorb significantly at 266 nm, photodissociation at this wavelength depends on intrinsic chromophore absorption from aromatic residues such as tyrosine and tryptophan and predominantly produces a, b, and y ions in addition to low abundances of c, x, and z ions with modest sequence coverage. As a result, attachment of chromophore specific for 266 nm absorption can be employed to help facilitate the production of diagnostic fragment ions and increase the sequence coverage of peptides. Presented in this thesis are methods to enhance the fragmentation of peptides activated by 266 nm UVPD and to identify the disulfide bond connectivities of proteins by 193 nm UVPD. Here peptides are derivatized with 4-acetamido-4-isothiocyanatostilbene-2-2 disulfonic acid (SITS), a chromophore well suited for absorption of 266 nm photons. This endows all tagged peptides with high absorptivity and facilitates bottom-up sequencing using 266 UVPD. Disulfide bound peptides are prepared by proteolytic digestion followed by partial reduction and alkylation. The resulting samples are separated using high performance liquid chromatography and analyzed with 193 nm UVPD resulting in spectra that lead to the unequivocal assignment of disulfide bonds.
Book Synopsis Characterization of Protein Therapeutics using Mass Spectrometry by : Guodong Chen
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.
Author :Alexander P. Demchenko Publisher :Springer Science & Business Media ISBN 13 :3642708471 Total Pages :323 pages Book Rating :4.6/5 (427 download)
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.
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.
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.
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.
Book Synopsis Structural Characterization and Quantification of Proteins and Peptides Using Mass Spectrometry by : Hari M. Kosanam
Download or read book Structural Characterization and Quantification of Proteins and Peptides Using Mass Spectrometry written by Hari M. Kosanam and published by . This book was released on 2008 with total page 318 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Protein Analysis using Mass Spectrometry by : Mike S. Lee
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 Synopsis Structural Characterization of Proteins and Peptides Using Mass Spectrometry by : Tarun Gheyi
Download or read book Structural Characterization of Proteins and Peptides Using Mass Spectrometry written by Tarun Gheyi and published by . This book was released on 2005 with total page 364 pages. Available in PDF, EPUB and Kindle. Book excerpt:
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 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.
