Read Books Online and Download eBooks, EPub, PDF, Mobi, Kindle, Text Full Free.
De Novo Sequencing And Peptide Characterization Via Ultraviolet Photodissociation Mass Spectrometry
Download De Novo Sequencing And Peptide Characterization Via Ultraviolet Photodissociation Mass Spectrometry full books in PDF, epub, and Kindle. Read online De Novo Sequencing And Peptide Characterization Via Ultraviolet Photodissociation Mass Spectrometry ebook anywhere anytime directly on your device. Fast Download speed and no annoying ads. We cannot guarantee that every ebooks is available!
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 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 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 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 Protein Sequencing and Identification Using Tandem Mass Spectrometry by : Michael Kinter
Download or read book Protein Sequencing and Identification Using Tandem Mass Spectrometry written by Michael Kinter and published by John Wiley & Sons. This book was released on 2005-04-12 with total page 321 pages. Available in PDF, EPUB and Kindle. Book excerpt: How to design, execute, and interpret experiments for protein sequencing using mass spectrometry The rapid expansion of searchable protein and DNA databases in recent years has triggered an explosive growth in the application of mass spectrometry to protein sequencing. This timely and authoritative book provides professionals and scientists in biotechnology research with complete coverage of procedures for analyzing protein sequences by mass spectrometry, including step-by-step guidelines for sample preparation, analysis, and data interpretation. Michael Kinter and Nicholas Sherman present their own high-quality, laboratory-tested protocols for the analysis of a wide variety of samples, demonstrating how to carry out specific experiments and obtain fast, reliable results with a 99% success rate. Readers will get sufficient experimental detail to apply in their own laboratories, learn about the proper selection and operation of instruments, and gain essential insight into the fundamental principles of mass spectrometry and protein sequencing. Coverage includes: * Peptide fragmentation and interpretation of product ion spectra * Basic polyacrylamide gel electrophoresis * Preparation of protein digests for sequencing experiments * Mass spectrometric analysis using capillary liquid chromatography * Techniques for protein identification by database searches * Characterization of modified peptides using tandem mass spectrometry And much more
Book Synopsis Methods for Proteomic Characterization of Antibody Repertoires and de Novo Peptide Sequencing by : Andrew Pitchford Horton
Download or read book Methods for Proteomic Characterization of Antibody Repertoires and de Novo Peptide Sequencing written by Andrew Pitchford Horton and published by . This book was released on 2016 with total page 180 pages. Available in PDF, EPUB and Kindle. Book excerpt: Driven by the increased performance and availability of protein mass spectrometry and next generation sequencing technologies, research in proteomics and systems biology has expanded far beyond the study of model organisms. This heralds a deeper understanding of biology, the world, and human health. However, it also brings significant new challenges to the interpretation of sequencing and mass spectrometry data, the current software tools ill-suited for many modern studies. The first half of this dissertation explores some of these challenges and solutions in the context of a particularly demanding domain – that of serological antibody proteomics. Our team has developed a combined sequencing and proteomics approach for profiling the human serum antibody repertoire. This opens an unprecedented view into the nature of the adaptive immune system and provides insight on antibody repertoire dynamics in both health and disease. The platform also provides effective means to evaluate vaccine efficacy and identify potential antibody therapeutics. Chapter 1 reviews recent advances in and results from such molecular level characterization of the serum antibody repertoire. Detailed in the second chapter, challenges specific to antibody repertoire proteomics preclude the use of standard analysis methods and motivated our development of novel tools and approaches for interpreting serum repertoire proteomic data. I will shift focus in chapters 3 and 4 to present an experimental and computational workflow for accurate and full-length de novo peptide sequencing. We applied 351 nm ultraviolet photodissociation (UVPD) on chromophore-tagged peptides and developed software for sequencing the resultant UVPD mass spectra. Improvements described in chapter 4 enable the software to automatically learn from and interpret new types and combinations of spectra from the same precursor peptide. We demonstrate the effectiveness of this machine learning framework on CID/UVPD spectral pairs and obtain results, from low resolution spectra, comparable to current state of the art. Continued development of these de novo interpretation and sequencing methods, in part or in whole, may sidestep many of the remaining challenges facing repertoire proteomics, and successful application of these efforts promises further advancement in antibody repertoire characterization and understanding.
Book Synopsis Development, Characterization, and Cleavage of On-Bead Cyclic Peptides for Expedient de Novo Sequencing Through Tandem Mass Spectrometry by : Madison Letendre
Download or read book Development, Characterization, and Cleavage of On-Bead Cyclic Peptides for Expedient de Novo Sequencing Through Tandem Mass Spectrometry written by Madison Letendre and published by . This book was released on 2021 with total page 578 pages. Available in PDF, EPUB and Kindle. Book excerpt:
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 Analysis of Peptides and Proteins by Mass Spectrometry by : C. J McNeal
Download or read book Analysis of Peptides and Proteins by Mass Spectrometry written by C. J McNeal and published by . This book was released on 1988-12-26 with total page 344 pages. Available in PDF, EPUB and Kindle. Book excerpt: Comprises the proceedings of the Fourth Texas Symposium on Mass Spectrometry, held at Texas A & M University, April 1988. Twenty-five papers, presented by an international gathering of mass spectroscopists and bioscientists, reveal the state of the art in the application of mass spectrometry to the life sciences. Discussed are instruments that are sensitive to femtomoles, how mass spectrometry can further peptide mapping, monitoring peptide synthesis, peptide sequencing, and much more.
Book Synopsis Mass Spectrometry of Peptides by : Dominic M. Desiderio
Download or read book Mass Spectrometry of Peptides written by Dominic M. Desiderio and published by CRC Press. This book was released on 1990-11-14 with total page 438 pages. Available in PDF, EPUB and Kindle. Book excerpt: The purpose of this book is to collect into one volume the research done on the mass spectrometry of peptides. It balances a range of topics including theory, instrumentation, analytical techniques, and biological applications. The scope of the work contains three major sections: ionization methods, instrumental developments, and analysis of peptides. It describes 252Cf plasma desorption and laser-induced multiphoton ionization methodology. This exciting resource covers many new areas, including continuous flow FAB, quantification of human neuropeptides, and peptide mapping. It also discusses Q-FTMS, cross-links, and metal ions.
Book Synopsis Characterization of Peptides, Proteins, and Protein Complexes Using Infrared Multiphoton Dissociation Spectroscopy, Ion Mobility Spectrometry, and Surface-induced Dissociation Mass Spectrometry by : Erin M. Panczyk
Download or read book Characterization of Peptides, Proteins, and Protein Complexes Using Infrared Multiphoton Dissociation Spectroscopy, Ion Mobility Spectrometry, and Surface-induced Dissociation Mass Spectrometry written by Erin M. Panczyk and published by . This book was released on 2021 with total page 210 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry-based techniques have emerged as powerful analytical tools to investigate the structure of proteins from the primary to quaternary levels. The advancement of mass spectrometry instrumentation and methods has allowed researchers to go beyond just measuring an analyte’s mass-to-charge ratio, but to also probe gas-phase dissociation behaviors and conformations of peptides, proteins, and protein complexes. The primary structure of a protein refers to the linear sequence of amino acids linked together via peptide bonds. The presence, and the order, of specific amino acids in a peptide can strongly influence how a peptide fragments in the gas-phase. Particular amino acids can direct where along the peptide backbone fragmentation is favored and the structure of the fragment ions formed. One method for probing the structure of peptide fragment ions is infrared multiphoton dissociation (IRMPD) mass spectrometry coupled with theoretical quantum chemical calculations. This approach is used to investigate the role of peptide bond conformation on the structure of b2+ fragment ions formed from proline and dimethylproline-containing peptides (Chapter 3). Additionally, IRMPD is used to study the fragmentation patterns of proline containing pentapeptides into b3+ ions (Chapter 4). Native mass spectrometry (nMS) analyzes the intact structures of proteins and protein complexes and offers complementary information to traditional biophysical methods, such as NMR or cryo-EM. Tandem mass spectrometry, specifically surface-induced dissociation (SID), provides information on protein complex connectivity, stoichiometry, and gas-phase structural rearrangement. SID is utilized to monitor deviation from native structure for protein complexes generated from submicrometer nanoelectrospray capillaries (Chapter 5), as well as to provide insight into connectivity of protein complexes selected by trapped ion mobility spectrometry (Chapter 6). In addition to SID, ion mobility spectrometry provides information on the gas-phase shape or conformation of biomolecules. Here, ion mobility spectrometry is utilized to separate multiple conformers of proline-containing peptides (Chapter 3), compare the collision cross sections of protein complexes generated from submicrometer and micrometer sized nanoelectrospray capillaries (Chapter 5), and select protein complexes and isomeric peptides prior to dissociation on an ultrahigh resolution mass spectrometry platform (Chapter 6). Finally, the development and optimization of Trapped Ion Mobility Spectrometry (TIMS) for native mass spectrometry applications is applied to the widely available timsTOF Pro mass spectrometry platform to promote the dissemination of native ion mobility technology.
Book Synopsis Strategies for Protein and Peptide Characterization and Quantification Using Electron-transfer Dissociation Mass Spectrometry and Intrinsic Fluorescence by :
Download or read book Strategies for Protein and Peptide Characterization and Quantification Using Electron-transfer Dissociation Mass Spectrometry and Intrinsic Fluorescence written by and published by . This book was released on 2012 with total page 442 pages. Available in PDF, EPUB and Kindle. Book excerpt: STRATEGIES FOR PROTEIN AND PEPTIDE CHARACTERIZATION AND QUANTIFICATION USING ELECTRON-TRANSFER DISSOCIATION MASS SPECTROMETRY AND INTRINSIC FLUORESCENCE Jason D. Russell Under the supervision of Associate Professor Joshua J. Coon At the University of Wisconsin-Madison The following chapters detail strategies for peptide and protein sequence analysis featuring electron-transfer dissociation (ETD) tandem mass spectrometry (MS/MS) and quantification using ultraviolet light-induced intrinsic fluorescence (UV-IF). Chapter 1 provides a brief background and overview. Chapter 2 discusses the optimization of the ETD MS/MS duty cycle for large-scale shotgun experiments. In Chapter 3, the first comprehensive analysis of peptide anions using negative ETD (NETD) is detailed. I report in Chapter 4 on the performance of an ion-ion reaction cell for intact protein analysis using large precursor populations for ETD MS/MS. The application of UV-IF for peptide detection and quantification using a custom fluorescence excitation and detection device is discussed in Chapter 5. An analysis of intact proteins from the 26S proteasome of Arabidopsis using top-down mass spectrometry and quantification by UV-IF is described in Chapter 6. In coordination with the Wisconsin Initiative for Science Literacy (WISL), Chapter 7 is a general description of my graduate research intended for non-specialists in an effort to promote science literacy in the broader community.
Book Synopsis Method Development for Qualitative and Quantitative Study in Mass Spectrometry by : Chang Xue
Download or read book Method Development for Qualitative and Quantitative Study in Mass Spectrometry written by Chang Xue and published by . This book was released on 2014 with total page 134 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry has become the method of choice for lots of areas in recent years including protein study and clinical research. In this thesis, work from three projects covering three very different domains in the big area of mass spectrometry has been described. Chapter 2 to Chapter 4 presented the project of methodology development in chemical derivatization of peptides. In this project, custom-tunable tags prepared through solid phase synthesis increased sequence coverage of peptide ions in electron transfer dissociation for de novo sequencing, thus simplifies the characterization of proteins. Additionally, the application of developed stable isotopic tags pair can be used for quantitation of the proteins as well as for the simplified characterization of proteins. Chapter 5 presented the project of newborn screening in clinical study. In this project, a novel tandem mass spectrometry duplex assay for the clinical diagnosis of neuronal ceroid lipofuscinoses (NCL) has been developed. The duplex assay recognized the deficient patients from healthy newborns by quantification of the enzyme activity in dried blood spot through the use of tandem mass spectrometer. Chapter 6 presented the result from protein-protein interaction project. Protein-protein interaction is essential to understand biological pathways. In this project, a second generation of protein interaction reporter BDD-PIR has been synthesized and applied to proteins for performance evaluation. This molecule has a low molecular weight and high solubility and is therefore expected to have improved performance than the first generation protein interaction reporter for in vivo application.
Book Synopsis Hybrid Activation for Intact Protein and Peptide Characterization by : Dustin Donald Holden
Download or read book Hybrid Activation for Intact Protein and Peptide Characterization written by Dustin Donald Holden and published by . This book was released on 2015 with total page 142 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometers may be utilized to generate and detect fragmentation patterns of peptides and proteins to acquire mass spectra that allow for accurate amino acid sequence characterization and localization of post-translational modifications. Conventional activation methods utilize collisions with neutral gas molecules to collisionally activate these ions but have some drawbacks, being dependent on charge state and amino acid sequence characteristics. More recent activation methods include radical transfer as well as ultraviolet photodissociation, the latter of which impart higher energy into target ions providing more extensive fragmentation patterns and more sequence information. In the following research all of these activation methods were explored with peptide and protein cations as well as various combinations of such methods to investigate their benefits and limitations. In summary, ultraviolet photodissociation provides the most diverse fragmentation patterns of all researched. Combining radical transfer and subsequent ultraviolet photodissociation termed “hybrid activation” generated interesting effects such as spectral simplification as well as constructive inclusion of fragment ions lacking in ultraviolet photodissociation. Included in this thesis are those interesting findings.
Book Synopsis Algorithms for Tandem Mass Spectrometry-based Proteomics by : Ari Michael Frank
Download or read book Algorithms for Tandem Mass Spectrometry-based Proteomics written by Ari Michael Frank and published by . This book was released on 2008 with total page 205 pages. Available in PDF, EPUB and Kindle. Book excerpt: Tandem mass spectrometry (MS/MS) has emerged as the leading technology for high-throughput proteomics analysis, making it possible to rapidly identify and characterize thousands of different proteins in complex biological samples. In recent years we have witnessed a dramatic increase in the capability to acquire proteomics MS/MS data. To avoid computational bottlenecks, this growth in acquisition power must be accompanied by a comparable improvement in analysis capabilities. In this dissertation we present several algorithms we developed to meet some of the major computational challenges that have arisen in MS/MS analysis. Throughout our work we continually address two (sometimes overlapping) problems: how to make MS/MS-based sequence identifications more accurate, and how to make the identification process work much faster. Much of the work we present revolves around algorithms for de novo sequencing of peptides, which aims to discover the amino acid sequence of protein digests (peptides), solely from their experimental mass spectrum. We start off by describing a new scoring model which is used in our de novo sequencing algorithm called PepNovo. Our scoring scheme is based on a graphical model decomposition that describes many of the conditions that determine the intensities of fragment ions observed in mass spectra, such as dependencies between related fragment ions and the influence of the amino acids adjacent to the cleavage site. Besides predicting whole peptide sequences, one of the most useful applications of de novo algorithms is to generate short sequence tags for the purpose of database filtration. We demonstrate how using these tags speeds up database searches by two orders of magnitude compared to conventional methods. We extend the use of tag filtration and show that with high-resolution data, our de novo sequencing is accurate enough to enable extremely rapid identification via direct hash lookup of peptide sequences. The vast amount of MS/MS data that has become available has made it possible to use advanced data-driven machine learning methods to devise more acute algorithms. We describe a new scoring function for peptide-spectrum matches that uses the RankBoost ranking algorithm to learn and model the influences of the many intricate processes that occur during peptide fragmentation. Our method's superior discriminatory power boosts PepNovo's performance beyond the current state-of-the-art de novo sequencing algorithms. Our score also greatly improves the performance of database search programs, significantly increasing both their speed and sensitivity. When we applied our method to the challenging task of a proteogenomic search against a six-frame translation of the human genome, we were able to significantly increase the number of peptide identifications compared to current techniques by 60\%. To help speed up MS/MS analysis, we developed a clustering algorithm that exploits the redundancy that is inherent in large mass spectrometry datasets (these often contain hundreds and even thousands of spectra of the same peptide). When applied to large MS/MS datasets on the order of ten million spectra, our clustering algorithm reduces the number of spectra by an order of magnitude, without losing peptide identifications. Finally, we touch upon sequencing of intact proteins (``top-down'' analysis), which from a computational perspective, is only in its infancy -- very few algorithms have been developed for analysis of this type of data. We developed MS-TopDown, which uses the Spectral Alignment algorithm to characterize protein forms (i.e., determine the modification/mutation sites). Our algorithm can handle heavily modified proteins and can also distinguish between several isobaric protein forms present in the same spectrum.
Book Synopsis Development of Photodissociation Methods for Biomolecule Analysis in a Quadupole Ion Trap Mass Spectrometer by : Jeffrey John Wilson
Download or read book Development of Photodissociation Methods for Biomolecule Analysis in a Quadupole Ion Trap Mass Spectrometer written by Jeffrey John Wilson and published by . This book was released on 2008 with total page 398 pages. Available in PDF, EPUB and Kindle. Book excerpt: Photodissociation methods have been implemented and compared to collision-induced dissociation (CID) in a quadrupole ion trap mass spectrometer for the structural analysis of peptides, proteins, oligosaccharides, DNA and DNA/drug complexes. Infrared multiphoton dissociation (IRMPD) was applied to N-terminally sulfonated peptides which offers efficient photo-fragmentation and detection of important low m/z fragments in comparison to CID. Upon IRMPD of these modified peptides a simplified MS/MS spectrum comprised of only characteristic y ions allows for better identification through de novo software analysis. Oligonucleotides can undergo highly efficient IRMPD due to the phosphate moiety located on along their backbone structure which yields excellent photon absorption at [lambda] = 10.6 [mu]m. IRMPD fragmentation pathways of DNA and DNA/drug complexes were shown to be comparable to CID, yielding cleavage at the [w / (a - B)] bond, except IRMPD allows for significantly improved MS/MS sensitivity through the secondary dissociation of uninformative duplex base losses which can further dissociate into useful fragment ions for sequencing. Ultraviolet photodissociation (UVPD) has been applied to chromophorederivatized peptides and oligosaccharides which retains the advantages associated with IRMPD, but also has additional benefits due to the greater energy per photon at 355 nm (3.5 eV / photon) in comparison to 10.6 [mu]m (0.12 eV / photon). Primarily, UVPD provides highly efficient secondary dissociation of chromophore-containing fragments allowing for simplified MS/MS spectra of chromophore-derivatized peptides. This concept was also implemented for the characterization of branched fluorescently-labeled oligosaccharides which produces different fragment ions complementary to CID experiments. Secondly, UVPD provides an ion activation method which is independent of the bath gas helium pressure in the ion trap in contrast to CID or IRMPD permits for optimal trap performance without compromise. Coordination of a chromogenic 18-crown-6 molecule to the lysine side chain of a peptide facilitates UVPD at both 266 nm and 355 nm. Energy absorbed by the crown ether is transferred intermolecularly to the peptide via the strong hydrogen bonds which hold the complex together, resulting in activation and fragmentation of the peptide. CID or IRMPD of these crown ether/peptide complexes results only in their disassembly without peptide fragmentation.
Book Synopsis Modern Proteomics – Sample Preparation, Analysis and Practical Applications by : Hamid Mirzaei
Download or read book Modern Proteomics – Sample Preparation, Analysis and Practical Applications written by Hamid Mirzaei and published by Springer. This book was released on 2016-12-14 with total page 525 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume serves as a proteomics reference manual, describing experimental design and execution. The book also shows a large number of examples as to what can be achieved using proteomics techniques. As a relatively young area of scientific research, the breadth and depth of the current state of the art in proteomics might not be obvious to all potential users. There are various books and review articles that cover certain aspects of proteomics but they often lack technical details. Subject specific literature also lacks the broad overviews that are needed to design an experiment in which all steps are compatible and coherent. The objective of this book was to create a proteomics manual to provide scientists who are not experts in the field with an overview of: 1. The types of samples can be analyzed by mass spectrometry for proteomics analysis. 2. Ways to convert biological or ecological samples to analytes ready for mass spectral analysis. 3. Ways to reduce the complexity of the proteome to achieve better coverage of the constituent proteins. 4. How various mass spectrometers work and different ways they can be used for proteomics analysis 5. The various platforms that are available for proteomics data analysis 6. The various applications of proteomics technologies in biological and medical sciences This book should appeal to anyone with an interest in proteomics technologies, proteomics related bioinformatics and proteomics data generation and interpretation. With the broad setup and chapters written by experts in the field, there is information that is valuable for students as well as for researchers who are looking for a hands on introduction into the strengths, weaknesses and opportunities of proteomics.
