Development and Application of Accurate Mass Measurements for Large-scale Protein Interaction and Proteome Studies PDF Download
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Author: Chad R. Weisbrod
Publisher:
ISBN:
Category :
Languages : en
Pages : 196
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Book Description
Traditionally and most commonly, accurate mass measurement is utilized to restrict peptide candidate search space on the precursor level to impart greater specificity in large peptide sequence databases. While use of accurate mass measurement is effective for discovery based proteomics, here we present novel applications of accurate mass measurement in proteomics to make further use of this expensive (monetarily and in time) acquisition attribute. This work began with the coupling and testing of a hybrid dual cell linear ion trap mass spectrometer (Velos) to a 7T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This combination provides high speed, efficient ion accumulation with the high resolution and mass accuracy of FTICR. Using this mass spectrometer (Velos-FT) and an LTQ-Orbitrap it was shown theoretically and empirically that high resolution product ion spectra alone can be utilized to detect and quantify peptides. A DIA acquisition and data processing pipeline called, FT-All Reaction Monitoring (FT-ARM), was designed to exploit this observation. Direct similarities of FT-ARM to targeted proteomics data allow for discovery and targeted proteomics to be conducted in a single pass with little to no assay development. A search algorithm developed to score and assess data acquired using this strategy enabled quantitation into the attomole range and enabled identification within a complex background matrix. Specificity of the peptide assignment is attributed to the requirement of the simultaneous observation of a minimum number of product ions from the target peptide at a required mass measurement accuracy. Real-time informatics analysis during mass spectrometry acquisition can serve to simplify post-acquisition analysis and more importantly, focus measurements on ions more likely to yield the desired information. Real-time informatics applied to engineered, cleavable cross-linkers allows known mass relationships to be used to direct subsequent experiments. Peptides released from cross-linked complexes can be directly targeted for MS3 acquisition to obtain sequence information within a single LC/MS/MS acquisition. This new mass spectrometry method called Real-time Analysis for Cross-linked peptide Technology (ReACT) has been developed to enable assignment of cross-linked peptides "on-the-fly". Using ReACT, 708 unique cross-linked (
Author: Chad R. Weisbrod
Publisher:
ISBN:
Category :
Languages : en
Pages : 196
Get Book Here
Book Description
Traditionally and most commonly, accurate mass measurement is utilized to restrict peptide candidate search space on the precursor level to impart greater specificity in large peptide sequence databases. While use of accurate mass measurement is effective for discovery based proteomics, here we present novel applications of accurate mass measurement in proteomics to make further use of this expensive (monetarily and in time) acquisition attribute. This work began with the coupling and testing of a hybrid dual cell linear ion trap mass spectrometer (Velos) to a 7T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This combination provides high speed, efficient ion accumulation with the high resolution and mass accuracy of FTICR. Using this mass spectrometer (Velos-FT) and an LTQ-Orbitrap it was shown theoretically and empirically that high resolution product ion spectra alone can be utilized to detect and quantify peptides. A DIA acquisition and data processing pipeline called, FT-All Reaction Monitoring (FT-ARM), was designed to exploit this observation. Direct similarities of FT-ARM to targeted proteomics data allow for discovery and targeted proteomics to be conducted in a single pass with little to no assay development. A search algorithm developed to score and assess data acquired using this strategy enabled quantitation into the attomole range and enabled identification within a complex background matrix. Specificity of the peptide assignment is attributed to the requirement of the simultaneous observation of a minimum number of product ions from the target peptide at a required mass measurement accuracy. Real-time informatics analysis during mass spectrometry acquisition can serve to simplify post-acquisition analysis and more importantly, focus measurements on ions more likely to yield the desired information. Real-time informatics applied to engineered, cleavable cross-linkers allows known mass relationships to be used to direct subsequent experiments. Peptides released from cross-linked complexes can be directly targeted for MS3 acquisition to obtain sequence information within a single LC/MS/MS acquisition. This new mass spectrometry method called Real-time Analysis for Cross-linked peptide Technology (ReACT) has been developed to enable assignment of cross-linked peptides "on-the-fly". Using ReACT, 708 unique cross-linked (
Author: Oscar Alzate
Publisher: CRC Press
ISBN: 1420076264
Category : Medical
Languages : en
Pages : 356
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Book Description
In this, the post-genomic age, our knowledge of biological systems continues to expand and progress. As the research becomes more focused, so too does the data. Genomic research progresses to proteomics and brings us to a deeper understanding of the behavior and function of protein clusters. And now proteomics gives way to neuroproteomics as we beg
Author: Peter James
Publisher: Springer Science & Business Media
ISBN: 9783540672562
Category : Science
Languages : en
Pages : 306
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Book Description
Recent advances in large scale DNA sequencing technology have made it possible to sequence the entire genome of an organism. Attention is now turning to the analysis of the product of the genome, the proteome, which is the set of proteins being expressed by a cell. Mass spectrometry is the method of choice for the rapid large-scale identification of these proteomes and their modifications. This is the first book to extensively cover the applications of mass spectrometry to proteome research.
Author: Institute of Medicine
Publisher: National Academies Press
ISBN: 0309224187
Category : Science
Languages : en
Pages : 354
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Book Description
Technologies collectively called omics enable simultaneous measurement of an enormous number of biomolecules; for example, genomics investigates thousands of DNA sequences, and proteomics examines large numbers of proteins. Scientists are using these technologies to develop innovative tests to detect disease and to predict a patient's likelihood of responding to specific drugs. Following a recent case involving premature use of omics-based tests in cancer clinical trials at Duke University, the NCI requested that the IOM establish a committee to recommend ways to strengthen omics-based test development and evaluation. This report identifies best practices to enhance development, evaluation, and translation of omics-based tests while simultaneously reinforcing steps to ensure that these tests are appropriately assessed for scientific validity before they are used to guide patient treatment in clinical trials.
Author: Richard B. Cole
Publisher: Wiley-Interscience
ISBN:
Category : Science
Languages : en
Pages : 610
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Book Description
Comprehensive, up-to-date coverage of a revolutionary technique Electrospray ionization mass spectrometry (ESI-MS) has completely changed the way physicists, chemists, and biologists view the study of large molecules. The technique derives detailed information about molecular weights and structures from extremely small sample quantities. ESI-MS can create highly charged forms of very high molecular weight compounds, it is naturally compatible with many types of separation techniques, and it allows noncovalent interactions between molecules in solution to be preserved in the gas phase. But many researchers may not use the technique to its full potential because they are unfamiliar with the different perspectives of its underlying processes, the varied approaches to implementation, and the wide-ranging utility of the technique. In this book, Richard B. Cole and an assemblage of leading researchers present a single-volume compilation of different approaches to the understanding and exploitation of ESI-MS. This comprehensive guide: * Examines the physical and chemical aspects of the electrospray process and describes the events involved in ion formation as well as the electro-chemical phenomena that are central to charged droplet formation during the process * Explores the coupling of electrospray ionization to various mass spectrometers, including quadrupole, magnetic, time-of-flight, quadrupole ion trap, and Fourier transform ion cyclotron resonance instruments * Describes recent progress in interfacing ESI with solution-based separation techniques, including liquid chromatography and capillary electrophoresis * Charts the rapid development of ESI applications and categorizes them by compound type: peptides and proteins, nucleic acids and their constituents, carbohydrates and lipids, small molecules related to pharmacology and drug metabolism, and organometallics and inorganic compounds Electrospray Ionization Mass Spectrometry is the indispensable handbook and reference for anyone who wishes to understand, implement, or apply this technique, including researchers in chemistry, metallochemistry, biochemistry, biology, pharmacology, and physics.
Author: Chhabil Dass
Publisher: John Wiley & Sons
ISBN: 0470118482
Category : Science
Languages : en
Pages : 512
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Book Description
Modern mass spectrometry - the instrumentation and applications in diverse fields Mass spectrometry has played a pivotal role in a variety of scientific disciplines. Today it is an integral part of proteomics and drug discovery process. Fundamentals of Contemporary Mass Spectrometry gives readers a concise and authoritative overview of modern mass spectrometry instrumentation, techniques, and applications, including the latest developments. After an introduction to the history of mass spectrometry and the basic underlying concepts, it covers: Instrumentation, including modes of ionization, condensed phase ionization techniques, mass analysis and ion detection, tandem mass spectrometry, and hyphenated separation techniques Organic and inorganic mass spectrometry Biological mass spectrometry, including the analysis of proteins and peptides, oligosaccharides, lipids, oligonucleotides, and other biological materials Applications to quantitative analysis Based on proven teaching principles, each chapter is complete with a concise overview, highlighted key points, practice exercises, and references to additional resources. Hints and solutions to the exercises are provided in an appendix. To facilitate learning and improve problem-solving skills, several worked-out examples are included. This is a great textbook for graduate students in chemistry, and a robust, practical resource for researchers and scientists, professors, laboratory managers, technicians, and others. It gives scientists in diverse disciplines a practical foundation in modern mass spectrometry.
Author: Nader Rifai
Publisher: Elsevier
ISBN: 0128160640
Category : Science
Languages : en
Pages : 218
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Book Description
Principles and Applications of Clinical Mass Spectrometry: Small Molecules, Peptides, and Pathogens is a concise resource for quick implementation of mass spectrometry methods in clinical laboratory work. Focusing on the practical use of these techniques, the first half of the book covers principles of chromatographic separations, principles and types of mass spectrometers, and sample preparation for analysis; the second half outlines the main applications of this technology within clinical laboratory settings, including determination of small molecules and peptides, as well as pathogen identification. A thorough yet succinct guide to using mass spectrometry technology in the clinical laboratory, Principles and Applications of Clinical Mass Spectrometry: Small Molecules, Peptides, and Pathogens is an essential resource for chemists, pharmaceutical and biotech researchers, certain government agencies, and standardization groups. - Provides concrete examples of the main applications of mass spectrometry technology - Describes current capabilities of the LC- and MS-based analytical methods - Details methods for successful analytical work in the field
Author:
Publisher:
ISBN:
Category :
Languages : en
Pages : 406
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Book Description
The work described in this dissertation highlights the versatility of mass spectrometry-based proteomics, detailing the development and/or application of several diverse methods that enable, and improve upon, the large-scale identification and/or quantification of whole proteomes. A broad overview of mass spectrometry-based proteomics and the technological innovations that have driven the field forward are presented in Chapter 1. Chapter 2 outlines a method that utilizes NeuCode SILAC labeling and machine learning algorithms to enable product ion annotation within tandem mass spectra, facilitating the implementation of both automated database searching and de novo sequencing. Chapter 3 presents a strategy for performing multiplexed quantification in the context of data-independent acquisition. Chapter 4 describes the extension of QuantMode, a strategy that utilizes gas-phase purification to improve the quantitative accuracy of isobaric tag-based methods, to an ETD-enabled ion trap system. Chapter 5 outlines a method that improves the sampling depth of label-free experiments without the use of offline fractionation or the significant increase in analysis time. In Chapter 6, both label-free and isobaric tag-based strategies are employed to evaluate the localization and functionality of proteins, protein phosphorylation, and protein acetylation within the various tissues of the model legume Medicago truncatula.
Author: Andrew J. Link
Publisher: Springer Science & Business Media
ISBN: 1592595847
Category : Science
Languages : en
Pages : 585
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Book Description
With the completion of sequencing projects and the advancement of a- lytical tools for protein identification, proteomics—the study of the expressed part of the genome—has become a major region of the burgeoning field of functional genomics. High-resolution 2-D gels can reveal virtually all p- teins present in a cell or tissue at any given time, including posttranslationally modified proteins. Changes in the expression and structure of most cellular proteins caused by differentiation or external stimuli can be displayed and eventually identified using 2-D protein gels. 2-D Proteome Analysis Protocols covers all aspects of the use of 2-D protein electrophoresis for the analysis of biological problems. The contri- tors include many of the leaders in the fields of biochemistry and analytical chemistry who were instrumental in the development of high-resolution 2-D gels, immobilized pH gradients, computer analysis, and mass spectromet- based protein identification methodologies. This book is intended as a benchtop manual and guide both for novices to 2-D gels and for those aficionados who wish to try the newer techniques. Any group using protein biochemistry—especially in the fields of molecular biology, biochemistry, microbiology, and cell biology—should find this book eminently useful. 2-D Proteome Analysis Protocols takes the researcher through the c- plete process of working with 2-D protein gels from making the protein - tract to finally identifying the proteins of interest. It includes protocols for generating 2-D protein extracts from most of the standard model organisms, including bacteria, yeast, nematode, Drosophila, plants, mouse, and human.
Author: Ievgen Motorykin
Publisher:
ISBN:
Category : Mass spectrometry
Languages : en
Pages : 147
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Book Description
Signal transduction within and between cells is at the core of biological activity in all living systems. Signaling networks are required for regulating biological functions, including growth, development and survival. Deregulation of signaling cascades has been linked to chronic and acute diseases and disorders This thesis focuses on mass spectrometry as a high resolution and high mass accuracy technique for the detection and characterization of proteins in biological systems. The thesis presents applications of contemporary mass-spectrometric methods to identify proteins, determine changes in their expression levels, and characterize post-translational modifications, in an effort to study changes in cell signaling in response to stress or disease. Various sample preparation methods to successfully suit needs of different biological questions were developed and applied: (1) extraction of the proteome, (2) chemical tagging and enrichment of the ATPome, a sub proteome comprising nucleotide binding proteins in particularly ATP-binding proteins including kinases, and (3) metal affinity complexation and enrichment of phosphopeptides. We used the following hybrid mass analyzer configurations: a quadrupole time-of-flight (qToF) instrument with ion mobility, a linear ion trap hyphenated with a FT-ICR mass spectrometry (LTQ-FT) and a hybrid ion trap-orbitrap mass spectrometer (Orbitrap Elite). The bioinformatic analysis of the proteomics data required multiple combinations of software packages to sequence proteins, perform absolute and relative quantification, statistically analyze and visualize data. Chapter 3 describes the use of zebrafish (Danio rerio) as one of the few vertebrate models that similar to humans cannot synthesize vitamin C to investigate the system-wide consequences of deficiencies in two essential micronutrients, vitamins E and C, on the proteome biology. A label-free proteomics workflow was applied to detect changes in protein abundance estimates dependent on vitamin regimes. The study reveals suppression in an energy metabolism cycle, glycolysis, in vitamin C and E deficient zebrafish. It was discovered that alternative energy cycle, glutaminolysis, is activated to fulfill energy requirement. Chapter 4 focuses on the determination of proteome differences that can be linked to the propensity of metastasis in osteosarcoma (OS), a bone cancer that predominantly targets the adolescent age group. OS has a high propensity to metastasize to the lungs, which is associated with a poor prognosis. The study utilizes canine osteosarcoma cell lines that were originally obtained from orthotropic primary OS and metastatic cells. Canis familiaris, the domestic dog, is an established large animal model of OS that recapitulate many biological and clinical features of the human malignancy. We applied a two-prone comparative proteomics approach that consisted of: (a) determination of protein abundance levels and (b) focus on kinases, a functional sub-proteome, using a chemical affinity tag for enrichment of ATP-binding proteins. Findings of this study indicated that in the highly metastasizing canine osteosarcoma cell line proteins associated with extracellular adhesion were deregulated, which may enhance metastogenesis. Mitogen activated protein kinases MAP2K6, MAP4K3, MAP4K4, MAP4K5, ZAK and v-akt murine thymoma viral oncogene homolog 1, AKT1, are among those expressed in significantly lower abundance in the highly metastasizing canine osteosarcoma cell line indicating changes in cell signaling. Chapter 5 describes the development and application of a multiple protease protocol (Trypsin, LysC, AspN, Chymotrypsin and GluC) for improving the number of phosphosite identifications in a large-scale phosphoproteomics studies. The method combines immobilized titanium ion affinity chromatography (Ti4+-IMAC) with a data-dependent, decision tree-based data acquisition technique utilizing two complementary fragmentation methods, namely collision induced dissociation (CID) and electron transfer dissociation. The multiple protease protocol was applied to human leukemic T cell lymphoblasts (Jurkat E6.1) and resulted in the detection of >11,000 unique phosphosites, the most comprehensive identification among methods that use similar phosphopeptide enrichment approach.
