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.

Download or read book Analysis of Protein Post Translational Modifications by Mass Spectrometry written by John R. Griffiths and published by John Wiley & Sons. This book was released on 2016-10-12 with total page 415 pages. Available in PDF, EPUB and Kindle. Book excerpt: Covers all major modifications, including phosphorylation, glycosylation, acetylation, ubiquitination, sulfonation and and glycation Discussion of the chemistry behind each modification, along with key methods and references Contributions from some of the leading researchers in the field A valuable reference source for all laboratories undertaking proteomics, mass spectrometry and post-translational modification research

Download or read book Development and Application of Mass Spectrometry Methods for Proteomic and Post translational Modification Analysis written by Danqing Wang (Ph.D.) and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins are essential biomolecules that perform a wide range of biological functions. Post-translational modifications (PTMs) substantially impact protein structure and function, making their characterization essential for understanding complex biological systems. This dissertation focuses on developing and applying novel mass spectrometry (MS)-based methodologies to address challenges in studying two common and important PTMs: phosphorylation and glycosylation. To this end, new enrichment materials and their corresponding workflows, including Cotton Ti-IMAC, epoxy-ATP-Ti4+ IMAC, and Very Weak Anion Exchange (VWAX) have been introduced for efficient phosphopeptide and glycopeptide enrichment. A strategy combining boronic acid enrichment, high-pH fractionation, and EThcD has been developed for comprehensive O-glycosylation profiling. Additionally, the Boost-DiLeu quantitative approach has been introduced to enhance glycopeptide quantification in size-limited samples, while a periodate oxidation-based SUGAR tag labeling method has been established for high-throughput, intact sialylated glycopeptide-specific quantification. These methods have been applied to study human diseases, such as Alzheimer's Disease, providing insights into dysregulated glycosylation patterns and their potential implications in disease pathogenesis. Overall, this work contributes to advancing MS-based proteomics strategies and broadening our understanding of the roles of PTMs in biological systems and is anticipated to inspire future research endeavors in related fields.

Download or read book Development and Application of Mass Spectrometry Methods for Proteomic and Post translational Modification Analysis written by Danqing Wang (Ph.D.) and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins are essential biomolecules that perform a wide range of biological functions. Post-translational modifications (PTMs) substantially impact protein structure and function, making their characterization essential for understanding complex biological systems. This dissertation focuses on developing and applying novel mass spectrometry (MS)-based methodologies to address challenges in studying two common and important PTMs: phosphorylation and glycosylation. To this end, new enrichment materials and their corresponding workflows, including Cotton Ti-IMAC, epoxy-ATP-Ti4+ IMAC, and Very Weak Anion Exchange (VWAX) have been introduced for efficient phosphopeptide and glycopeptide enrichment. A strategy combining boronic acid enrichment, high-pH fractionation, and EThcD has been developed for comprehensive O-glycosylation profiling. Additionally, the Boost-DiLeu quantitative approach has been introduced to enhance glycopeptide quantification in size-limited samples, while a periodate oxidation-based SUGAR tag labeling method has been established for high-throughput, intact sialylated glycopeptide-specific quantification. These methods have been applied to study human diseases, such as Alzheimer's Disease, providing insights into dysregulated glycosylation patterns and their potential implications in disease pathogenesis. Overall, this work contributes to advancing MS-based proteomics strategies and broadening our understanding of the roles of PTMs in biological systems and is anticipated to inspire future research endeavors in related fields.

Download or read book Mass Spectrometry Data Analysis in Proteomics written by Rune Matthiesen and published by Springer Science & Business Media. This book was released on 2008-02-02 with total page 322 pages. Available in PDF, EPUB and Kindle. Book excerpt: This is an in-depth guide to the theory and practice of analyzing raw mass spectrometry (MS) data in proteomics. The volume outlines available bioinformatics programs, algorithms, and databases available for MS data analysis. General guidelines for data analysis using search engines such as Mascot, Xtandem, and VEMS are provided, with specific attention to identifying poor quality data and optimizing search parameters.

Download or read book New Methods for the Analysis of Noncovalent Biological Complexes and Protein Posttranslational Modifications written by Yonghao Yu and published by . This book was released on 2006 with total page 480 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Advancing Quantitative Proteomics and Protein Post translational Modification Analyses by Multi dimensional Mass Spectrometric Approaches written by Zihui Li and published by . This book was released on 2021 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins are important molecules that are involved in numerous critical biological processes. Consequently, understanding the functional roles and dysregulation of proteins constitutes a vital task in both basic and clinical research. However, it is very challenging to define the full complement of proteins due to their large degree of variations, especially considering that many proteins can undergo numerous post-translational modifications (PTMs) which dramatically affect protein conformations and functions. Recently, mass spectrometry (MS) has evolved as a powerful tool to characterize proteins and their PTMs benefiting from its high speed, sensitivity and the ability to monitor thousands of analytes simultaneously. This dissertation is devoted to the development and application of various MS-based strategies to facilitate the detection of proteins and their PTMs qualitatively and quantitatively. A portion of this dissertation describes the investigation of proteome-wide alterations with the development and maturation of human pancreas using custom-developed isobaric tags. This study focuses on the extracellular matrix proteins and provides information on the localization changes of selected proteins in islet or acinar compartments as well. The normal baseline data across the lifespan of human pancreas will become a valuable resource and benefit future studies into the pancreatic pathologies. Switching gear to protein PTMs, this dissertation documents the design and development of a novel biotin thiol tag to greatly improve the in-depth profiling of two important PTMs, citrullination and homocitrullination, from complex biological samples. The utility of the approach is demonstrated by providing the first tissue-specific citrullination and homocitrullination atlas in mice. The first high-throughput quantitative analysis pipeline of protein citrullination is subsequently developed by the integration with isobaric labeling strategy. We also further improve the protein citrullination detection by using a combination of various digestion and fragmentation techniques. Citrullination alteration during the progression of Alzheimer's disease is further explored using human cerebrospinal fluid to shed light on the disease pathogenesis. This dissertation also highlights the application of a novel sub-atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry platform for the in situ imaging of N-glycans. Spatial distribution of N-glycans in ovarian cancer tissue section may indicate potential association of this modification with tumor progression. Collectively, we expect the biological insights and technological advancements presented in this work will lead to improved understanding of microenvironment in both healthy and disease conditions, and the new knowledge gained from these studies will inspire and benefit future research effort in related fields.

Download or read book Mass Spectrometry Modified Proteins and Glycoconjugates written by A.L. Burlingame and published by Gulf Professional Publishing. This book was released on 2005-12-13 with total page 482 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume provides comprehensive treatment of tools and proper usage for the identification of proteins, affinity chromatography and studies the complexity of protein machines and assemblages, assignment of the most common protein posttranslational modifications (phosphorylation and glycosylation) and glycolipidomics. *Part 2 of 2 volumes about Mass Spectrometry *Discusses peptide and protein cleanup and preparation requirements for mass spectrometry *Explains protein enzymic and chemical digestion strategies *Includes case studies of protein assemblages and machines

Download or read book Mass Spectrometry of Proteins and Peptides written by Mary S. Lipton and published by Humana Press. This book was released on 2008-11-01 with total page 470 pages. Available in PDF, EPUB and Kindle. Book excerpt: When the last edition of this book was published in 2000, the field of proteomics was in its infancy. At that time, multidimensional liquid chromatographic separations were being introduced as an alternative to traditional gel-based techniques for separating complex protein and peptide mixtures prior to mass spectrometric detection. Today, this approach – referred to as shotgun proteomics – is considered routine for lar- scale global analyses of protein mixtures. Now in its adolescence, proteomics is fundamentally transforming biological and medical research. Much of this transformation can be attributed to technological advancements, particularly in mass spectrometry. Much wider accessibility of hi- resolution and mass measurement accuracy instrumentation in recent years has ini- ated a new revolution in the field by providing more reliable data and shifting the focus from cataloging proteins to precisely quantifying changes in protein abundance over time and in response to stimuli. Advanced mass spectrometers and novel ion d- sociation schemes such as electron transfer/capture dissociation make it possible to venture boldly into the maze of protein posttranslational modifications, which are an integral component of understanding functional proteomics in the spatial and t- poral domains. Another area that has benefited from these advancements is top-down proteomics, an emerging method essential for characterizing various protein variants that has potentially high impact in biomedical research.

Download or read book Advancing Intact Protein Analysis by Top down Mass Spectrometry written by Bifan Chen and published by . This book was released on 2019 with total page 215 pages. Available in PDF, EPUB and Kindle. Book excerpt: The study of proteins is critical for understanding cellular functions at the molecular level. Top-down mass spectrometry (MS) has emerged as a premier tool for global and comprehensive analysis of proteoforms. The top-down approach retains intact mass information, providing a "bird's-eye" view of the proteome and allowing for identification of novel proteoforms, in-depth sequence characterization, and quantification of disease associated post-translational modifications (PTMs). However, many technical challenges still exist. The research described here involves analytical development in top-down MS, particularly in the areas of enrichment, separation, and characterization of samples ranging from standard proteins and complex lysates, to large therapeutic biomolecules. Chapter 1 provides an introduction and review of recent advances in different aspects of top-down proteomics. Chapters 2 and 3 are related to the study of intact phosphoproteins. Specifically, chapter 2 describes the use of functionalized nanoparticles for enrichment and the subsequent coupling of online liquid chromatography (LC)-MS for characterizing endogenous phosphoproteins from complex cell lysates. Chapter 3 investigates how phosphorylation moieties might influence the efficiency of electron capture dissociation (ECD). Chapters 4 and 5 focus on the development of hydrophobic interaction chromatography (HIC) that could be coupled online directly with MS and its applications to therapeutic molecules (monoclonal antibodies). Chapter 6 describes a middle-down approach to obtain multi-attribute of both cysteine and lysine conjugated antibody-drug conjugates, which overcomes some current challenges using HIC-MS and the top-down approach. Overall, these analytical developments expand the toolbox of the top-down approach and generally facilitate the analysis of intact proteins.

Download or read book Mass Spectrometry Based Method Development for Elucidation of Protein Sequences Structures and Post Translational Modifications written by Qingyu Sun and published by . This book was released on 2011 with total page 152 pages. Available in PDF, EPUB and Kindle. Book excerpt: The advanced development of mass spectrometry (MS) makes MS a powerful technique for proteomics study. The increasing demands for proteomics study stimulate creation of more applicable MS-based methods. This dissertation focuses on development of novel MS-based methods to characterize three different aspects of proteins: primary sequence, post-translational modifications (PTMs) and three-dimensional (3D) structure.

Download or read book Evaluation and Improvement of Mass Spectrometry Based Strategies for Protein Post translational Modification Analysis written by Li Cui and published by . This book was released on 2014 with total page 242 pages. Available in PDF, EPUB and Kindle. Book excerpt:

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

Download or read book Method Development for the Comprehensive Analysis of Post Translational Modifications by Mass Spectometry written by and published by . This book was released on 2007 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Signal Transduction is mediated by protein complexes whose spatial- and temporal-distribution, composition and function within cells are often regulated by different post-translational modifications (PTM). As PTMs add or subtract a specific mass difference to a protein, mass spectrometry becomes very amenable for modification analysis. These modifications have conventionally been monitored by fragmenting the modified protein or peptide by collision induced dissociation (CID) within the mass spectrometer, and then screening for the characteristic neutral fragment or fragment ion (marker ion), which is particular to the modification in question. Unfortunately, there are two major issues with respect to the traditional mass spectrometric analysis of PTMs: (1) as there are over 300 known types of modifications, the characteristic fragmentation of only a fraction of these modifications has been studied and (2) the traditional mass spectrometric approaches can only monitor these modifications sequentially, and thus comprehensive modification analysis would be unfeasible considering the breadth of PTMs. The following work aims to address these issues by (1) analyzing PTMs that have never been characterized mass spectrometrically and (2) developing a multiplexed technique for comprehensive PTM monitoring by simultaneously screening for all known characteristic fragments. With respect to the first issue, the characteristic fragmentation of lipid modifications and HNO-induced modifications was investigated. The most prevalent indicator(s) of the modification within the mass spectra are as follows: fragmentation of N-terminal myristoylated peptides produced marker ions at 240 and 268 Th, fragmentation of cysteine farnesylated peptides produced a marker ion at 205 Th and a neutral fragment of 204 Da, and fragmentation of cysteine palmitoylated peptides produced a neutral fragment of 272 Th. For HNO-induced modifications, fragmentation of the sulfinamide- and sulfinic acid-modif.

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.

Download or read book Development of Ultraviolet Photodissociation Mass Spectrometry Strategies for the Characterization of Biomolecular Structure written by Luis Antonio Macias and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ultraviolet photodissociation (UVPD) is an alternative high-energy ion activation technique implemented to produce information rich tandem mass spectra. Dissociation of biomolecules by UVPD results in structure dependent fragmentation to reveal molecular details that are otherwise undiscernible by traditional tandem mass spectrometry techniques, providing an avenue to rapidly interrogate the structure-function relationship of biologically relevant species. Applied to glycerophospholipids, UVPD is capable of resolving locations of unsaturation and stereospecific numbering of acyl chains, subtle structural features that are traditionally challenging to resolve. In the analysis of intact proteins, UVPD produces excellent sequence coverage that can pinpoint sites of post translational modifications, while providing conformation sensitive fragmentation that also informs changes in higher-order structure that occur upon ligand binding or mutations. Studies covered in this work extend the unique capabilities of UVPD to characterize increasingly complex molecules, explore associations between UVPD resolved structure and disease, and develop an understanding of dissociation mechanisms that govern fragmentation induced by 193 nm photons. Here, the high versatility of this technique was applied to the detailed structural characterization of cardiolipins at the double bond and stereochemistry level by utilizing hybrid techniques that combine collisional activation with UVPD; similarly, UVPD was integrated to both imaging and chromatographic workflows to evaluate fatty acid structure and phosphatidylcholine structure, respectively, as a function of disease state; furthermore, fragmentation of intact proteins was evaluated to discern mechanisms that influence photon-induced dissociation and leveraged to assign paratopes and interpret complex top-down spectra of proteins with disulfide bonds

Download or read book Mass Spectrometry of Proteins written by Caroline A. Evans and published by Humana. This book was released on 2020-08-14 with total page 264 pages. Available in PDF, EPUB and Kindle. Book excerpt: New insights into modern medicine and systems biology are enabled by innovative protocols and advanced technologies in mass spectrometry-based proteomics. This volume details new pipelines, workflows, and ways to process data that allow for new frontiers in proteomics to be pushed forward. With applications to biomarker discovery, interactions between proteins, between biological systems, dynamics of post-translational modifications among others, new protocols have been developed and iteratively refined to probe the endless complexity of the proteome in ever greater details. This volume deals with methods for data dependent and data independent mass spectrometry analyses. Valuable, first-hand information is provided from designing experiments, sample preparation and analysis, exploitation of public datasets and carrying out reproducible data pipelines, using modern computational tools such as Galaxy or Jupyter. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Mass Spectrometry of Proteins: Methods and Protocols aims to ensure successful results in the further study of this vital field.