Download or read book Complex Proteoform Identification Using Top Down Mass Spectrometry written by Qiang Kou and published by . This book was released on 2018 with total page 198 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteoforms are distinct protein molecule forms created by variations in genes, gene expression, and other biological processes. Many proteoforms contain multiple primary structural alterations, including amino acid substitutions, terminal truncations, and posttranslational modifications. These primary structural alterations play a crucial role in determining protein functions: proteoforms from the same protein with different alterations may exhibit different functional behaviors. Because top-down mass spectrometry directly analyzes intact proteoforms and provides complete sequence information of proteoforms, it has become the method of choice for the identification of complex proteoforms. Although instruments and experimental protocols for top-down mass spectrometry have been advancing rapidly in the past several years, many computational problems in this area remain unsolved, and the development of software tools for analyzing such data is still at its very early stage. In this dissertation, we propose several novel algorithms for challenging computational problems in proteoform identification by top-down mass spectrometry. First, we present two approximate spectrum-based protein sequence filtering algorithms that quickly find a small number of candidate proteins from a large proteome database for a query mass spectrum. Second, we describe mass graph-based alignment algorithms that efficiently identify proteoforms with variable post-translational modifications and/or terminal truncations. Third, we propose a Markov chain Monte Carlo method for estimating the statistical signi ficance of identified proteoform spectrum matches. They are the first efficient algorithms that take into account three types of alterations: variable post-translational modifications, unexpected alterations, and terminal truncations in proteoform identification. As a result, they are more sensitive and powerful than other existing methods that consider only one or two of the three types of alterations. All the proposed algorithms have been incorporated into TopMG, a complete software pipeline for complex proteoform identification. Experimental results showed that TopMG significantly increases the number of identifications than other existing methods in proteome-level top-down mass spectrometry studies. TopMG will facilitate the applications of top-down mass spectrometry in many areas, such as the identification and quantification of clinically relevant proteoforms and the discovery of new proteoform biomarkers.

Download or read book Proteoform Identification written by Liangliang Sun and published by Humana. This book was released on 2023-06-18 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume discusses the latest mass spectrometry (MS)-based technologies for proteoform identification, characterization, and quantification. Some of the topics covered in this book include sample preparation, proteoform separation, proteoform gas-phase fragmentation, and bioinformatics tools for MS data analysis. 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. Cutting-edge and comprehensive, Proteoform Identification: Methods and Protocols is a valuable resource for researchers in both academia and the biopharmaceutical industry who are interested in proteoform analysis using MS.

Download or read book Development of Methods to Improve the Efficiency of Proteoform Identification by Mass Spectrometry in Complex Systems written by John Gerrit Pavek and published by . This book was released on 2024 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The gap between genotype and phenotype is incredibly difficult to bridge. The complexity of the intervening processes is the reason for this challenge. We believe that proteoforms, the specific molecular products of genes, hold the key to bridging the genotype-phenotype gap with detailed molecular understanding. However, comprehensive proteoform characterization is incredibly difficult due to the wide range in both physicochemical properties and abundances of proteoforms in complex systems. Most commonly, the broad characterization of proteoforms is achieved by top-down proteomics, a technique in which intact proteoforms are analyzed by tandem mass spectrometry. To move towards comprehensive characterization of proteoforms in complex systems requires the development of novel techniques at all aspects of top-down proteomics, from sample preparation to data analysis.Even as deeper proteoform coverage becomes achievable, the practicality of the technique for routine biological analysis remains limited. This is because many of the strategies used to improve proteoform coverage also come with a significant time cost. In this work, I demonstrate our efforts toward developing techniques to make proteoform identification by mass spectrometry more efficient while also achieving reasonable depth. This work centers around the development of strategies for proteoform identification without the need for tandem MS. First, I present a NeuCode chemical labeling strategy that enables the experimental determination of tissue proteoform cysteine counts, which can be used to improve the confidence of proteoform identifications made sans-fragmentation. I then present a proof-of-concept study, where an E.coli proteoform atlas is constructed, and subsequently used to enable efficient proteoform re-identification.

Download or read book Proteomics Sample Preparation written by Jörg von Hagen and published by John Wiley & Sons. This book was released on 2011-08-24 with total page 498 pages. Available in PDF, EPUB and Kindle. Book excerpt: This long-awaited first guide to sample preparation for proteomics studies overcomes a major bottleneck in this fast growing technique within the molecular life sciences. By addressing the topic from three different angles -- sample, method and aim of the study -- this practical reference has something for every proteomics researcher. Following an introduction to the field, the book looks at sample preparation for specific techniques and applications and finishes with a section on the preparation of sample types. For each method described, a summary of the pros and cons is given, as well as step-by-step protocols adaptable to any specific proteome analysis task.

Download or read book Leveraging Capillary Zone Electrophoresis mass Spectrometry for Multi level Proteomics written by Xiaojing Shen and published by . This book was released on 2020 with total page 194 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry (MS) coupled with online liquid-phase separation is the major tool for large-scale bottom-up proteomics (peptide-centric), top-down proteomics (proteoform-centric), and native proteomics (protein complex-centric). While liquid chromatography (LC)-MS is the dominant method for proteomics at different levels, capillary zone electrophoresis (CZE)-MS has emerged as a valuable and complementary technique, which provides high-capacity separation and highly sensitive detection of peptides, proteoforms and even protein complexes under native conditions. This work focuses on developing novel CZE-MS/MS methods for multi-level proteomics (bottom-up, top-down, and native).In Chapter 2, a high-throughput bottom-up proteomics workflow was developed by coupling immobilized trypsin-based speedy protein digestion with fast CZE-MS/MS. Immobilized trypsin produced almost the same digestion performance as free trypsin for complex proteomes with about 50-times higher speed (15 min vs. 12 h). Integration of immobilized trypsin (IM)-based rapid protein cleavage and fast CZE-MS/MS enables the identification of thousands of proteins from the mouse brain proteome in only 3 h, which is significantly faster than the typical LC-MS-based bottom-up proteomics workflow (3 h vs. >12 h). The high-throughput workflow was expected to be useful for bottom-up proteomics of human clinical samples (e.g., serum and urine).Chapter 3 presents the first example of CZE-MS/MS with activated ion-electron capture dissociation (AI-ECD) on a high-end quadrupole-time-of-flight (Q-TOF) mass spectrometer for top-down proteomics, enabling high-resolution separation, highly sensitive detection, and extensive gas-phase backbone cleavages of proteoforms. The CZE-AI-ECD method will be useful to the top-down proteomics community for the comprehensive characterization of proteoforms in complex proteomes. Chapter 4 and 5 focus on the development of novel CZE-MS methods for native proteomics, delineating proteins and protein complexes under native conditions. In Chapter 4, a native CZE-MS/MS platform with an Orbitrap mass spectrometer was established for native proteomics of a complex proteome (E. coli), leading to the identification of 23 protein complexes in discovery mode. The work represents the first example of native proteomics via coupling online liquid-phase separation to native MS and MS/MS. The characterization of large protein complexes (up to 200 kDa) was also achieved with a new CZE-MS system on a high-end Q-TOF mass spectrometer.In Chapter 5, a novel native capillary isoelectric focusing (cIEF)-assisted CZE-MS method is presented for the characterization of monoclonal antibodies (mAbs) with large sample loading capacity and high separation resolution. Using the method, the potential separations of different conformations of the SigmaMAb and the detection of its various glyco-proteoforms and homodimer were documented. The method separated the NISTmAb into three peaks with a microliter sample loading volume, corresponding to its different proteoforms. In addition, eight glyco-proteoforms of the NISTmAb and its homodimer were detected. The results demonstrate the potential of the native cIEF-assisted CZE-MS method for advancing the characterization of large proteins (i.e., mAbs) and protein complexes under native conditions.

Download or read book Advancing Capillary Electrophoresis mass Spectrometry for Top down Proteomics written by Tian Xu and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Top-down proteomics (TDP) enables the proteome profiling of biological subjects at the proteoform level and understanding of differential functions associated with proteoform heterogeneity, such as sequence variation, post-translational modifications (PTMs), etc. Drastic advances on TDP technologies (e.g. sample preparation, separation/fractionation, fragmentation, bioinformatics, etc.) have been achieved in the past decades. Further improvements in separation remain desired for better analysis throughput and deeper proteome coverage. Capillary electrophoresis (CE), including capillary zone electrophoresis (CZE) and capillary isoelectric focusing (cIEF), provide superior separation performance for proteoforms. This dissertation focuses on the advancement of CE-MS-based tools on throughput, separation resolution, and capacity for TDP and utility of these tools for biological applications.In Chapter 2, we developed high-throughput and high-capacity cIEF-MS/MS platforms. The high-throughput platform enables efficient identification and quantification of proteoforms (less than one hour per run), whereas the high-capacity cIEF-MS/MS provides large number of proteoform identifications (IDs, more than 700 proteoforms in a single shot analysis) which is valuable for deep TDP. In Chapter 3, we further improved the stability and robustness of cIEF-MS platform using optimized linear polyacrylamide (LPA) capillary coating and catholyte with lower pH (pH~10). The work achieved high-resolution characterization and accurate isoelectric point (pI) determination of charge variants (~0.1 pI difference) of monoclonal antibodies (mAbs). In Chapter 4, we developed a nondenaturing cIEF-MS platform for ultrahigh resolution characterization of microheterogeneity of a variety of protein complexes. Typically, pI determinations of variants in protein complexes allow us to decipher how sequence or PTM variations modulate the pIs of the protein complexes. In Chapter 5, while CZE-MS/MS is a well-developed approach, for the first time, we coupled FAIMS to CZE-MS/MS to facilitate online gas-phase fractionation of proteoforms. The FAIMS greatly enhanced the sensitivity of the system and expanded the number of proteoform IDs, especially large proteoform IDs. The work renders CZE-FAIMS-MS/MS as a new powerful multidimensional platform for deep TDP.In Chapters 6 and 7, we applied cIEF-MS/MS and CZE-MS/MS for studying the sexual dimorphism of zebrafish brains and proteoform-level differences between metastatic and nonmetastatic colorectal cancer (CRC) cells, respectively. In Chapter 6, quantitative TDP of thousands of proteoforms from male and female zebrafish brains by cIEF-MS/MS based approach discovered various overexpressed proteoforms in male or female brains that are closely associated with hormone activity. In Chapter 7, We performed deep TDP study of non-metastatic and metastatic CRC cells (SW480 and SW620) using CZE-MS/MS based multidimensional platform and identified more than 20,000 proteoforms of over 2,000 proteins from the two cell lines, which presents around 5-folds higher number of proteoform IDs in comparison with previous TDP studies of human cancer cells. The work revealed significant discrepancies between the two isogenic cell lines regarding proteoform and single amino acid variant (SAAV) profiles. Quantitative data disclosed differentially expressed proteoforms between the two cell lines and their corresponding genes were connected to cancer pathways and networks.

Download or read book Novel Automated Platform for Proteoform Driven Top down Mass Spectrometry Proteomics written by John Rawson Corbett and published by . This book was released on 2017 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Top-Down proteomics studies protein complexity at the intact proteoform level in order to study chemical modifications, such as co-post translational modifications and non-enzymatic protein processing (e.g., redox active modifications, glycation). With this approach, information content associated with the diversity of chemical/biological processes, such as glycosylation, lipidation, and proteolysis that occur in vivo, is captured facilitating an enhanced representative observation of biological complexity. To obtain this information, a traditional Top-Down approach uses liquid chromatography separations in conjunction with mass spectrometry and database querying techniques in order to identify proteoforms. For example, this approach was used in a study highlighting differentially expressed levels of phosphor-proteoforms within cardiac myofilaments and their association with different degrees of congestive heart failure. Although these strategies have been well characterized, such an approach is not applicable towards large scale proteome analysis due to the high heterogeneity of expressed proteoforms. For this type of analysis, multiple dimensions of orthogonal chromatographic separations are used to antagonize proteoform complexity, with prior attempts identifying over 3,000 unique proteoforms from the HeLa S3 cell line. These Top-Down platforms have also been used towards completing proteome scale label-free quantitative studies; however, such approaches have often struggled due to limited quantitative dynamic range. Additionally, chromatographic separation strategies have been protein driven reducing proteoform observation to only the most abundant species, and in some cases a complete loss of proteoform information (i.e., related glycoproteoforms) due to limitations associated with charging/ionization efficiency, ion transfer, and mass spectrometer resolving power. To address these obstacles, a novel platform that utilizes the concept of isoelectric point separation has been implemented in order to complete chromatographic separations at the proteoform level. Utilizing high resolution in solution isoelectric focusing with superficially porous liquid chromatography and Fourier-transform mass spectrometry, a ~5x improvement of observed proteoforms from cardiac myofibril tissue (1D: 112 vs. 2D: 582 proteoforms) was determined with species ranging from 3 – 230 kDa in size. In addition, novel data processing strategies that are capable of distinguishing related proteoform information content separated into different mass spectra have been implemented with the objective to establish the three quantitative levels of Top-Down proteomics (proteoform, protein, and proteoform ratios). Standard proteins with different physiochemical properties and modification classes were studied to create calibration curves under non-spiked and spiked conditions (i.e., E. coli matrix effect) with a linear dynamic range of 102 – 103 and low femtomole limits of detection values established. Additionally, results indicate that proteoform ratio information content, outside of matrix effects, is independent of protein loading. To aid in automating the data processing strategies associated with mass spectral deconvolution and data binning procedures, triplicate E. coli proteome analyses have been completed with a sliding window approach illustrating reproducible spectral intensity values (~15.1% relative standard deviation) and chromatographic precision tolerances of ± 0.2 pI units and ± 12 seconds for weighted pI and hydrophobicity calculations respectively. Using this platform, Lipocalin-type Prostaglandin D-Synthase, a highly glycosylated cerebrospinal fluid (CSF) protein, was fully characterized with 200+ proteoforms identified, a 65x improvement compared to other non-pI based Top-Down platforms that are chromatographically protein driven. In the future, the completion of CSF proteome profiling investigations will contribute to the interpretation of changes in proteoform modifications and expression levels and the correlation to unique pathobiology associated with different neurodegenerative and neuroinflammatory diseases.

Download or read book New Front end Separation Approaches for Top down Proteomics written by Eli Larson and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Top-down mass spectrometry (MS) and top-down proteomics have become indispensable tools to characterize and identify unique proteoforms. Proteoforms are defined as all protein products of a single gene, including splicing variants, mutants, and post-translationally modified forms. Although the development of new MS capabilities has exploded in recent years, the comparative underdevelopment of intact protein separations and data processing solutions has prevented full realization of the benefits of top-down. To address these challenges, I have developed new front-end separation approaches for top-down proteomics, beginning with targeted separations for multi-attribute analysis of antibody-drug conjugates (ADCs) and later developing an online two-dimensional liquid chromatography (2DLC) method to expand global proteome coverage by top-down proteomics. Chapter 1 focuses on recent advances in front-end separations and data processing solutions for top-down proteomics and introduces top-down applications to antibody-based therapeutic analysis. Chapter 2 and chapter 3 detail new targeted separation approaches for monoclonal antibodies and ADCs. Chapter 2 reports reversed phase liquid chromatography (RPLC) coupled to high-resolution Fourier transform ion cyclotron resonance MS for top-down analysis of a reduced cysteine-linked ADC. Chapter 3 details the development of a native complex-down workflow using trapped ion mobility spectrometry-MS with a cysteine-linked ADC and parent mAb under non-denaturing conditions (Chapter 3). Chapter 4 reports a new software package designed to address the challenges associated with native top-down proteomics, MASH Native. Chapter 5 focuses on the development of a new online 2DLC method coupling serial size exclusion and RPLC to expand global top-down proteome coverage, with application to human heart extract. Appendix I reports a shotgun proteomic approach to characterize the impact of splicing factor RNA binding motif 20 knockout on the rat heart proteome and identifies targets for follow-up analysis by top-down proteomics. The developed techniques detailed here will address key challenges to front-end separation in the field of top-down proteomics, expanding analytical capabilities for future targeted and discovery studies.

Download or read book Coupling Liquid Chromatography to Capillary Zone Electrophoresis Tandem Mass Spectrometry for Deep Top down Proteomics written by Elijah Neal McCool and published by . This book was released on 2021 with total page 182 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteomes are very complex with a large number of unique proteoforms spread across a wide concentration dynamic range. This means that an MS-based platform with highly efficient separation and highly sensitive detection of proteoforms is required. Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has been suggested as one such platform. When coupled to offline liquid chromatography-based fractionation, CZE-MS/MS has proven to be invaluable to the TDP community.In Chapter 2, the first optimization of dynamic pH junction-based sample stacking for TDP is provided along with one of the first comparisons of reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS) and CZE-MS/MS. Optimization of dynamic pH junction is performed with a standard protein mixture, and this platform was ultimately applied to an Eschericia coli (E. coli) whole cell lysate. This resulted in the largest TDP dataset for single-shot CZE-MS/MS. The comparison of RPLC-MS/MS and CZE-MS/MS also included analysis of an E. coli cell lysate and resulted in high numbers of identifications and highlighted the various pros and cons of each method.In Chapter 3, two dimensional LC fractionation (size exclusion chromatography (SEC) and RPLC) was coupled to CZE-MS/MS for deep TDP of E. coli cells. This study resulted in the largest TDP dataset, at the time, for E. coli, identifying 5700 proteoforms and 850 proteins. We were also able to identify and localize various interesting PTMs and estimate protein abundances using a spectral counting method. From this study it was clear thatour platform was comparable to other RPLC-MS/MS methods for deep TDP in terms of number of proteoform identifications and total instrument time.In Chapter 4, we applied our TDP platform to two isogenic colorectal cancer (CRC) cell lines, SW480 and SW620, from primary and metastatic tumors. Genetic changes have been known for a long time to affect CRC progression but this was the first proteoform-level deep TDP study of CRC metastasis. In total, we identified over 23000 proteoforms and over 2000 proteins, for the largest TDP dataset of any cell type and was a 400% increase in terms of identifications over previous deep TDP studies. We used a special database searching tool to identify single amino acid variants (SAAVs) for the largest dataset of proteoforms containing SAAVs. Quantitative analysis identified 460 proteoforms with significant differences in abundance between SW480 and SW620. Several of these proteoforms were also phosphorylated which could further impact disease progression and outcome for a specific patient phenotype and could serve as biomarkers for deciding how to treat a patient or for drug development.In Chapter 5, both activated ion electron transfer dissociation (AI-ETD) and ultraviolet photodissociation (UVPD) at 213 nm were coupled to CZE for deep TDP of E. coli and zebrafish brain samples, respectively. Optimized CZE-AI-ETD and CZE-UVPD resulted in large numbers of proteoform identifications, and many important modifications were identified and localized using these effective fragmentation techniques. This included N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation.In Chapter 6, a variety of insights into the future of TDP are provided. This includes important applications for TDP, such as personalized medicine, drug development, embryonic development, and pathogen identification. Also, a few advancements to the TDP workflow that may have increased focus on in the future are mentioned.

Download or read book Proteomics for Biomarker Discovery written by Virginie Brun and published by Humana Press. This book was released on 2019-05-04 with total page 293 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume presents modern and enhanced methods that detail techniques to perform proteomics analyses dedicated to biomarker discovery for human health. Chapters guide readers through pre/post analytical factors, protocols for the preparation of extracellular vesicles and exosomes, and various analytical pipelines including Data Independent Acquisition (DIA), discovery, as well as targeted and top-down proteomics analysis workflows. Bioinformatics tools and workflows to select and evaluate candidate biomarkers or combinations of biomarkers are also presented. 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, Proteomics for Biomarker Discovery: Methods and Protocols aims to ensure successful results in the further study of this vital field.

Download or read book Proteomic and Metabolomic Approaches to Biomarker Discovery written by Haleem J. Issaq and published by Academic Press. This book was released on 2013-05-20 with total page 489 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteomic and Metabolomic Approaches to Biomarker Discovery demonstrates how to leverage biomarkers to improve accuracy and reduce errors in research. Disease biomarker discovery is one of the most vibrant and important areas of research today, as the identification of reliable biomarkers has an enormous impact on disease diagnosis, selection of treatment regimens, and therapeutic monitoring. Various techniques are used in the biomarker discovery process, including techniques used in proteomics, the study of the proteins that make up an organism, and metabolomics, the study of chemical fingerprints created from cellular processes. Proteomic and Metabolomic Approaches to Biomarker Discovery is the only publication that covers techniques from both proteomics and metabolomics and includes all steps involved in biomarker discovery, from study design to study execution. The book describes methods, and presents a standard operating procedure for sample selection, preparation, and storage, as well as data analysis and modeling. This new standard effectively eliminates the differing methodologies used in studies and creates a unified approach. Readers will learn the advantages and disadvantages of the various techniques discussed, as well as potential difficulties inherent to all steps in the biomarker discovery process. A vital resource for biochemists, biologists, analytical chemists, bioanalytical chemists, clinical and medical technicians, researchers in pharmaceuticals, and graduate students, Proteomic and Metabolomic Approaches to Biomarker Discovery provides the information needed to reduce clinical error in the execution of research. - Describes the use of biomarkers to reduce clinical errors in research - Includes techniques from a range of biomarker discoveries - Covers all steps involved in biomarker discovery, from study design to study execution

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 Development and Application of Top down and Middle down Proteomics for Comprehensive Protein Characterization in the Muscle Proteome written by Yutong Jin and published by . This book was released on 2019 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry (MS)-based proteomics, including top-down, middle-down and bottom-up approaches, has become the method of choice for protein identification, quantification, and characterization. Top-down proteomics represents a superior approach for comprehensive protein characterization by analyzing intact proteins, providing an overview of all the proteoforms present in a sample and allowing for identification of various proteoforms and in-depth characterization of sequences and post-translational modifications (PTMs). However, the protein mass range that can be analyzed by top-down approach is normally limited to 100 kDa. Middle-down proteomics, analyzing polypeptides from proteolysis, is a complementary strategy to top-down proteomics for characterization of large proteins (>100 kDa) and complex PTMs. In this dissertation, I developed and applied top-down and middle-down proteomics to comprehensively characterize the sarcomeric proteins from striated muscle tissues. Sarcomeric proteins are expressed in different isoforms produced by homologous genes, in the presence of various proteoforms produced by a single gene via genetic variations, alternative RNA splicing, and PTMs. The various isoforms and PTMs of sarcomeric proteins are associated with muscle contractile properties and thus affects the entire muscle function. However, a comprehensive characterization of sarcomeric protein isoforms and PTMs is still challenging due to the high complexity of the muscle proteome and the limitations in techniques for protein separation and characterization. The first part of this dissertation focuses on characterizing sarcomeric proteins below 100 kDa using top-down proteomics. Sarcomeric proteins from multiple skeletal muscle tissues were characterized by liquid chromatography and mass spectrometry plus (LC-MS+) strategy which combines online LC-MS and offline tandem MS (MS/MS) analysis. I also developed a reversed-phase chromatography method using monolithic column for highly efficient separation of sarcomeric proteins and combined it with high-resolution MS for top-down proteomics. The second part of this dissertation describes the development of middle-down proteomics for characterization of large sarcomeric proteins (>100 kDa). I developed a MS-compatible size-exclusion chromatography (SEC) method to purify myosin heavy chain from cardiac muscle tissue and characterize this protein by refining a middle-down MS strategy. I also developed a middle-down proteomics strategy to study the phosphorylation status of a serine-arginine-rich protein RNA-binding motif 20. Finally, combining top-down and middle-down proteomics, I described a strategy for the comprehensive characterization of a monoclonal antibody. Collectively, this dissertation provides comprehensive analysis of the isoforms and PTMs of sarcomeric proteins that will help elucidate molecular event in muscle contraction and disease pathologies. Furthermore, this dissertation presents a variety of novel LC and MS methods for protein separation and characterization, enabling the further characterization of other proteomes with deep proteome coverage.

Download or read book Advancing Technologies for the Study of Proteoforms and Protein nucleic Acid Interactomes written by Katherine B. Henke and published by . This book was released on 2021 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins are critical actors within the cell, enabling complex biological processes essential for cell survival, development, and homeostasis. Generally, proteins function via their interactions with other biomolecules, such as nucleic acids, making knowledge of these interactions and the players involved essential to our understanding of even basic biological function. This dissertation describes the advancement of technologies for the identification of proteins interacting with target nucleic acid sequences (e.g., genomic loci or RNA transcripts), as well as the development of approaches for better understanding the diversity of proteins expressed in human cells. Chapter 2 presents the application of HyCCAPP (Hybridization Capture of Chromatin-Associated Proteins for Proteomics), a technology developed in the Smith lab for the study of protein-DNA interactions in a locus-specific manner, to identify the protein interactome of human centromeric alpha satellite DNA. We identified 90 proteins as enriched in alphoid chromatin, and this list included many known centromere-binding proteins in addition to multiple novel alpha satellite-binding proteins. This work represents the first application of the HyCCAPP technology in mammalian cells and is the first DNA-centric examination of human protein-alpha satellite interactions. In Chapter 4, we present a detailed and comprehensive guide for the application of HyPR-MS (Hybridization Purification of RNA-protein complexes followed by Mass Spectrometry), a technology developed in the Smith lab for the identification of proteins interacting with target RNA transcripts. It is our hope that the practical advice provided in this chapter will enable the widespread utilization of this technology. In Chapter 3, we explored how different types of proteomics data could be integrated to maximize proteoform identifications from a human cell line. Proteoforms are the specific molecular forms of proteins expressed in the cell, accounting for genetic variation, alternative splicing, and post-translational modifications. Through the integration of intact-mass, top-down, and bottom-up proteomics data, we were able to identify ~1,200 proteoforms representing 484 genes from the human Jurkat cell line. Finally, in Chapter 5, we present the current status of our work to combine proteoform analysis with HyPR-MS to enable the first ever study of the proteoforms bound to a target RNA transcript.

Download or read book Proteoforms written by Xianquan Zhan and published by BoD – Books on Demand. This book was released on 2020-07-15 with total page 92 pages. Available in PDF, EPUB and Kindle. Book excerpt: A proteoform is the basic unit in a proteome, defined as its amino acid sequence + post-translational modifications + spatial conformation + localization + cofactors + binding partners + a function, which is the final functional performer of a gene. Studies on proteoforms offer in-depth insights and can lead to the discovery of reliable biomarkers and therapeutic targets for effective prediction, diagnosis, prognostic assessment, and therapy of disease. This book focuses on the concept, study, and applications of proteoforms. Chapters cover such topics as methodologies for identifying and preparing proteoforms, proteoform pattern alteration in pituitary adenomas, and proteoforms in leukemia.

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

Download or read book Subcellular Proteomics written by Eric Bertrand and published by Springer Science & Business Media. This book was released on 2007-08-29 with total page 397 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume summarizes the new developments that made subcellular proteomics a rapidly expanding area. It examines the different levels of subcellular organization and their specific methodologies. In addition, the book includes coverage of systems biology that deals with the integration of the data derived from these different levels to produce a synthetic description of the cell as a system.