› › Defining The Human Heart Proteoform Landscape With Top Down Proteomics

Defining the Human Heart Proteoform Landscape with Top-Down Proteomics

Author : Trisha Tucholski

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Page : 0 pages

File Size : 12,26 MB

Release : 2020

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Functional diversity in the heart proteome is attributed to the vast array of proteoforms arising from molecular processing events such as alternative splicing and post-translational modifications (PTMs). Given the importance of isoform switching and PTMs in the regulation of heart function and dysfunction, it is important to study the heart proteome at the proteoform level in our effort to understand heart health and disease. Top-down proteomics featuring high-resolution mass spectrometry (MS) is a premier tool for studying proteoforms in biological systems. However, the high complexity and wide dynamic range of the heart proteome have precluded in-depth top-down proteomic analysis using conventional platforms. Moreover, studying large proteoforms (>60 kDa) with top-down proteomics is especially challenging due to reduced MS signal-to-noise ratio with increasing size combined with signal suppression caused by the co-elution of smaller, more abundant proteoforms. To improve coverage of the heart proteoform landscape and access large proteoforms by top-down proteomics, serial size-exclusion chromatography (sSEC) was introduced for size-based fractionation of complex protein mixtures. A two-dimensional separation platform coupling sSEC with reversed-phase chromatography and high-resolution MS expanded coverage of heart proteoforms and enabled the detection of those up to 223 kDa from heart tissue lysate (Chapter 3). Despite improved MS detection of large proteoforms, challenges associated with tandem MS (MS/MS) fragmentation and limitations in mass resolution still prevented their identification. Intact-mass analysis software relating experimental proteoform mass to a list of candidate proteoforms enabled interpretation of complex top-down proteomic data and facilitated the identification of large heart proteoforms, including the 140-kDa myosin binding protein C (Chapter 4). Fourier transform ion cyclotron resonance (FT-ICR) MS has also provided a versatile platform for characterization of proteoforms, given its high mass resolving power and mass accuracy combined with multi-faceted fragmentation capabilities (Chapter 5). sSEC fractionation paired with top-down FT-ICR MS allowed for straightforward characterization of metabolic enzymes extracted from heart tissue (Chapter 6). Finally, top-down proteomics was applied to investigate the genotype-proteoform phenotype relationship in human hypertrophic cardiomyopathy (HCM), the most common heritable heart disease (Chapter 7). We quantified contractile proteoforms in septal myectomy tissues from HCM patients and observed converging proteoform phenotypes, regardless of disease-causing mutation, suggesting that common pathways are activated during disease progression and underscoring the importance of proteoform-level analysis. Overall, the studies detailed in this dissertation have charted new territory in the heart proteoform landscape, especially for heart proteoforms with molecular weight above 60 kDa. sSEC-based top-down proteomics facilitated cataloging of heart proteoforms to aid future studies seeking to investigate proteoforms in heart health and disease (Chapter 8). Future applications of the technology detailed herein will expand on SEC-based techniques for top-down proteomics and adapt the technology to accommodate the study of protein interactomes and large proteoforms from minute samples, such as those obtained from patients with heart disease.



Proteoforms

Author : Xianquan Zhan

Publisher : BoD – Books on Demand

Page : 92 pages

File Size : 29,96 MB

Release : 2020-07-15

Category : Science

ISBN : 1838800336

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Book Description

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.



Novel Proteomic Approaches to Characterize Endogenous Membrane Proteins

Author : Kyle Brown

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File Size : 20,86 MB

Release : 2020

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Biological information flows as DNA is transcribed into mRNA and then translated into proteins. However, sequence variations from mutations and alternative splicing events combined with post-translational modification (PTMs) of proteins result in a diversity of protein forms (referred to as proteoforms) that can arise from a single gene. Mass spectrometry (MS)-based proteomics provides an unprecedented opportunity to understand the role of proteoforms in health and disease; however, many challenges remain. For example, despite their importance as drug targets (>50% of current drugs), membrane proteins are traditionally underrepresented using MS-based proteomics because of their lower expression level, hydrophobicity, and lack of established protocols. To address these challenges, I developed a novel photocleavable surfactant, Azo, which can effectively solubilize proteins and is compatible with MS analysis (Chapter 2). We demonstrated Azo-aided top-down proteomics (the analysis of intact proteins by MS) enabled the solubilization of important membrane proteins from biological samples, including heart tissues, for comprehensive characterization of their proteoforms. Moreover, Azo is simple to synthesize and can be used as a surfactant in polyacrylamide gel electrophoresis. We next incorporated the surfactant technology to facilitate high-throughput bottom-up proteomics (the analysis of digested proteins by MS) for more extensive proteome coverage and protein expression quantification. Furthermore, we established simple, high-throughput membrane and extracellular matrix proteomic methods using Azo (Chapter 3-4). Combining Azo-aided bottom-up and top-down proteomics, we established a powerful integrated strategy to extensively characterize proteins from biological samples. Finally, a novel membrane protein enrichment and multidimensional liquid chromatography separation strategy was developed to further expand the scope of MS-based top-down proteomics for characterizing the membrane proteoform landscape (Chapter 4). Future development and applications of MS-based approaches for the characterization of membrane proteoforms are discussed in Chapter 5.



Novel Strategies to Address the Challenge of Sensitivity in Top-down Proteomics

Author : Jake Melby

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Page : 0 pages

File Size : 41,7 MB

Release : 2023

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The cellular effectors of biological function are proteoforms - the diversity of protein variants that arise due to events such as genetic mutations, alternative splicing, and post-translational modifications (PTMs). Top-down mass spectrometry (MS)-based proteomics is the premier technology to decipher biological processes that are occurring at the proteoform level. However, top-down proteomics faces several challenges (Chapter 1) before it reaches the same depth of coverage and utility than that of the more developed bottom-up proteomics. I have devoted my thesis to developing strategies to address the challenge of sensitivity in top-down proteomics. First, we demonstrated that top-down proteomics is a robust and high-throughput approach to characterize the molecular heterogeneity of myofilament proteoforms from seven different skeletal muscle tissues (Chapter 2). We then established an integrated method that permits sequential assessment of functional properties and top-down proteomics from the same stem cell-derived engineered cardiac tissue, enabling the connection of kinetics measurements with proteoforms (Chapter 3). The development of a highly sensitive top-down proteomics platform capable of measuring proteoforms from single muscle fibers (SMFs), multinucleated single cells, found fiber-to-fiber heterogeneity among the sarcomeric proteoforms (Chapter 4). We reproducibly detected multiple isoforms of myosin heavy chain (~223 kDa), a motor protein that drives muscle contraction, with high mass accuracy enabling the classification of individual fiber types. Finally, we developed a workflow termed "small-scale serial size exclusion chromatography", or s3SEC, for the analysis of large proteoforms from minimal amounts of cardiac tissue (Chapter 5). We implemented s3SEC using a cohort of control and heart failure cardiac tissue (~1 mg) to facilitate the measurement of high MW proteoforms in addition to showing differences in PTMs in control and disease samples. Future applications of the technology detailed herein will enable proteoform measurements from biological systems of interest once thought to be inaccessible by top-down proteomics analysis (Chapter 6). Further development will continue to push the boundaries of sensitivity in top-down proteomics to enable the measurement of proteoforms from single mononucleated cells with an emphasis on creating new technology for the analysis of large proteoforms.



Glyco-Engineering

Author : Alexandra Castilho

Publisher : Humana

Page : 0 pages

File Size : 14,46 MB

Release : 2015-06-17

Category : Science

ISBN : 9781493927593

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Book Description

Conceived with the intention of providing an array of strategies and technologies currently in use for glyco-engineering distinct living organisms, this book contains a wide range of methods being developed to control the composition of carbohydrates and the properties of proteins through manipulations on the production host rather than in the protein itself. The first five sections deal with host-specific glyco-engineering and contain chapters that provide protocols for modifications of the glycosylation pathway in bacteria, yeast, insect, plants and mammalian cells, while the last two sections explore alternative approaches to host glyco-engineering and selected protocols for the analysis of the N-glycans and glyco-profiling by mass spectrometry. Written for the highly successful Methods in Molecular Biology series, 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 extensive, Glyco-Engineering: Methods and Protocols offers vast options to help researchers to choose the expression system and approach that best suits their intended protein research or applications.



Subcellular Proteomics

Author : Eric Bertrand

Publisher : Springer Science & Business Media

Page : 397 pages

File Size : 29,10 MB

Release : 2007-08-29

Category : Science

ISBN : 1402059434

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Book Description

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.



Proteoform Identification

Author : Liangliang Sun

Publisher : Humana

Page : 0 pages

File Size : 35,1 MB

Release : 2023-06-18

Category : Science

ISBN : 9781071623275

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Book Description

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.



Recoding: Expansion of Decoding Rules Enriches Gene Expression

Author : John F. Atkins

Publisher : Springer Science & Business Media

Page : 473 pages

File Size : 50,69 MB

Release : 2010-03-10

Category : Science

ISBN : 0387893822

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Book Description

The literature on recoding is scattered, so this superb book ?lls a need by prov- ing up-to-date, comprehensive, authoritative reviews of the many kinds of recoding phenomena. Between 1961 and 1966 my colleagues and I deciphered the genetic code in Escherichia coli and showed that the genetic code is the same in E. coli, Xenopus laevis, and guinea pig tissues. These results showed that the code has been c- served during evolution and strongly suggested that the code appeared very early during biological evolution, that all forms of life on earth descended from a c- mon ancestor, and thus that all forms of life on this planet are related to one another. The problem of biological time was solved by encoding information in DNA and retrieving the information for each new generation, for it is easier to make a new organism than it is to repair an aging, malfunctioning one. Subsequently, small modi?cations of the standard genetic code were found in certain organisms and in mitochondria. Mitochondrial DNA only encodes about 10–13 proteins, so some modi?cations of the genetic code are tolerated that pr- ably would be lethal if applied to the thousands of kinds of proteins encoded by genomic DNA.



Protein-Nanoparticle Interactions

Author : Masoud Rahman

Publisher : Springer Science & Business Media

Page : 95 pages

File Size : 23,90 MB

Release : 2013-06-24

Category : Science

ISBN : 3642375553

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Book Description

In recent years, the fabrication of nanomaterials and exploration of their properties have attracted the attention of various scientific disciplines such as biology, physics, chemistry, and engineering. Although nanoparticulate systems are of significant interest in various scientific and technological areas, there is little known about the safety of these nanoscale objects. It has now been established that the surfaces of nanoparticles are immediately covered by biomolecules (e.g. proteins, ions, and enzymes) upon their entrance into a biological medium. This interaction with the biological medium modulates the surface of the nanoparticles, conferring a “biological identity” to their surfaces (referred to as a “corona”), which determines the subsequent cellular/tissue responses. The new interface between the nanoparticles and the biological medium/proteins, called “bio-nano interface,” has been very rarely studied in detail to date, though the interest in this topic is rapidly growing. In this book, the importance of the physiochemical characteristics of nanoparticles for the properties of the protein corona is discussed in detail, followed by comprehensive descriptions of the methods for assessing the protein-nanoparticle interactions. The advantages and limitations of available corona evaluation methods (e.g. spectroscopy methods, mass spectrometry, nuclear magnetic resonance, electron microscopy, X-ray crystallography, and differential centrifugal sedimentation) are examined in detail, followed by a discussion of the possibilities for enhancing the current methods and a call for new techniques. Moreover, the advantages and disadvantages of protein-nanoparticle interaction phenomena are explored and discussed, with a focus on the biological impacts.



Peptidomics

Author : Michael Schrader

Publisher : Humana

Page : 423 pages

File Size : 43,99 MB

Release : 2019-06-04

Category : Science

ISBN : 9781493985142

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Book Description

This volume describes protocols for basic state-of-the-art approaches in the field of peptidomics. Most of these approaches are independent of the instruments used for analysis and can easily be adapted for equipment that is available in a typical proteomics facility. Chapters detail many of the basic techniques used to detect and identify peptides, methods for the relative quantitation of peptides between samples using isotopic labels or label-free approaches, and biological species as well as sample types. Written in the highly successful format of the Methods in Molecular Biology series, each chapter includes an introduction to the topic, a list of the necessary materials and reagents, reproducible step-by-step laboratory protocols, and tips on troubleshooting common problems and avoiding pitfalls. Authoritative and practical, Peptidomics: Methods and Strategies provides useful guidance for studies in the rapidly growing field of peptidomics.