Proteoform Identification PDF Download

Author: Liangliang Sun
Publisher: Humana
ISBN: 9781071623275
Category : Science
Languages : en
Pages : 0

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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.

Author: Qiang Kou
Publisher:
ISBN:
Category :
Languages : en
Pages : 198

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Book Description
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.

Author: Xianquan Zhan
Publisher: BoD – Books on Demand
ISBN: 1838800336
Category : Science
Languages : en
Pages : 92

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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.

Author: Olga Lima Tavares Machado
Publisher:
ISBN:
Category : Science
Languages : en
Pages : 0

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Book Description
The term proteoform is used to denote all the molecular forms in which the protein product of a single gene can be found. The most frequent processes that lead to transcript modification and the biological implications of these changes observed in the final protein product will be discussed. Proteoforms arising from genetic variations, alternatively spliced RNA transcripts and post-translational modifications will be commented. This chapter will present an evolution of the techniques used to identify the proteoforms and the importance of this identification for understanding of biological processes. This chapter highlights the fundamental concepts in the field of top-down mass spectrometry (TDMS), and provides numerous examples for the use of knowledge obtained from the identification of proteoforms. The identification of mutant proteins is one of the emerging areas of proteogenomics and has the potential to recognize novel disease biomarkers and may point to useful targets for identification of therapeutic approaches.

Author: Leah Schaffer
Publisher:
ISBN:
Category :
Languages : en
Pages : 200

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Book Description
Diversity at the protein level accounts for much of our biological complexity. A proteoform family consists of all of the different forms of a protein (proteoforms) arising from a single gene, including sequence variants, splice variants, and posttranslationally modified forms. It is important to identify and quantify proteoforms to understand biological systems because different proteoforms from the same family can exhibit different functions. The established technique to identify proteoforms is top-down proteomics, where intact proteins are analyzed by mass spectrometry. Available top-down search software programs require quality fragmentation spectra to identify proteoforms, limiting the number of proteoforms that can be identified due to instrument time and spectral complexity. The subset of proteoforms identified is typically in the highly abundant and low molecular weight portion of the proteome. This dissertation describes the development of integrated proteomic strategies to address these challenges. We created a software program that constructs proteoform families by grouping together observed proteoforms based on differences in intact mass, enabling the identification of proteoforms by intact-mass analysis. Chapter 1 provides an overview of mass spectrometry-based proteomics and outlines current challenges in proteoform analysis. Chapter 2 describes the integration of proteoform family construction into a typical top-down proteomic workflow, resulting in a 40% increase in the number of proteoform identifications in an analysis of yeast lysate. Chapter 3 demonstrates the application of this integrated intact-mass and top-down strategy to identify and quantify murine mitochondrial proteoforms. Chapter 4 presents the construction of proteoform families in a human breast cancer cell line using data acquired on the 21 tesla FT-ICR mass spectrometry platform. Chapter 5 describes augmenting intact-mass analysis to determine candidate identifications for isotopically unresolved proteoforms, facilitating identification of human heart proteoforms >50 kDa. Chapter 6 describes the integration of bottom-up peptide data and top-down proteoform data to improve proteoform identifications and to infer potential proteoform candidates with peptide-level evidence. Chapter 7 explains the research in this dissertation to a broader nonscientific audience. Finally, Chapter 8 discusses remaining challenges and future directions for integrated proteomic strategies towards the goal of comprehensive proteoform analysis.

Author: Katherine B. Henke
Publisher:
ISBN:
Category :
Languages : en
Pages :

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Book Description
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.

Author: Liang Qiao
Publisher: Royal Society of Chemistry
ISBN: 183767034X
Category : Science
Languages : en
Pages : 299

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Book Description
In the human body, there are millions of living microorganisms involved in protecting the body from invaders, helping digestion and regulating moods, but there are also harmful pathogens that cause infectious diseases. For instance, the coronavirus (COVID-19) has caused considerable loss of life since its outbreak. Comprehensive analysis and characterization of microbes is of significant importance to understand the function and role of microorganisms, and rapid detection and identification of unknown pathogens are essential in early diagnosis, treatment monitoring and personalized medicine. Mass spectrometry is a technique to ionize molecules and detect the mass-to-charge ratio of the generated ions. The technique is widely used in hospitals for pathogenic bacteria identification, as well as in environmental science and food science for biosafety control. This book summarizes the most recent development of mass spectrometry techniques in microbial analysis, including mass spectrometry-based microbial identification, bacterial antimicrobial resistance study, data mining algorithm development, omics for microbial research, applications in clinical diagnosis, environmental science and food science, and more. It will guide researchers in the field, and those who are about to enter the field, in the most appropriate methods to characterize microbes and enable their detection.

Author: John Rawson Corbett
Publisher:
ISBN:
Category : Automation
Languages : en
Pages :

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Book Description
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.

Author: Martin Frith
Publisher: Springer
ISBN: 3319436813
Category : Computers
Languages : en
Pages : 336

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Book Description
This book constitutes the refereed proceedings of the 16th International Workshop on Algorithms in Bioinformatics, WABI 2016, held in Aarhus, Denmark. The 25 full papers together with 2 invited talks presented were carefully reviewed and selected from 54 submissions. The selected papers cover a wide range of topics from networks, tophylogenetic studies, sequence and genome analysis, comparative genomics, and mass spectrometry data analysis.

Author: Jörg von Hagen
Publisher: John Wiley & Sons
ISBN: 3527644695
Category : Science
Languages : en
Pages : 498

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Book Description
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.