Integrated Computational and Experimental Platform for Characterizing Protein Isoforms and PTMs in Microbial Systems by Top-down FT-ICR Mass Spectrometry PDF Download
Are you looking for read ebook online? Search for your book and save it on your Kindle device, PC, phones or tablets. Download Integrated Computational and Experimental Platform for Characterizing Protein Isoforms and PTMs in Microbial Systems by Top-down FT-ICR Mass Spectrometry PDF full book. Access full book title Integrated Computational and Experimental Platform for Characterizing Protein Isoforms and PTMs in Microbial Systems by Top-down FT-ICR Mass Spectrometry by Heather Marie Connelly. Download full books in PDF and EPUB format.
Author: Heather Marie Connelly
Publisher:
ISBN:
Category :
Languages : en
Pages : 251
Get Book Here
Book Description
The goals of this dissertation research were to develop an integrated computational and experimental platform for characterizing protein isoforms and post translational modifications (PTMs) in microbial systems by top-down FT-ICR mass spectrometry. To accomplish this goal, we employed methodologies of microbial growth, intact protein and protein complex extractions, followed by sample preparation and then progressed to identification of the instrumentation needed to integrate the top-down and bottom-up proteomics methodologies used in these studies. Emphasis is placed on the development of integrated top-down and bottom-up informatics and the challenges faced in the integration of these two large mass spectrometry data sets and extraction of relevant biological data. We then illustrate how top-down and bottom-up methods can be applied to the analysis of complex protein mixtures, protein complexes, and microbial proteomes. Through the work of this dissertation we have contributed to the advancement of top-down proteomics by providing an experimental platform which will aid in the analysis of intact proteins and their associated PTMs and isoforms, as well as providing a computational method that allows for the integration of top-down and bottom-up data sets.
Author: Heather Marie Connelly
Publisher:
ISBN:
Category :
Languages : en
Pages : 251
Get Book Here
Book Description
The goals of this dissertation research were to develop an integrated computational and experimental platform for characterizing protein isoforms and post translational modifications (PTMs) in microbial systems by top-down FT-ICR mass spectrometry. To accomplish this goal, we employed methodologies of microbial growth, intact protein and protein complex extractions, followed by sample preparation and then progressed to identification of the instrumentation needed to integrate the top-down and bottom-up proteomics methodologies used in these studies. Emphasis is placed on the development of integrated top-down and bottom-up informatics and the challenges faced in the integration of these two large mass spectrometry data sets and extraction of relevant biological data. We then illustrate how top-down and bottom-up methods can be applied to the analysis of complex protein mixtures, protein complexes, and microbial proteomes. Through the work of this dissertation we have contributed to the advancement of top-down proteomics by providing an experimental platform which will aid in the analysis of intact proteins and their associated PTMs and isoforms, as well as providing a computational method that allows for the integration of top-down and bottom-up data sets.
Author: Ritin Sharma
Publisher:
ISBN:
Category : Eukaryotic cells
Languages : en
Pages : 186
Get Book Here
Book Description
Over the last decade, mass spectrometry based proteomics has been established as the front-runner in systems-level protein expression studies. However, with the field progressing into research of more and more complex samples, novel challenges have been raised with respect to efficient protein extraction and computational matching. In this dissertation, various aspects in the proteomics workflow, including experimental and computational approaches, have been developed, optimized and systematically evaluated. In this work, some of the critical factors with respect to proteomics sample preparation, like available biomass, detergent removal methods, and intact protein fractionation to achieve deeper proteome measurements were evaluated. The presented work will help the broader scientific community to carefully design proteomics experiments especially in biomass limited samples. A second major area of focus in this dissertation is comprehensive characterization of post-translational modifications (PTMs) in different biological systems. PTMs are critical for functioning of both the prokaryotic and eukaryotic species and this dissertation will highlight some of the experimental strategies to explore the diversity of PTMs in microbial isolates via application of alternate protease and multiple fragmentation schemes. The PTM discovery approach will be further extended into a complex eukaryotic model trees species Populus trichocarpa using recently developed sequence tagging methods to carryout broad scale PTM search and a complete blind PTM search. Although the work presented in this dissertation mainly revolves around prokaryotic and eukaryotic species involved in environmental proteomics, the general considerations outlined in this work can be extended to every proteomics pipeline. Thus this dissertation will benefit the scientific community in carefully designing experiments before embarking on any research project involving mass spectrometry.
Author: Chongle Pan
Publisher:
ISBN: 9781109893304
Category :
Languages : en
Pages : 283
Get Book Here
Book Description
In the research described here, a substantial advancement in both qualitative and quantitative proteomic measurements was achieved using an integrated experiment and computational approach. The improved proteomic measurements can help elucidate a range of biological processes.
Author: Samantha Jo Knott
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
Get Book Here
Book Description
The completion of the human genome project allowed scientists to gain a wealth of information and quickly led to the acknowledgement that the complexity of disease phenotypes cannot be explained by genomic information alone. This observation can be attributed to the diversity of the proteome, where sequence variants, alternative splicing of mRNA transcripts and post translational modifications (PTMs), mean a singular gene can result in an enormous number of protein variants or proteoforms. Proteoforms have been shown to influence numerous biological processes and correlate well to disease phenotypes. Thus, it is critical to map and characterize tissues at the proteoform level to increase our understanding of disease mechanisms and identify new therapeutic targets. Mass spectrometry (MS) based proteomics plays an integral role in bridging our gaps in understanding. However, technological challenges still exist, and many areas have yet to be fully explored.The development of new platforms for in-depth tumor tissue characterization is needed, and despite the immense potential of top-down MS, few platforms have been developed. While bottom-up (peptide level) MS is routinely employed to identify thousands of proteins in tissues, the platform is inferior for the characterization of proteoforms and associated PTMs. On the other hand, top-down MS looks at intact proteoforms and is a powerful method for the unambiguous identification of isoforms and PTMs. In this thesis work, I have met the need and developed a top-down proteomics platform to inventory the proteoform landscape of tumor tissues harvested from a well-established mouse model of metastatic breast cancer. Using a conventional platform, few large proteins >35 kDa were observed due to the challenge and reduced signal-to-noise high-MW proteoforms present for top-down MS. In this thesis, I utilized MS-compatible size-exclusion chromatography to provide greater separation power and expanded the coverage of large tumor proteoforms 5-fold, with the detection of species up to 104 kDa. Another under explored area in proteomic analysis of tumor tissues is the characterization and quantification of the extracellular matrix (ECM), primarily due to the inherent insolubility. I developed a quantitative bottom-up MS approach to effectively solubilize, digest, and identify ECM proteins in both tumor and cardiac tissues enabled by a MS-compatible photocleavable surfactant. Importantly, the method is more accessible and takes just eight hours, compared to the previous multi-day process.
Author: Jörg von Hagen
Publisher: John Wiley & Sons
ISBN: 3527644695
Category : Science
Languages : en
Pages : 498
Get Book Here
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.
Author: John R. Griffiths
Publisher: John Wiley & Sons
ISBN: 1119045851
Category : Science
Languages : en
Pages : 414
Get Book Here
Book Description
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
Author: John Rawson Corbett
Publisher:
ISBN:
Category : Automation
Languages : en
Pages :
Get Book Here
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: Ryan Tal Fellers
Publisher:
ISBN:
Category :
Languages : en
Pages : 80
Get Book Here
Book Description
Author:
Publisher:
ISBN:
Category :
Languages : en
Pages :
Get Book Here
Book Description
With the advent of whole genome sequencing, a new era of biology was ushered in allowing for "systems-biology" approaches to characterizing microbial systems. The field of systems biology aims to catalogue and understand all of the biological components, their functions, and all their interactions in a living system as well as communities of living systems. Systems biology can be considered an attempt to measure all of the components of a living system and then produce a data-driven model of the system. This model can then be used to generate hypotheses about how the system will respond to perturbations, which can be tested experimentally. The first step in the process is the determination of a microbial genome. This process has, to a large extent, been fully developed, with hundreds of microbial genome sequences completed and hundreds more being characterized at a breathtaking pace. The developments of technologies to use this information and to further probe the functional components of microbes at a global level are currently being developed. The field of gene expression analysis at the transcript level is one example; it is now possible to simultaneously measure and compare the expression of thousands of mRNA products in a single experiment. The natural extension of these experiments is to simultaneously measure and compare the expression of all the proteins present in a microbial system. This is the field of proteomics. With the development of electrospray ionization, rapid tandem mass spectrometry and database-searching algorithms, mass spectrometry (MS) has become the leader in the attempts to decipher proteomes. This research effort is very young and many challenges still exist. The goal of the work described here was to build a state-of-the-art robust MSbased proteomics platform for the characterization of microbial proteomes from isolates to communities. The research presented here describes the successes and challenges of this objective. Proteome analyses of the metal-reducing bacteria Shewanella oneidensis and the metabolically versatile bacteria Rhodopseudomonas palustris are given as examples of the power of this technology to elucidate proteins important to different metabolic states at a global level. The analysis of microbial proteomes from isolates is only the first step of the challenge. In nature, microbial species do not act alone but are always found in mixtures with other species where their intricate interactions are critical for survival. These studies conclude with some of the first efforts to develop methodologies to measure proteomes of simple controlled mixtures of microbial species and then present the first attempt at measuring the proteome of a natural microbial community, a biofilm from an acid mine drainage system. This microbial system illustrates life at the extreme of nature where life not only exists but flourishes in very acidic conditions with high metal concentrations and high temperatures. The technologies developed through these studies were applied to the first deep characterization of a microbial community proteome, the deciphering of the expressed proteome of the acid mine drainage biofilm. The research presented here has led to development of a state-of-the-art robust proteome pipeline, which can now be applied to the proteome analysis of any microbial isolate for a sequenced species. The first steps have also been made toward developing methodologies to characterize microbial proteomes in their natural environments. These developments are key to integrating proteome technologies with genome and transcriptome technologies for global characterizations of microbial species at the system level. This will lead to understanding of microbial physiology from a global view where instead of analyzing one gene or protein at a time, hundreds of genes/proteins will be interrogated in microbial species as the adapt and survive in the environment.
Author: Haroun N. Shah
Publisher: John Wiley & Sons
ISBN: 1118960238
Category : Science
Languages : en
Pages : 850
Get Book Here
Book Description
This book highlights the triumph of MALDI-TOF mass spectrometry over the past decade and provides insight into new and expanding technologies through a comprehensive range of short chapters that enable the reader to gauge their current status and how they may progress over the next decade. This book serves as a platform to consolidate current strengths of the technology and highlight new frontiers in tandem MS/MS that are likely to eventually supersede MALDI-TOF MS. Chapters discuss: Challenges of Identifying Mycobacterium to the Species level Identification of Bacteroides and Other Clinically Relevant Anaerobes Identification of Species in Mixed Microbial Populations Detection of Resistance Mechanisms Proteomics as a biomarker discovery and validation platform Determination of Antimicrobial Resistance using Tandem Mass Spectrometry
