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Author: John Rawson Corbett Publisher: ISBN: Category : Automation Languages : en Pages :
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: John Rawson Corbett Publisher: ISBN: Category : Automation Languages : en Pages :
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: Austin Vincent Carr Publisher: ISBN: Category : Languages : en Pages : 0
Book Description
Proteomics technologies enable the large-scale, high-throughput study of proteoforms inbiological systems. The foremost approach, bottom-up proteomics, can identify and quantify thousands of proteins. However, the analysis of proteoforms is hindered by the required enzymatic digestion. Top-down proteomics does not require enzymatic digestion, enabling proteoform-level analysis. The technical challenge of analyzing proteoforms has thwarted the proliferation of top-down approaches, particularly compared to the ubiquity of bottom-up proteomics. Recent advances in sample preparation have enabled the intact mass measurements of classes of proteoforms traditionally considered challenging for top-down proteomics. Consequently, key barriers to broader applicability remaining in top-down proteomics are data acquisition and data processing. An additional challenge to proteomics in general is data interpretation. Lists of thousands of quantified proteins and/or proteoforms per experiment are not interpretable without software solutions capable of converting the raw output of proteomics experiments into biologically meaningful and actionable insight. This dissertation describes software-based solutions designed to improve the acquisition, processing, and interpretation of proteomics data. Chapter 1 provides an overview of mass spectrometry and the three key challenges addressed by methods in this thesis. Chapter 2 address challenges in data acquisition by introducing automated database searching of a data independent acquisition method for top-down proteomics using a hybrid database search strategy. Chapter 3 describes a new deconvolution method for low-resolution mass spectrometry data processing, taking top-down proteomics beyond high-resolution instruments. Chapter 4 also describes improvements in top-down proteomics data processing, applying astronomical averaging with outlier rejection algorithms to mass spectrometry data. In Chapter 5, a description of the application MetaNetwork, a computer program designed to facilitate systems biology insights for proteomics data is given.
Author: Xiaojing Shen Publisher: ISBN: Category : Electronic dissertations Languages : en Pages : 194
Book Description
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.
Author: Tian Xu Publisher: ISBN: Category : Electronic dissertations Languages : en Pages : 0
Book Description
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.
Author: Samantha Jo Knott Publisher: ISBN: Category : Languages : en Pages : 0
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: Liangliang Sun Publisher: Humana ISBN: 9781071623275 Category : Science Languages : en Pages : 0
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: Eli Larson Publisher: ISBN: Category : Languages : en Pages : 0
Book Description
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.
Author: Laura Heikaus Publisher: ISBN: Category : Languages : en Pages :
Book Description
A characteristic of many proteoforms, derived from a single gene, is their similarity regarding the composition of atoms, making their analysis very challenging. Many overexpressed recombinant proteins are strongly associated with this problem, especially recombinant therapeutic glycoproteins from large-scale productions. In contrast to small molecule drugs, which consist of a single defined molecule, therapeutic protein preparations are heterogenous mixtures of dozens or even hundreds of very similar species. With mass spectrometry, currently high-quality spectra of intact proteoforms can be obtained only, if the complexity of the mixture of individual proteoform-ions, entering the gas phase at the same time is low. Thus, prior to mass spectrometric analysis, an effective separation is required for getting fractions with a low number of individual proteoforms. This is especially true not only for recombinant therapeutic proteins, because of their huge heterogeneity, but also relevant for top-down proteomics. Purification of proteoforms is the bottleneck in analyzing intact proteoforms with mass spectrometry. This review is focusing on the current state of the art, especially of liquid chromatography for preparing proteoforms for mass spectrometric top-down analysis. The topic of therapeutic proteins has been chosen, because this group of proteins is most challenging regarding their proteoform analysis.
Author: Hartmut Schl√oter Publisher: ISBN: Category : Science Languages : en Pages : 0
Book Description
A characteristic of many proteoforms, derived from a single gene, is their similarity regarding the composition of atoms, making their analysis very challenging. Many overexpressed recombinant proteins are strongly associated with this problem, especially recombinant therapeutic glycoproteins from large-scale productions. In contrast to small molecule drugs, which consist of a single defined molecule, therapeutic protein preparations are heterogenous mixtures of dozens or even hundreds of very similar species. With mass spectrometry, currently high-quality spectra of intact proteoforms can be obtained only, if the complexity of the mixture of individual proteoform-ions, entering the gas phase at the same time is low. Thus, prior to mass spectrometric analysis, an effective separation is required for getting fractions with a low number of individual proteoforms. This is especially true not only for recombinant therapeutic proteins, because of their huge heterogeneity, but also relevant for top-down proteomics. Purification of proteoforms is the bottleneck in analyzing intact proteoforms with mass spectrometry. This review is focusing on the current state of the art, especially of liquid chromatography for preparing proteoforms for mass spectrometric top-down analysis. The topic of therapeutic proteins has been chosen, because this group of proteins is most challenging regarding their proteoform analysis.
Author: John Rawson Corbett
Author: John Rawson Corbett
Author: Austin Vincent Carr
Author: Steven M. Patrie
Author: Xiaojing Shen
Author: Tian Xu
Author: Samantha Jo Knott
Author: Liangliang Sun
Author: Eli Larson
Author: Laura Heikaus
Author: Hartmut Schl√oter