Download or read book Method Development for the Comprehensive Analysis of Post Translational Modifications by Mass Spectometry written by and published by . This book was released on 2007 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Signal Transduction is mediated by protein complexes whose spatial- and temporal-distribution, composition and function within cells are often regulated by different post-translational modifications (PTM). As PTMs add or subtract a specific mass difference to a protein, mass spectrometry becomes very amenable for modification analysis. These modifications have conventionally been monitored by fragmenting the modified protein or peptide by collision induced dissociation (CID) within the mass spectrometer, and then screening for the characteristic neutral fragment or fragment ion (marker ion), which is particular to the modification in question. Unfortunately, there are two major issues with respect to the traditional mass spectrometric analysis of PTMs: (1) as there are over 300 known types of modifications, the characteristic fragmentation of only a fraction of these modifications has been studied and (2) the traditional mass spectrometric approaches can only monitor these modifications sequentially, and thus comprehensive modification analysis would be unfeasible considering the breadth of PTMs. The following work aims to address these issues by (1) analyzing PTMs that have never been characterized mass spectrometrically and (2) developing a multiplexed technique for comprehensive PTM monitoring by simultaneously screening for all known characteristic fragments. With respect to the first issue, the characteristic fragmentation of lipid modifications and HNO-induced modifications was investigated. The most prevalent indicator(s) of the modification within the mass spectra are as follows: fragmentation of N-terminal myristoylated peptides produced marker ions at 240 and 268 Th, fragmentation of cysteine farnesylated peptides produced a marker ion at 205 Th and a neutral fragment of 204 Da, and fragmentation of cysteine palmitoylated peptides produced a neutral fragment of 272 Th. For HNO-induced modifications, fragmentation of the sulfinamide- and sulfinic acid-modif.
Download or read book Analysis of Protein Post Translational Modifications by Mass Spectrometry written by John R. Griffiths and published by John Wiley & Sons. This book was released on 2016-10-18 with total page 377 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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
Download or read book Development and Application of Mass Spectrometry Methods for Proteomic and Post translational Modification Analysis written by Danqing Wang (Ph.D.) and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins are essential biomolecules that perform a wide range of biological functions. Post-translational modifications (PTMs) substantially impact protein structure and function, making their characterization essential for understanding complex biological systems. This dissertation focuses on developing and applying novel mass spectrometry (MS)-based methodologies to address challenges in studying two common and important PTMs: phosphorylation and glycosylation. To this end, new enrichment materials and their corresponding workflows, including Cotton Ti-IMAC, epoxy-ATP-Ti4+ IMAC, and Very Weak Anion Exchange (VWAX) have been introduced for efficient phosphopeptide and glycopeptide enrichment. A strategy combining boronic acid enrichment, high-pH fractionation, and EThcD has been developed for comprehensive O-glycosylation profiling. Additionally, the Boost-DiLeu quantitative approach has been introduced to enhance glycopeptide quantification in size-limited samples, while a periodate oxidation-based SUGAR tag labeling method has been established for high-throughput, intact sialylated glycopeptide-specific quantification. These methods have been applied to study human diseases, such as Alzheimer's Disease, providing insights into dysregulated glycosylation patterns and their potential implications in disease pathogenesis. Overall, this work contributes to advancing MS-based proteomics strategies and broadening our understanding of the roles of PTMs in biological systems and is anticipated to inspire future research endeavors in related fields.
Download or read book Development and Application of Mass Spectrometry Methods for Proteomic and Post translational Modification Analysis written by Danqing Wang (Ph.D.) and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins are essential biomolecules that perform a wide range of biological functions. Post-translational modifications (PTMs) substantially impact protein structure and function, making their characterization essential for understanding complex biological systems. This dissertation focuses on developing and applying novel mass spectrometry (MS)-based methodologies to address challenges in studying two common and important PTMs: phosphorylation and glycosylation. To this end, new enrichment materials and their corresponding workflows, including Cotton Ti-IMAC, epoxy-ATP-Ti4+ IMAC, and Very Weak Anion Exchange (VWAX) have been introduced for efficient phosphopeptide and glycopeptide enrichment. A strategy combining boronic acid enrichment, high-pH fractionation, and EThcD has been developed for comprehensive O-glycosylation profiling. Additionally, the Boost-DiLeu quantitative approach has been introduced to enhance glycopeptide quantification in size-limited samples, while a periodate oxidation-based SUGAR tag labeling method has been established for high-throughput, intact sialylated glycopeptide-specific quantification. These methods have been applied to study human diseases, such as Alzheimer's Disease, providing insights into dysregulated glycosylation patterns and their potential implications in disease pathogenesis. Overall, this work contributes to advancing MS-based proteomics strategies and broadening our understanding of the roles of PTMs in biological systems and is anticipated to inspire future research endeavors in related fields.
Download or read book Advancement of Photodissociation Mass Spectrometry Methods for the Analysis of Protein Post translational Modifications written by Michelle Renee Robinson and published by . This book was released on 2016 with total page 410 pages. Available in PDF, EPUB and Kindle. Book excerpt: Post-translational modifications (PTMs) are important for regulating protein structure and function. Despite significant progress for PTM analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS), opportunities for new method development remain. The research presented in this dissertation promotes 193 nm ultraviolet photodissociation (UVPD) as an alternative activation technique for PTM analysis with specific utility for phosphorylated and sulfated peptides. A novel de novo sequencing method with applications for unbiased PTM discovery was developed utilizing Lys-N proteolysis, N-terminal imidazolinylation, and UVPD to direct fragmentation for the formation of N-terminal ions. The N-terminal a, b, and c ions generated by UVPD were differentiated from one another by characteristic mass shifts. Sets of triplet peaks were used to distinguish N-terminal ions from confounding C-terminal ions and improve the accuracy of de novo sequencing. UVPD was evaluated for the analysis of phosphopeptide cations and anions. Negative mode analysis was advantageous for the detection of casein peptides in high phosphorylation states, while positive mode proved more robust for global phosphoproteomic analysis of HeLa and HCC70 cell lysates. Compared to collisional activation, the depth of coverage was lower using UVPD yet more extensive fragmentation and improved phosphate retention on products ions was achieved. Phosphorylation mapping by LC-UVPD-MS was carried out in the C-terminal domain (CTD) of RNA polymerase II as a function of kinase treatment, ERK2 or TFIIH, and organism, yeast or fruit fly. Single phosphorylations on Ser2 or Ser5 in the consensus heptad, YSPTSPS, were observed across all experimental conditions. Analysis of the non-consensus fruit fly CTD revealed the significance of Tyr1 and Pro residues in the +1 position relative to Ser for phosphorylation to occur. For sulfated peptides, negative mode UVPD yielded a and x ions that largely retained the labile sulfate modification, facilitating peptide sequencing and PTM localization. With appropriate MS/MS tools established, the next step towards global sulfoproteomics was the development of enrichment methods. Weak anion exchange (WAX) was applied for this purpose. Following carbamylation to neutralize primary amines which otherwise repel the anion exchanger; improved WAX retention was observed for sulfopeptides relative to a complex mixture of unmodified bovine serum albumin peptides.
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 Multidimensional Liquid Chromatography Mass Spectrometric Analysis of Selected Post Translationally Modified Peptides written by 全泉 and published by . This book was released on 2017-01-26 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: This dissertation, "Multidimensional liquid chromatography/mass spectrometric analysis of selected post-translationally modified peptides: from fundamentals to shotgun proteomics" by Quan, Quan, 全泉, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: The continuing evolution of multidimensional liquid chromatography/mass spectrometry (MDLC-MS)-based proteomics is an important element of the developing field of shotgun proteomics for peptide sequencing, protein identification and quantification. The first part of this thesis, Chapter 2, demonstrates the development of a comprehensive automated MDLC platform capable of performing both quantitative proteomics analyses and post-translational modifications analysis-in particular, of protein tyrosine nitration and protein phosphorylation. The current multidimensional reversed-phase (RP) liquid chromatography design was employed with the addition of strong anion exchange (SAX) and cation exchange (SCX) columns. The inclusion of the complementary S(A/C)X column chemistries in the RP-SA(C)X-RP system allowed the retention of deprotonated peptides in the SAX trap column, followed by diversion of non-retained peptides to an online SCX trap column, thereby allowing identification of both anionic and cationic peptides from a single injection event. This MDLC RP-SA(C)X-RP platform provided more extensive protein and proteome coverage, thereby leading to improved protein quantification from analyses of Saccharomyces cerevisiae tryptic digests, a prototypical model proteome, as well as those of various other complex biological samples. Phosphorylated and 3-nitrotyrosyl-containing peptides-two important and biologically relevant post-translational modifications-were efficiently retained in this newly developed platform, in some cases without the need for any pre-enrichment steps. This RP-SA(C)X-RP technology performed well, as judged by the mapped protein inventory from the global collection of endogenous protein tyrosine nitration, the phosphoproteome, and its associated proteomics networks of permanent cerebral ischemia of Macaca fascicularis. The goal of the subsequent study was to gain insight into various aspects of the gas phase radical ion chemistry of phosphorylated peptides; these findings should provide an underlying scientific basis for the development of peptide sequencing strategies, because the general guidelines governing phosphorylated peptide radical cation dissociation remain poorly understood. No previous reports have described the successful generation of radical cationic phosphopeptides under low-energy collision-induced dissociation (CID). Chapters 3 and 4 describe a systematic investigation into the effect of the structures of the metal complexes on the efficient generation of radical phosphopeptide cations. To examine the mechanisms, energetics, and kinetics of these reactions, a combined experimental and computational approach was undertaken to facilitate a greater understanding of their dissociation behavior. Several model phosphopeptide radical cations were synthesized and characterized to formulate the fragmentation rules. The findings suggest that the dissociations of isomeric peptide radical cations can be more efficient than their isomerizations. In a situation similar to the dissociations of analogous even-electron protonated peptides, the losses of H3PO4 from both even- and odd-electron peptide cations are due preferentially to charge-driven mechanisms; the charge-driven loss of H3PO4 is favored as a result of the distonic radical character of the α-radical cation, enhancing the...
Download or read book Method Development and Application of Mass Spectrometry for Better Understanding of Disease Mechanisms and Diagnosis written by Yuan Liu (Ph. D.) and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry is a very powerful tool for identifying and quantifying biomolecules of interest, including metabolites, drug compounds, proteins, and post-translational modifications of (PTMs) of proteins. In mass spectrometry (MS)-based analytical techniques, liquid-chromatography coupled with tandem mass spectrometry (LC-MS/MS) is widely used for biomolecule analysis, including relative and absolute quantification of metabolites, proteins and PTMs. Data dependent acquisition (DDA) and data-independent acquisition (DIA) are two main methods for quantification of biomolecules in LC-MS/MS. By integrating isotopic and/or isobaric tags labeling, the DDA method enables precise absolute quantification and high-throughput analysis of biomolecules, facilitating biomarker discovery and elucidation of disease mechanisms. In contrast, DIA has garnered significant interest due to its exceptional reproducibility and depth in identifying and quantifying biomolecules. In this dissertation work, I apply these isotopic/isobaric tag labeling and label-free strategies to investigate potential biomarkers in the serum of Alzheimer's disease (AD) and colorectal cancer (CRC). Additionally, I explore the crosstalk between pancreatic stellate cells and pancreatic cancer cells using multi-faceted MS approaches.In addition to LC-MS/MS, mass spectrometry imaging (MSI) offers unparalleled insights into the spatial distribution of biomolecules across organs, tissues, and cell cultures, further enhancing our understanding of biological systems. We develop a tagging method to enhance the identification, quantification, and visualization of amine-containing metabolites by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Furthermore, I optimize a workflow for rapid sample preparation and high throughput MSI of biomolecule distribution in a widely used three-dimensional cell culture system - spheroids. In summary, the integration of various mass spectrometry techniques and method development discussed in this dissertation demonstrates that combining different mass spectrometry approaches can provide a more comprehensive molecular picture of the complex biological system, ultimately enhancing our understanding of disease mechanisms and advancing diagnosis and treatment options.
Download or read book Development of Enabling Tools for Global Profiling and Quantitative Analysis of Protein Post translational Modifications written by Yatao Shi (Ph.D.) and published by . This book was released on 2020 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Protein post-translational modifications (PTMs) involve the covalent chemical modifications of specific amino acid residues in proteins, which play crucial roles in protein physiochemical properties, structures and their physiological functions. Alterations in protein PTMs have been implicated in many serious diseases. Comprehensive study of disease-related protein PTMs is critical to explore their roles in the pathogenesis of diseases, contributing to the diagnosis and clinical treatment of diseases. Recently, given the capability of monitoring and identifying thousands of peptides simultaneously, mass spectrometry (MS) has evolved as a powerful tool in bottom-up proteomics, especially the protein PTM analysis. This dissertation is devoted to the development and application of novel electrospray ionization mass spectrometry (ESI-MS) based strategies for the in-depth profiling and quantitative analysis of several important protein PTMs as well as matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) technique for the in-situ imaging of biomolecules. A portion of this dissertation describes the development of a novel biotin tag-assisted MS-based method, as the first method, for the in-depth profiling of citrullinated and homocitrullinated proteins in biological samples. Its utility is demonstrated through its application in mouse tissue-specific citrullination and homocitrullination analysis. We further demonstrate the combination of the biotin tag-assisted MS-based method with different quantitative techniques, enabling the simultaneously qualitative and quantitative analysis of citrullinated and homocitrullinated proteins from different biological samples for the first time. Meanwhile, quantitative proteomics and phosphoproteomics was performed to reveal the signaling pathways involved in the macrophages treated by lipopolysaccharides (LPS) and thapsigargin (TPG). This dissertation also highlights two projects focusing on in-situ imaging of biomolecules from tissue sections using MALDI MS imaging (MSI). MALDI MSI of mouse arteries undergoing restenosis revealed the involvement of many bioactive lipids in the progress of restenosis. In addition, a novel subatmospheric pressure ionization source has been coupled with MS for the high-resolution imaging of N-glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections. Application of this new SubAP/MALDI MS platform to FFPE mouse ovarian cancer tissue section unraveled the specific distribution of high-mannose N-glycans in the tumor region, suggesting potential association of this type of N-glycans with tumor progression.
Download or read book Mass Spectrometry Based Method Development for Elucidation of Protein Sequences Structures and Post Translational Modifications written by Qingyu Sun and published by . This book was released on 2011 with total page 152 pages. Available in PDF, EPUB and Kindle. Book excerpt: The advanced development of mass spectrometry (MS) makes MS a powerful technique for proteomics study. The increasing demands for proteomics study stimulate creation of more applicable MS-based methods. This dissertation focuses on development of novel MS-based methods to characterize three different aspects of proteins: primary sequence, post-translational modifications (PTMs) and three-dimensional (3D) structure.
Download or read book Neuroproteomics written by Oscar Alzate and published by CRC Press. This book was released on 2009-10-26 with total page 356 pages. Available in PDF, EPUB and Kindle. Book excerpt: In this, the post-genomic age, our knowledge of biological systems continues to expand and progress. As the research becomes more focused, so too does the data. Genomic research progresses to proteomics and brings us to a deeper understanding of the behavior and function of protein clusters. And now proteomics gives way to neuroproteomics as we beg
Download or read book Development of Enabling Tools for Global Profiling and Quantitative Analysis of Protein Post translational Modifications written by Yatao Shi (Ph.D.) and published by . This book was released on 2020 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Protein post-translational modifications (PTMs) involve the covalent chemical modifications of specific amino acid residues in proteins, which play crucial roles in protein physiochemical properties, structures and their physiological functions. Alterations in protein PTMs have been implicated in many serious diseases. Comprehensive study of disease-related protein PTMs is critical to explore their roles in the pathogenesis of diseases, contributing to the diagnosis and clinical treatment of diseases. Recently, given the capability of monitoring and identifying thousands of peptides simultaneously, mass spectrometry (MS) has evolved as a powerful tool in bottom-up proteomics, especially the protein PTM analysis. This dissertation is devoted to the development and application of novel electrospray ionization mass spectrometry (ESI-MS) based strategies for the in-depth profiling and quantitative analysis of several important protein PTMs as well as matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) technique for the in-situ imaging of biomolecules. A portion of this dissertation describes the development of a novel biotin tag-assisted MS-based method, as the first method, for the in-depth profiling of citrullinated and homocitrullinated proteins in biological samples. Its utility is demonstrated through its application in mouse tissue-specific citrullination and homocitrullination analysis. We further demonstrate the combination of the biotin tag-assisted MS-based method with different quantitative techniques, enabling the simultaneously qualitative and quantitative analysis of citrullinated and homocitrullinated proteins from different biological samples for the first time. Meanwhile, quantitative proteomics and phosphoproteomics was performed to reveal the signaling pathways involved in the macrophages treated by lipopolysaccharides (LPS) and thapsigargin (TPG). This dissertation also highlights two projects focusing on in-situ imaging of biomolecules from tissue sections using MALDI MS imaging (MSI). MALDI MSI of mouse arteries undergoing restenosis revealed the involvement of many bioactive lipids in the progress of restenosis. In addition, a novel subatmospheric pressure ionization source has been coupled with MS for the high-resolution imaging of N-glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections. Application of this new SubAP/MALDI MS platform to FFPE mouse ovarian cancer tissue section unraveled the specific distribution of high-mannose N-glycans in the tumor region, suggesting potential association of this type of N-glycans with tumor progression.
Download or read book Analysis of Protein Post Translational Modifications by Mass Spectrometry written by John R. Griffiths and published by John Wiley & Sons. This book was released on 2016-10-12 with total page 415 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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
Download or read book Studies of Protein Post translational Modifications Using High Resolution Tandem Mass Spectrometry written by Huilin Li and published by . This book was released on 2012 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Development of Methods for the Analysis of Protein Post translational Modifications written by Min Liu and published by . This book was released on 2014 with total page 23 pages. Available in PDF, EPUB and Kindle. Book excerpt: Analysis of protein posttranslational modifications (PTMs) plays pivotal roles for the understanding of their biological importance. Isoaspartic acid (isoAsp) as the smallest PTM is observed "in vivo" and "in vitro". No mass difference and subtle difference in physiochemical property between isoAsp and Asp pose a great challenging for sensitive detection and ambiguous location of isoAsp site in complex samples. A novel assay of isoAsp by exploiting methylation specificity of protein isoaspartate methyltransferase (PIMT) at isoAsp and subsequent 18O-incorporation during methyl ester hydrolysis is presented for sensitive detection and unambiguous site location of several isoAsp residues in IgG1 ("Anal Chem" 2012, 84, 1056-1062). The method can be applied to biological samples to understand the isoAsp process and identify biomarkers. Ubiquitous protein crosslinks in biological systems and biopharmaceuticals are reported to result in loss of bioactivity and immunogenicity, but their characterization is poor, especially when the crosslink chemistry is undefined, due to their intrinsic structural complexity and a lack of a systematic analytical approach. A comprehensive methodology, XChem-Finder, has been developed to break down the analytical challenge via 18O labeling and mass spectrometry, leading to the discovery of a total of 14 cross-linked thioether peptides in IgG2, including those that have not been previously reported ("Anal Chem" 2013, 85, 5900-5908). Furthermore, a novel Histidine-Histidine (His-His) crosslink in IgG1 was successfully discovered and characterized via our XChem-Finder ("Anal Chem" 2014, 86, 4940-4948). This again demonstrates the broad applicability and utility of our XChem-Finder. The further improvement of XChem-Finder is discussed. The discovery of more novel crosslinks in protein by XChem-Finder will be successful without any doubt.
Download or read book Mass spectrometric Methods for Quantitative Proteomics and Post translational Modification Mapping written by Ievgen Motorykin and published by . This book was released on 2015 with total page 147 pages. Available in PDF, EPUB and Kindle. Book excerpt: Signal transduction within and between cells is at the core of biological activity in all living systems. Signaling networks are required for regulating biological functions, including growth, development and survival. Deregulation of signaling cascades has been linked to chronic and acute diseases and disorders This thesis focuses on mass spectrometry as a high resolution and high mass accuracy technique for the detection and characterization of proteins in biological systems. The thesis presents applications of contemporary mass-spectrometric methods to identify proteins, determine changes in their expression levels, and characterize post-translational modifications, in an effort to study changes in cell signaling in response to stress or disease. Various sample preparation methods to successfully suit needs of different biological questions were developed and applied: (1) extraction of the proteome, (2) chemical tagging and enrichment of the ATPome, a sub proteome comprising nucleotide binding proteins in particularly ATP-binding proteins including kinases, and (3) metal affinity complexation and enrichment of phosphopeptides. We used the following hybrid mass analyzer configurations: a quadrupole time-of-flight (qToF) instrument with ion mobility, a linear ion trap hyphenated with a FT-ICR mass spectrometry (LTQ-FT) and a hybrid ion trap-orbitrap mass spectrometer (Orbitrap Elite). The bioinformatic analysis of the proteomics data required multiple combinations of software packages to sequence proteins, perform absolute and relative quantification, statistically analyze and visualize data. Chapter 3 describes the use of zebrafish (Danio rerio) as one of the few vertebrate models that similar to humans cannot synthesize vitamin C to investigate the system-wide consequences of deficiencies in two essential micronutrients, vitamins E and C, on the proteome biology. A label-free proteomics workflow was applied to detect changes in protein abundance estimates dependent on vitamin regimes. The study reveals suppression in an energy metabolism cycle, glycolysis, in vitamin C and E deficient zebrafish. It was discovered that alternative energy cycle, glutaminolysis, is activated to fulfill energy requirement. Chapter 4 focuses on the determination of proteome differences that can be linked to the propensity of metastasis in osteosarcoma (OS), a bone cancer that predominantly targets the adolescent age group. OS has a high propensity to metastasize to the lungs, which is associated with a poor prognosis. The study utilizes canine osteosarcoma cell lines that were originally obtained from orthotropic primary OS and metastatic cells. Canis familiaris, the domestic dog, is an established large animal model of OS that recapitulate many biological and clinical features of the human malignancy. We applied a two-prone comparative proteomics approach that consisted of: (a) determination of protein abundance levels and (b) focus on kinases, a functional sub-proteome, using a chemical affinity tag for enrichment of ATP-binding proteins. Findings of this study indicated that in the highly metastasizing canine osteosarcoma cell line proteins associated with extracellular adhesion were deregulated, which may enhance metastogenesis. Mitogen activated protein kinases MAP2K6, MAP4K3, MAP4K4, MAP4K5, ZAK and v-akt murine thymoma viral oncogene homolog 1, AKT1, are among those expressed in significantly lower abundance in the highly metastasizing canine osteosarcoma cell line indicating changes in cell signaling. Chapter 5 describes the development and application of a multiple protease protocol (Trypsin, LysC, AspN, Chymotrypsin and GluC) for improving the number of phosphosite identifications in a large-scale phosphoproteomics studies. The method combines immobilized titanium ion affinity chromatography (Ti4+-IMAC) with a data-dependent, decision tree-based data acquisition technique utilizing two complementary fragmentation methods, namely collision induced dissociation (CID) and electron transfer dissociation. The multiple protease protocol was applied to human leukemic T cell lymphoblasts (Jurkat E6.1) and resulted in the detection of >11,000 unique phosphosites, the most comprehensive identification among methods that use similar phosphopeptide enrichment approach.
Download or read book Protein Modificomics written by Tanveer Ali Dar and published by Academic Press. This book was released on 2019-05-21 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Protein Modificomics: From Modifications to Clinical Perspectives comprehensively deals with all of the most recent aspects of post-translational modification (PTM) of proteins, including discussions on diseases involving PTMs, such as Alzheimer's, Huntington's, X-linked spinal muscular atrophy-2, aneurysmal bone cyst, angelman syndrome and OFC10. The book also discusses the role PTMs play in plant physiology and the production of medicinally important primary and secondary metabolites. The understanding of PTMs in plants helps us enhance the production of these metabolites without greatly altering the genome, providing robust eukaryotic systems for the production and isolation of desired products without considerable downstream and isolation processes.
