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Development Of Mass Spectrometric Techniques For The Analysis Of Neuropeptides
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Book Synopsis Development of Mass Spectrometric Techniques for the Analysis of Neuropeptides by : Stephanie Shannon Dekeyser
Download or read book Development of Mass Spectrometric Techniques for the Analysis of Neuropeptides written by Stephanie Shannon Dekeyser and published by . This book was released on 2007 with total page 252 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Analysis of Neuropeptides by Liquid Chromatography and Mass Spectrometry by : D.M. Desiderio
Download or read book Analysis of Neuropeptides by Liquid Chromatography and Mass Spectrometry written by D.M. Desiderio and published by Elsevier. This book was released on 2000-04-01 with total page 255 pages. Available in PDF, EPUB and Kindle. Book excerpt: Analysis of Neuropeptides by Liquid Chromatography and Mass Spectrometry
Book Synopsis Analysis of Neuropeptides by Liquid Chromatography and Mass Spectrometry by : Dominic M. Desiderio
Download or read book Analysis of Neuropeptides by Liquid Chromatography and Mass Spectrometry written by Dominic M. Desiderio and published by . This book was released on 1984 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Mass Spectrometry and Hyphenated Techniques in Neuropeptide Research by : Jerzy Silberring
Download or read book Mass Spectrometry and Hyphenated Techniques in Neuropeptide Research written by Jerzy Silberring and published by John Wiley & Sons. This book was released on 2002-05-02 with total page 576 pages. Available in PDF, EPUB and Kindle. Book excerpt: The first authoritative guide to the application of this vital analytical technique Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds, to quantify known materials, and to elucidate the structural and chemical properties of molecules. In analyzing the effects of experimental drugs on the brain, it is the sole technique for identifying the presence and structure of neuropeptides-substances that indicate the effect of the drug. Mass Spectrometry and Hyphenated Techniques in Neuropeptide Research specifically explains how to apply the technology to this process. Because the book is written by mass spectrometry users, as opposed to mass spectrometrists, the focus remains on practical applications of the technique. The authors demonstrate how mass spectrometry works, how to apply the technique to research, which types of instrumentation should be used for particular requirements, and how to plan experiments. Readers will learn why mass spectrometry provides more outcome features than other techniques in neuropeptide analysis, including simultaneous detection, identification of substances present in mixtures, and sequence information even when the residues are modified, blocked, or unusual. Among the chapters in this comprehensive text are: * Sequencing of Peptides by Nanospray Mass Spectrometry * Laser-Machined Microdevices for Mass Spectrometry * Electron Capture Dissociation of Peptides * Synthesis of Combinatorial Peptide Libraries * Analysis of Tissues That Reflect Nervous System Disease Doctoral students, researchers, and industry professionals in pharmacology, chemistry, biochemistry/biotechnology, and medicine will find Mass Spectrometry and Hyphenated Techniques in Neuropeptide Research to be an indispensable starting point for understanding peptides, their function, and identification.
Book Synopsis Probing Neuropeptidomics by Mass Spectrometry by :
Download or read book Probing Neuropeptidomics by Mass Spectrometry written by and published by . This book was released on 2012 with total page 548 pages. Available in PDF, EPUB and Kindle. Book excerpt: In this thesis, an array of biological mass spectrometry (MS) based methods were developed and employed to enable the identification and functional analysis of neuropeptides in the crustacean nervous system. Neuropeptides encompass the largest and most diverse group of signaling molecules in the nervous system. They are necessary for the initiation and regulation of numerous physiological processes such as feeding, reproduction and development. However, the discovery and identification of these important signaling molecules presents unique challenges due to the vast complexity and minute quantities of endogenously expressed peptides. Further functional analysis of neuropeptides needs to be conducted for a comprehensive neuropeptidomic study. Using the decapod crustacean nervous system and associated neuroendocrine organs as experiment model, this dissertation research focuses on the development of an integrated analytical platform for large-scale identification and discovery of neuropeptides, as well as investigation of the functional roles of neuropeptides. The work presented here contributes insight into neuropeptidome of a commercially and scientifically important crustacean species, blue crab /italicCallinectes sapidus/italic and the possible functionality of certain peptides. A multifaceted mass spectrometry approach combining complementary sample preparation techniques, advanced separation strategies, alternative fragmentation methods, mass spectral imaging (MSI) and stable isotopic labeling was used for large-scale identification and to provide comprehensive information on peptide expression level changes under different physiological states. Several new analytical methodologies and tools have been developed to enable enhanced neuropeptidome coverage and improved /italicde novo/italic sequencing. A two dimensional liquid chromatography (2D-LC) method coupling reversed-phase liquid chromatography (RPLC) with nano-LC-ESI-Q-TOF MS/MS was used for neuronal tissue extract analysis and greatly improved the neuropeptidome coverage. A homemade capillary isoelectric focusing (cIEF) system was used for separation of the most acidic neuropeptide family Orcokinins in the sinus gland (SG). Additionally, electron transfer dissociation (ETD) was employed for the identification and sequencing of intact crustacean hyperglycemic hormone precursor related peptides (CPRPs), a group of multiply charged peptides in the SG. More than 150 neuropeptides including over 40 novel ones were identified in blue crab's nervous system. Furthermore, a comparative peptidomic study utilized mass spectral imaging (MSI), stable isotopic labeling and electrophysiological study was developed to provide comprehensive information about peptide expression level changes after food intake. Moreover, a MS-based platform was developed to study the differential degradation of neuropeptides by extracellular peptidases via monitoring degradation rate and degradation products. Collectively, this body of work extends the capability of mass spectrometry as a bioanalytical tool for neuropeptide analysis and advances the basic understanding of neuronal signaling.
Book Synopsis Mass Spectral Analysis of Crustacean Signaling Peptides Using a Multi-dimensional Strategy by :
Download or read book Mass Spectral Analysis of Crustacean Signaling Peptides Using a Multi-dimensional Strategy written by and published by . This book was released on 2013 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: By definition, neuropeptides are a class of signaling molecules either secreted by or that have an effect on neurons. They are present at minute concentrations in complex biological matrices. Their expression levels vary both temporally and spatially as they regulate several biological functions. Our limited knowledge of their regulatory processes has resulted in the creation or understanding of several therapeutic treatments with more advancements hinging on future neuropeptidomic breakthroughs. A multi-dimensional strategy has been implemented for the analysis of neuropeptides in the crustacean model system. Due to their dynamic nature, biological mass spectrometry is the instrumental platform used for neuropeptide analysis. It is coupled with analytical methodologies primarily focused on improving neuropeptide detection, identification, and quantitation. Simple protocols for rapid, robust, and reproducible analysis have been developed by modifying previous techniques for novel application on newer technologies. Several protocols were developed by acknowledging both the benefits and limitations of the techniques implemented. While this work utilizes both soft ionization techniques, more emphasis is given to the development of MALDI analysis. Relative quantitative analysis by 4-plex N,N-dimethyl leucine (DiLeu) labels was applied to study the neuropeptide expression level changes in relation to feeding. A neuropeptide database was compiled for automated analysis of large datasets using popular protein identification and quantitation software. Improving mass spectrometry de novo sequencing was achieved by modifying a simple acid-driven deamidation protocol to determine the presence and location of amides. Also, the formation of multiply charged peptides by MALDI ionization was explored to improve fragmentation spectra. Finally, to further analysis by MALDI ionization, proof-of-principle projects were initiated. The first was established to improve detection of hemolymph samples on newer MALDI instruments to determine expression level changes of circulating neuropeptides in relation to the circadian rhythm cycle. The second was the application of isotopic DiLeu (iDiLeu) labels to generate a five point standard curve for absolute quantitation. Primary interests of these labels are in their application for imaging experiments. In conclusion, all projects were developed by implementing a multi-dimensional strategy to strengthen the mass spectrometric platform for advancing neuropeptidomic research.
Book Synopsis Neuropeptides in Crustaceans by : Chuanzi OuYang
Download or read book Neuropeptides in Crustaceans written by Chuanzi OuYang and published by . This book was released on 2016 with total page 341 pages. Available in PDF, EPUB and Kindle. Book excerpt: In this thesis, multifaceted biological mass spectrometry (MS) based methods were developed and employed to enable comprehensive characterization of neuropeptides. Neuropeptides encompass the largest and most diverse group of signaling molecules in the nervous system. They are necessary for the initiation and regulation of numerous physiological processes such as feeding, reproduction and development. Due to the complexity and inaccessibility of human systems, many invertebrate model nervous systems have been utilized as test bed for neuropeptidome research. Of these, the decapod crustacean nervous system provides an excellent test bed to facilitate technology development and investigate the biological functions of neuropeptides. A review of the current status and progress in the field of invertebrate neuropeptide is included in this thesis. Among various MS based technologies, we focused on developing new methodologies in mass spectral imaging (MSI) to provide high throughput tools for multiplexed distribution characterization. Current development of MSI and microdialysis based MS approaches have been summarized as the background of MSI related projects. To this rapidly expanding body of literature, the work presented here contributes insight into the possible functionality of certain peptides by identifying neuropeptides found to be differentially expressed in correlation with food intake and changes of environmental conditions. This comparative peptidomic study utilized direct tissue analysis, MSI and stable isotopic labeling to provide comprehensive information regarding to peptide expression level changes under different physiological states. In addition, sample preparation method was developed to detect trace level of neuropeptides from crustacean circulating fluid hemolymph. Furthermore, optimized sample preparation for imaging mass spectrometry was developed to greatly increase the coverage of peptidome and the accuracy of identification. Finally, using established techniques, novel neuropeptides were identified from the neuroendocrine organs in Dungeness crab and rock crab, and their distribution was also investigated. Collectively, this body of work extends the capability of mass spectrometry as a bioanalytical tool for neuropeptide analysis and advances the basic understanding of neuronal signaling.
Book Synopsis Discovery and Quantification of Crustacean Neuropeptides Using Mass Spectrometry and Informatics Approaches by : Nhu Vu
Download or read book Discovery and Quantification of Crustacean Neuropeptides Using Mass Spectrometry and Informatics Approaches written by Nhu Vu and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The study of endogenous peptides is important for discovering critical elements involved in many physiological processes and disorders. Neuropeptides are endogenous peptide hormones that regulate these processes by binding to receptors to initiate cellular signaling pathways. Discovery of neuropeptide function is an increasingly popular avenue for disease therapeutics research, resulting in increased need for improved neuropeptide characterization methods. Although mass spectrometry (MS) is well-suited for neuropeptide analysis due to its high sensitivity and capacity for untargeted, global neuropeptide measurements, there are still technical challenges that must be overcome. For example, neuropeptide sequences within a peptide family are highly homologous and therefore must be analyzed in their intact form to obtain high sequence coverage for confident identification and often require high resolution instrumentation. This becomes more challenging as sequence length and post-translational modification complexity increases. In this thesis, I will discuss my efforts in improving MS-based neuropeptidomic workflows, specifically in relation to "top-down" protein and peptide characterization, while highlighting improvements in identification and quantification of peptides through optimized fragmentation and cysteine reactive labeling. I will also discuss my contributions to our group's novel software, HyPep, enabling utility for confident neuropeptide identification. Altogether, these methods have contributed to increased neuropeptide coverage and can be translated to the analysis of other peptide classes.This thesis begins with two reviews of recent developments in MS-based analysis of neuropeptides. Each review outlines examples of how advancements in MS sample preparation, MS acquisition, data analysis, and technology has increased our understanding of neuropeptides, and provides insight on promising directions for further development within each section. My research contributions in Professor Lingjun Li's research group started by applying peptidomics workflows to quantify changes in neuropeptide expression due to hypoxia stress in a crustacean animal model, C. sapidus. Using the same animal model, we also increased the number of detectable neuropeptides using matrix-assisted laser desorption/ionization (MALDI)-MS imaging by incorporating novel tissue wash steps during sample preparation. Over 100 putative peptidomic biomarkers for different levels of hypoxia severity and duration were discovered within multiple tissue types. Four neuropeptide biomarkers were examined for functional activity and two neuropeptides were discovered to increase cardiac output ex vivo, including the first-ever reported function for a crustacean hyperglycemic hormone precursor-related peptide (CPRP). Since CPRP (3.5 kDa) is co-released with crustacean hyperglycemic hormone (CHH) neuropeptide (8.5 kDa), an important neuropeptide that regulates glucose metabolism, we did not detect CHH in these hypoxia studies because its larger size prevented confident detection using typical neuropeptidomic MS workflow and data acquisition methods. Previous members of our research group have developed MS methods to sequence CHH, but limitations of those methods included requiring multiple MS instruments or reliance on targeted MS strategies in which information of potential novel neuropeptides is lost. I built upon those MS acquisition and data analysis methods and was able to sequence novel CHH isoforms and proteoforms, without assistance from genomic or transcriptomic-based sequence prediction, using a Thermo Orbitrap Fusion Lumos instrument. Additionally, I developed novel sample preparation methods to reduce the disulfide bonds and alkylate all six cysteine residues on CHH at high efficiency (83-89% labeling efficiency). While developing methods for CHH are beneficial for analyzing mid-size (8-10 kDa) peptides, I sought to also advance general neuropeptidomics data analysis pipeline. A novel concept for neuropeptide identification software, called HyPep, was created 10 years ago but was not finished and could not be used. To make the software usable, I incorporated features typically used in peptide-spectrum matching (PSM)-based algorithms in proteomics software, such as a false discovery rate. I optimized the software parameters which resulted in HyPep outperforming an analogous commercial software in identifying neuropeptides from four crustacean neuronal tissue types. Collectively, this dissertation is devoted to advancing sample preparation, data acquisition, and data analysis strategies for neuropeptidomics, and these methods are also potentially translatable to analyze other classes of endogenous peptides.
Book Synopsis Mass Spectrometry Methods and Applications for Functional Characterization of the Crustacean Neuropeptidome by : Kellen Lynn DeLaney
Download or read book Mass Spectrometry Methods and Applications for Functional Characterization of the Crustacean Neuropeptidome written by Kellen Lynn DeLaney and published by . This book was released on 2020 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Neuropeptides are a diverse class of signaling molecules that are responsible for modulating numerous functions within the nervous system. They are challenging to study because, like most signaling molecules, they are present in low abundances in vivo and prone to rapid degradation. In this dissertation, multifaceted mass spectrometry (MS) methods are developed and implemented to characterize neuropeptides within crustacean model organisms. Decapod crustaceans are a useful model for understanding circuit dynamics due to their simplified nervous system, robust central pattern generators, and homology to higher-order organisms, including mammals. This work involves developing methods to improve the coverage of neuropeptides detected by implementing complementary separation and ionization techniques, the identification and reproducibility of neuropeptide analysis with optimized mass spectral acquisition parameters, and the accuracy of sequence information with software specifically designed for neuropeptide analysis. These methods are utilized to profile the neuropeptides present in the cardiac neuromuscular system that may be responsible for modulating heartbeat, as well as feeding-related neuropeptides in tissue and circulating fluid that may be implicated in generating rhythms related to chewing and filtering of food. Overall, this dissertation research develops improved methods for analyzing neuropeptides, demonstrates the utility of these methods for better understanding their functional roles within crustacean model organisms, and provides deeper insight into how these neuropeptides may be involved in modulating key central pattern generators and influencing circuit dynamics.
Book Synopsis Neuropeptide Signaling in Crustaceans Probed by Mass Spectrometry by :
Download or read book Neuropeptide Signaling in Crustaceans Probed by Mass Spectrometry written by and published by . This book was released on 2016 with total page 564 pages. Available in PDF, EPUB and Kindle. Book excerpt: Neuropeptides are one of the most diverse classes of signaling molecules whose identities and functions are not yet fully understood. They have been implicated in the regulation of a wide range of physiological processes, including feeding-related and motivated behaviors, and also environmental adaptations. In this work, improved mass spectrometry-based analytical platforms were developed and applied to the crustacean systems to characterize signaling molecules. This dissertation begins with a review of mass spectrometry-based neuropeptide studies from both temporal- and spatial-domains. This review is then followed by several chapters detailing a few research projects related to the crustacean neuropeptidomic characterization and comparative analysis. The neuropeptidome of crayfish, Orconectes rusticus is characterized for the first time using mass spectrometry-based tools. In vivo microdialysis sampling technique offers the capability of direct sampling from extracellular space in a time-resolved manner. It is used to investigate the secreted neuropeptide and neurotransmitter content in Jonah crab, Cancer borealis, in this work. A new quantitation strategy using alternative mass spectrometry data acquisition approach is developed and applied for the first time to quantify neuropeptides. Coupling of this method with microdialysis enables the study of neuropeptide dynamics concurrent with different behaviors. Proof-of-principle experiments validating this approach have been carried out in Jonah crab, Cancer borealis to study feeding- and circadian rhythm-related neuropeptide changes using micoridialysis in a time-resolved manner. This permits a close correlation between behavioral and neurochemical changes, providing potential candidates for future validation of regulatory roles. In addition to providing spatial information, mass spectrometry imaging (MSI) technique enables the characterization of signaling molecules while preserving the temporal resolution. A novel MSI-based platform is developed by interfacing with microdialysis sample collection and is applied to the study of in vivo neuropeptide degradation profiles in an off-line ‘real-time’ fashion. This unique platform provides novel insights into neuropeptide inactivation/elimination process, which is an essential step to understand peptidegic signaling pathway. In addition to neuropeptides, this platform has been further modified to study secreted neurotransmitters, and small molecule metabolites that might interact with neuropeptides in crustacean for the first time. This work not only reports improvements upon neuropeptides identification and quantitation by developing improved analytical strategies, but also provides important evidence for the potential roles of several neuropeptides in regulating food intake and circadian rhythm behaviors. Novel platform that integrates MALDI-MSI with microdialysis in exploring neurochemical changes in dynamic biological events is also presented, which could be potentially applied to many other systems in future research. Collectively this dissertation research develops new analytical methods and improves our fundamental understanding of neuropeptide signaling.
Book Synopsis Sampling Strategies for Characterization of Neuropeptides Using Mass Spectrometry and Functional Implications Into Cell-cell Signaling by : Adriana Bora
Download or read book Sampling Strategies for Characterization of Neuropeptides Using Mass Spectrometry and Functional Implications Into Cell-cell Signaling written by Adriana Bora and published by . This book was released on 2010 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: In this dissertation, there are developed different analytical strategies to discover and characterize mammalian brain peptides using small amount of tissues. The magnocellular neurons of rat supraoptic nucleus in tissue and cell culture served as the main model to study neuropeptides, in addition to hippocampal neurons and mouse embryonic pituitaries. The neuropeptidomcis studies described here use different extraction methods on tissue or cell culture combined with mass spectrometry (MS) techniques, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). These strategies lead to the identification of multiple peptides from the rat/mouse brain in tissue and cell cultures, including novel compounds One of the goals in this dissertation was to optimize sample preparations on samples isolated from well-defined brain regions for mass spectrometric analysis. Here, the neuropeptidomics study of the SON resulted in the identification of 85 peptides, including 20 unique peptides from known prohormones. This study includes mass spectrometric analysis even from individually isolated magnocellular neuroendocrine cells, where vasopressin and several other peptides are detected. At the same time, it was shown that the same approach could be applied to analyze peptides isolated from a similar hypothalamic region, the suprachiasmatic nucleus (SCN). Although there were some overlaps regarding the detection of the peptides in the two brain nuclei, different peptides were detected specific to each nucleus. Among other peptides, provasopressin fragments were specifically detected in the SON while angiotensin I, somatostatin-14, neurokinin B, galanin, and vasoactive-intestinal peptide (VIP) were detected in the SCN only. Lists of peptides were generated from both brain regions for comparison of the peptidome of SON and SCN nuclei. Moving from analysis of magnocellular neurons in tissue to cell culture, the direct peptidomics of the magnocellular and hippocampal neurons led to the detection of 10 peaks that were assigned to previously characterized peptides and 17 peaks that remain unassigned. Peptides from the vasopressin prohormone and secretogranin-2 are attributed to magnocellular neurons, whereas neurokinin A, peptide J, and neurokinin B are attributed to cultured hippocampal neurons. This approach enabled the elucidation of cell-specific prohormone processing and the discovery of cell-cell signaling peptides. The peptides with roles in the development of the pituitary were analyzed using transgenic mice. Hes1 KO is a genetically modified mouse that lives only e18.5 (embryonic days). Anterior pituitaries of Hes1 null mice exhibit hypoplasia due to increased cell death and reduced proliferation and in the intermediate lobe, the cells differentiate abnormally into somatotropes instead of melanotropes. These previous findings demonstrate that Hes1 has multiple roles in pituitary development, cell differentiation, and cell fate. AVP was detected in all samples. Interestingly, somatostatin [92-100] and provasopressin [151-168] were detected in the mutant but not in the wild type or heterozygous pituitaries while somatostatin-14 was detected only in the heterozygous pituitary. In addition, the putative peptide corresponding to m/z 1330.2 and POMC [205-222] are detected in the mutant and heterozygous pituitaries, but not in the wild type. These results indicate that Hes1 influences the processing of different prohormones having possible roles during development and opens new directions for further developmental studies. This research demonstrates the robust capabilities of MS, which ensures the unbiased direct analysis of peptides extracted from complex biological systems and allows addressing important questions to understand cell-cell signaling in the brain.
Download or read book Peptidomics written by Mikhail Soloviev and published by John Wiley & Sons. This book was released on 2007-12-21 with total page 432 pages. Available in PDF, EPUB and Kindle. Book excerpt: The definitive guide to peptidomics- a hands-on lab reference The first truly comprehensive book about peptidomics for protein and peptide analysis, this reference provides a detailed description of the hows and whys of peptidomics and how the techniques have evolved. With chapters contributed by leading experts, it covers naturally occurring peptides, peptidomics methods and new developments, and the peptidomics approach to biomarker discovery. Explaining both the principles and the applications, Peptidomics: Methods and Applications: * Features examples of applications in diverse fields, including pharmaceutical science, toxicity biomarkers, and neuroscience * Details the successful peptidomic analyses of biological material ranging from plants to mammals * Describes a cross section of analytical techniques, including traditional methodologies, emerging trends, and new techniques for high throughput approaches An enlightening reference for experienced professionals, this book is sufficiently detailed to serve as a step-by-step guide for beginning researchers and an excellent resource for students taking biotechnology and proteomics courses. It is an invaluable reference for protein chemists and biochemists, professionals and researchers in drug and biopharmaceutical development, analytical and bioanalytical chemists, toxicologists, and others.
Book Synopsis Mass Spectrometry Method Development for Targeted and Discovery Neuroproteomics and Peptidomics by :
Download or read book Mass Spectrometry Method Development for Targeted and Discovery Neuroproteomics and Peptidomics written by and published by . This book was released on 2012 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry is a powerful analytical technique that facilitates the identification of hundreds to thousands of compounds within one experiment. Many research fields have emerged or have been revitalized due to advances in this technology; among these the fields of peptidomics and proteomics have experienced exponential growth in the past decade. This thesis introduces new methods to more confidently identify neuropeptides in complex biological samples by simplifying mass spectral complexity through immunoaffinity enrichment of neuropeptides of a certain neuropeptide family and elimination of putative post-mortem protein fragments in crustacean neuroendocrine organs through the use of heat treatment. Matrix-free mass spectrometry imaging (MSI) of neuropeptides in crustacean brain tissue is also explored as a solution to analyte delocalization associated with matrix application in MALDI MSI experiments. Beyond neuropeptidomics, a targeted absolute quantification (AQUA) assay of the infectious conformer of prion protein (PrPTSE) was developed utilizing the specificity of the enzyme chymotrypsin. The method has several advantages over a tryptic method including: (1) the use of a signature peptide lacking chemically reactive residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. The method has been applied to the detection and quantification of PrPTSE in several animal species. Complementary to targeted protein quantitation, my dissertation research also explored large-scale discovery neuroproteomics. An analytical workflow has been established to facilitate the identification of proteins secreted from human embryonic stem cell (hESC) derived astrocytes using a combination of shotgun proteomics and bioinformatics. Lastly, in an attempt to improve the quantification accuracy and solve problems inherent with the use of isobaric tags, ion mobility spectrometry (IMS) has been applied successfully on a quadrupole time-of-flight (Q-TOF) mass analyzer to reduce interfering species and improve the quantitative accuracy of multiplex isobaric tagging experiments. Taken together, these newly developed analytical methods improve the confidence of identification and the quantitative accuracy for neuropeptidomic and neuroproteomic mass spectrometry based experiments.
Download or read book MS Informatics written by and published by . This book was released on 2012 with total page 426 pages. Available in PDF, EPUB and Kindle. Book excerpt: Nowadays, mass spectrometry (MS)-based proteomics is playing a leading role to enable deep investigation of cellular proteomes. However, the explosive amount of data generated by mass spectrometry makes it difficult to map these complex proteomics data to biological processes. This dissertation research focuses on the development of bioinformatic tools to accelerate and enhance MS-based neuropeptidomics and proteomics. Representing the largest group of signaling molecules, neuropeptides regulate many biological processes such as locomotion, feeding, learning and memory and etc. Characterization of neuropeptides is the first step towards understanding how the neural circuitry functions. An integrated analytical platform combining multi-faceted mass spectrometric approaches and in silico data mining techniques has been developed to discover neuropeptides in Callinectes sapidus and Carcinus maenas. Multiple ionization techniques coupled to various fractionation methods are used for neuronal tissue extract analysis and greatly improved the neuropeptidome coverage. Additionally, in silico data mining techniques including in silico transcriptomics and public database mining facilitate the discovery of previously known neuropeptides and large neuropeptides. A database is constructed for the storage and query of neuropeptides. Meanwhile, two algorithms, prescreening precursors prior to de novo sequencing (PRESnovo) and post-treatment to select potential neuropeptide candidates (HyPep) are developed to interpret low-quality MS/MS spectra and thus enhance neuropeptide discovery. With these algorithms, many new neuropeptides are discovered from mass spectral data. In proteomics, accurate peptide and protein identification and quantification is a key step to understand the role of proteins in biological processes. However, traditional database search strategy lacks sensitivity and accuracy for peptide identification. To address this issue, a bioinformatic tool (RT-SVR + q) is developed in which retention time is employed to improve peptide identification while q value metric is used to replace false discovery rate (FDR) for identification evaluation. Finally, spectral counting, a label-free protein quantification technique, is optimized to demonstrate high performance for large-scale quantitative proteomic analysis. Collectively, this body of work develops and applies bioinformatics tools in MS-based neuropeptidomics and proteomics, accelerating proteomic data analysis while enabling extraction of biological insights from complex mass spectral data via computational techniques.
Book Synopsis Mass Spectrometric Application in Neuroscience by : Jingxin Wang
Download or read book Mass Spectrometric Application in Neuroscience written by Jingxin Wang and published by . This book was released on 2016 with total page 430 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry based comparative proteomics and peptidomics for neuroscience application have been reported in this thesis using different animal and human models. These work are enabled by improved methods that have been developed for neuropeptide characterization and shotgun proteomics. Chapter 1 presents an overview of the background information of each project described in this thesis. In Chapter 2 we employed a combination of cryostat dissection, heat stabilization, neuropeptide extraction and label-free quantitative neuropeptidomics via a liquid chromatography-high resolution mass spectrometry platform to monitor the feeding-induced changes in neuropeptide expression levels within the nucleus accumbens (Acb). We showed feeding caused the expression level changes of ProSAAS. We further performed a microinjection using ProSAAS neuropeptides to the rat Acb. We showed that at high concentration, big LEN significantly increased rats' food and water intake. In addition, both big LEN and PEN at high concentration altered locomotion and rearing. Chapter 3 details my work in Alzheimer's disease (AD) biomarker discovery. By optimized molecular weight cutoff filtration and in house-constructed database, 645 peptides were identified. Interestingly, 42 proSAAS peptides discovered were exclusively cleaved either from N- or C-termini of the proSAAS protein precursor. Glycoproteins were enriched by lectin affinity chromatography, enabling identification of 795 glycoproteins. One way ANOVA was applied for statistical analysis, a total 15 proteins were found differentially expressed among the three groups. The dynamic changes of transthyretin that exhibited initial increase in MCI followed by decrease in AD were reported for the first time. In Chapter 4, a comparative study of membrane protein in mouse hippocampi was conducted by shotgun proteomics. In this study, AT-1 Tg (acetyltransferase) mouse model that selectively overexpresses human AT-1 in neurons. We found a large set of differentially expressed proteins that are involved in neuronal migration and adhesion, neurite outgrowth, transport of synaptic vesicles, assembly of synaptic connections, and regulation of synaptic activity. Chapters 5 and 6 reported on our method development for neuropeptide characterization and detection. Conclusions and research summaries are presented in Chapter 7. Collectively, this dissertation develops several improved mass spectrometric methods for neuropeptide analysis and showcases several important applications in neuroscience.
Book Synopsis Development and Application of Quantitative and Qualitative Mass Spectrometry Techniques to Probe Crustacean Neuropeptides and Beyond by : Amanda Rae Buchberger Jones
Download or read book Development and Application of Quantitative and Qualitative Mass Spectrometry Techniques to Probe Crustacean Neuropeptides and Beyond written by Amanda Rae Buchberger Jones and published by . This book was released on 2018 with total page 553 pages. Available in PDF, EPUB and Kindle. Book excerpt: Biomolecules, especially neuropeptides, are highly involved in the stress response. Due to their biological and chemical complexity, studying neuropeptides in mammals is challenging. To address this challenge, this thesis employs both model organisms and mass spectrometry (MS) to better characterize neuropeptides and their role in stress. For example, crustaceans provide a simple, well-characterized network for method development while also being ecological relevant to stressors of interest (e.g., hypoxia and salinity). With MS, we can probe perturbations at a global level both quantitatively and qualitatively. Quantitatively, several tagging agents have been explored to improve multiplexing and extend to novel applications. For example, MS imaging, a powerful technique capable of determining the localization of hundreds of biomolecules without prior knowledge of their structure, is inherently qualitative. While useful for relative comparisons between conditions, the application of quantitative tagging reagents to MS imaging explored in this work could advance quantitative MS imaging techniques. Overall, this work improves methodology for probing biomolecules, such as neuropeptides, while expanding biological information about neuropeptides and their roles in stress.
Book Synopsis A Mass Spectrometry-based Neuropeptide Discovery Pipeline by :
Download or read book A Mass Spectrometry-based Neuropeptide Discovery Pipeline written by and published by . This book was released on 2013 with total page 272 pages. Available in PDF, EPUB and Kindle. Book excerpt: Neuropeptides represent the most complex group of endogenous molecules in the nervous system that plays important roles in the regulation of physiological process. Elucidation of these signaling molecules is a crucial step towards understanding the underlying mechanism of neuromodulation. My thesis research focuses on development of an efficient peptide discovery pipeline that accelerates neuropeptidomic analysis in crustacean models. We established a high-definition MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, which was achieved by combining bottom-up, off-line top-down, and on-line top-down tandem MS methods. We further refined and incorporated this high-definition approach into a multi-scale strategy for simultaneous and confident sequence elucidation of various sizes of peptides in the crustacean nervous system. A wide range of neuropeptides with various molecular weights (0.9-8.2 kDa) were fully sequenced. The well-characterized primary sequences of neuropeptides provide opportunities for elucidation of their structural information. To rapidly and precisely localize D-amino acids in bioactive peptides, we developed a novel site-specific strategy by ion mobility spectrometry (IMS) analysis of MS-generated epimeric fragment ions. Our results indicate that the isomerization of L- to D-Phe occurs in the American lobster CHHs. In addition to characterization of conformational isomers of neuropeptides, our method allows elucidating structural isomers in gas phase. We report on a novel strategy for qualitative and quantitative analysis of b-type ion isomers by combining electron transfer dissociation, IMS and formaldehyde labeling. The analysis results provide evidence to support the proposed fragmentation mechanism of peptide sequence scrambling in gas phase. The final goal of our study is to understand the roles of neuropeptides play in neural circuits. To investigate if CHHs are involved in regulation of food intake, a new isotopic labeling-assisted top-down MS strategy was developed to directly monitor the abundance changes of endogenous large neuropeptides. Comparative analysis in unfed and fed crabs revealed that the CHH abundance in the sinus glands was significantly increased after food intake. In addition, label-free quantitative analysis reveals that the orcokinin and RFamide peptides were down-regulated after food intake, suggesting that the release of these neuropeptides might be altered by feeding behavior.
