Development Of Metabolomics And Lipidomics Workflows For Phosphorylated Metabolites Using Ultra High Performance Liquid Chromatography With Tandem Mass Spectrometry
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Development Of Metabolomics And Lipidomics Workflows For Phosphorylated Metabolites Using Ultra High Performance Liquid Chromatography With Tandem Mass Spectrometry
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Author : Peng Li
language : en
Publisher:
Release Date : 2022
Development Of Metabolomics And Lipidomics Workflows For Phosphorylated Metabolites Using Ultra High Performance Liquid Chromatography With Tandem Mass Spectrometry written by Peng Li and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2022 with categories.
Over the last decades, system biology and multi-omics have become an emerging field in life science. With the development of analytical instruments, especially the mass spectrometry (MS), MS based metabolomics are widely utilized to understanding the metabolic status. Due to significantly different properties of lipids, lipidomics became more and more an independent research discipline. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the most popular analytical platform in metabolomics and lipidomics. Recent progress in bioinformatical approaches has also facilitated the bottlenecking data processing steps. Although some widely used methods were established for general metabolomics and lipidomics analysis, there are still some challenging analytes like phosphorylated metabolites/lipids, which showed compromised performance. Through highly regulated phosphorylation and dephosphorylation by enzymes, phosphorylated metabolites/lipids regulate many important cellular processes. Despite their importance, the performance of their current analytical methods is not satisfying. Generally, analysis of phosphorylated analytes by LC-MS/MS face the following challenges, including low abundance, low extraction recovery, instability, regioisomers, availability of standards, etc. In order to address these issues, we developed new methods based on phosphate methylation. Accordingly, complete workflows from sample preparation to data processing were developed and validated in biological matrix. In the 1st project, a targeted LC-MS/MS method of fatty acyl-CoAs (acyl-CoAs) with different chain lengths was established. Acyl-CoAs are central metabolic intermediates in numerous biological processes. Due to diverse fatty acyl compositions, acyl-CoAs vary greatly in polarity, which poses great challenges to their chromatographic separation. Currently, short chain acyl-CoAs are separated by HILIC or acidic RP-LC, while medium-, long- and very long-chain acyl-CoAs are analysed in basic RP-LC. Additionally, carryover in LC-MS/MS instrumentation requires extra flushing/cleaning procedures. To address these problems, we developed phosphate methylation strategy for acyl-CoAs. In the sample preparation, mixed-mode SPE was optimized to be compatible with methylation. SRM transitions in MS detection was constructed with fragmentation rule for all methylated acyl-CoAs. Uniformly 13C labelled yeast extract was used as internal standards for accurate quantification. Odd chain and stable isotope labelled analogs were used as surrogate calibrators. The LOQs were between 16.9 nM and 4.2 nM. The method was applied in cultured HeLa cells and human platelets of coronary artery disease patients. The results showed that the established method can be used to profile acyl-CoAs in biological samples. In the 2nd project, an isomer selective LC-MS/MS method of PIPx was developed. PIPx is a family of low abundant phospholipids commonly found in eukaryotic cells. Starting from phosphoinositol (PI), PIPx family consists of three lipid classes (PIP, PIP2, and PIP3) according to phosphorylation degree. There are three distinct regioisomers in each class of PIP and PIP2. As phospholipids, PIPx has two fatty acyl groups at the sn1 and sn2 position of a glycerol backbone. Currently, methods capable of regioisomer separation and fatty acyl coverage are still missing. To develop a comprehensive method, we utilized polysaccharide chiral column with data independent acquisition technique. Stable isotope labelled PIPx were characterized and used as internal standards. The established method was applied to several biological samples, including NIST SRM1950 plasma, Pichia Pastoris, and cultured HeLa cells. The results indicate this method could effectively monitor PIPx profiles in real samples and facilitate our understanding of physiological and pathological conditions. In the 3rd and 4th project, improved LC-MS/MS methods of IPx and SPx were developed. Compared to PIPx and acyl-CoAs, IPx and SPx are more polar and hydrophilic. To apply the phosphate methylation strategy, a new derivatization solvent mixture was developed and optimized. To improve extraction recovery, an extraction kit compromised of TiO2 and centrifuge filter was established. Differential isotope labelling methylation was utilized to generate stable isotope labelled analogs in both projects (internal standards in IPx project, surrogate calibrators in SPx project). Cholesterol-ether bonded RP column was used to separate IPx regioisomers, especially I(1,4,5)P3. Porous graphite carbon column was used to separate SPx in glycolysis pathway. After validation in biological matrix, the methods were applied in different biological samples, including NIST SRM1950 plasma, cultured HeLa cells, and human platelets.
Processing Metabolomics And Proteomics Data With Open Software
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Author : Robert Winkler
language : en
Publisher: Royal Society of Chemistry
Release Date : 2020-03-19
Processing Metabolomics And Proteomics Data With Open Software written by Robert Winkler and has been published by Royal Society of Chemistry this book supported file pdf, txt, epub, kindle and other format this book has been release on 2020-03-19 with Science categories.
Metabolomics and proteomics allow deep insights into the chemistry and physiology of biological systems. This book expounds open-source programs, platforms and programming tools for analysing metabolomics and proteomics mass spectrometry data. In contrast to commercial software, open-source software is created by the academic community, which facilitates the direct interaction between users and developers and accelerates the implementation of new concepts and ideas. The first section of the book covers the basics of mass spectrometry, experimental strategies, data operations, the open-source philosophy, metabolomics, proteomics and statistics/ data mining. In the second section, active programmers and users describe available software packages. Included tutorials, datasets and code examples can be used for training and for building custom workflows. Finally, every reader is invited to participate in the open science movement.
Metabolic Profiling
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Author : Georgios A. Theodoridis
language : en
Publisher: Humana
Release Date : 2019-04-30
Metabolic Profiling written by Georgios A. Theodoridis and has been published by Humana this book supported file pdf, txt, epub, kindle and other format this book has been release on 2019-04-30 with Science categories.
This volume explores the different approaches and techniques used by researchers to study the recent challenges and developments in metabolic profiling. This book is divided into IV parts. Part I contains chapters that highlight basic concepts, such as experimental design, data treatment, metabolite identification, and harmonization. Part II describes experimental protocols for both targeted and untargeted metabolomics covering the basic analytical technologies: LC-MS, GC-MS, NMR and CE-MS. In addition the protocols describe methods for the study of tissues, feces, blood and other types of biological samples as well as the application of chemical derivatization for GC-MS. Parts III and IV present the use of metabolomics in the study of food, plants and the life sciences, with examples from the quest for the discovery of disease biomarkers, physical exercise omics and metabolic profiling of food, fruit and wine. 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. Authoritative and thorough, Metabolic Profiling: Methods and Protocols is a valuable resource for researchers who are interested in expanding their knowledge of this rapidly developing field.
Ultra Performance Liquid Chromatography Mass Spectrometry
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Author : Mu Naushad
language : en
Publisher: CRC Press
Release Date : 2014-03-18
Ultra Performance Liquid Chromatography Mass Spectrometry written by Mu Naushad and has been published by CRC Press this book supported file pdf, txt, epub, kindle and other format this book has been release on 2014-03-18 with Science categories.
This book presents a unique collection of up-to-date UPLC-MS/MS (ultra performance liquid chromatography-tandem mass spectrometric) methods for the separation and quantitative determination of pesticides, capsaicinoids, heterocyclic amines, aflatoxin, perfluorochemicals, acrylamide, procyanidins and alkaloids, lactose content, phenolic compounds, vitamins, and aroma and flavor compounds in a wide variety of foods and food products. With contributions by experts in interdisciplinary fields, this reference offers practical information for readers in research and development, production, and routing analysis of foods and food products.
Mass Spectrometry Based Lipidomics
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Author : Fong-Fu Hsu
language : en
Publisher: Humana
Release Date : 2021-05-29
Mass Spectrometry Based Lipidomics written by Fong-Fu Hsu and has been published by Humana this book supported file pdf, txt, epub, kindle and other format this book has been release on 2021-05-29 with Science categories.
This detailed volume covers conventional MS-based “shotgun lipidomics” by which samples are introduced by infusion or loop injection, as well as LC-MS-based lipidomics, which are becoming increasingly important due to the ever-increasing demand for a complete and precise lipid analysis of the complex and diversified lipids in nature. The volume features protocols applying chemical reactions, the on-line photochemical reactions combined with various MS methods for comprehensive characterization of various lipid classes, and quantification of specific and rare lipids. Written for the highly successful Methods in Molecular Biology series, 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. Authoritative and practical, Mass Spectrometry-Based Lipidomics: Methods and Protocols serves as an invaluable guide for biochemists and mass spectroscopists who are interested in lipid studies.
Metabolomics
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Author : Wolfram Weckwerth
language : en
Publisher: Springer Science & Business Media
Release Date : 2008-02-04
Metabolomics written by Wolfram Weckwerth and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2008-02-04 with Science categories.
Metabolomics: Methods and Protocols examines the state-of-the-art in metabolomic analysis. Leading researchers in the field present protocols for the application of complementary analytical methods, such as gas chromatography-mass spectrometry (GC-MS). Metabolomics: Methods and Protocols contains forward-looking protocols, which provide the essential groundwork for future efforts in elucidating the structure of the unknowns detected in metabolomic studies.
Sample Preparation In Metabolomics
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Author : Julia Kuligowski
language : en
Publisher: MDPI
Release Date : 2021-04-07
Sample Preparation In Metabolomics written by Julia Kuligowski and has been published by MDPI this book supported file pdf, txt, epub, kindle and other format this book has been release on 2021-04-07 with Medical categories.
Metabolomics is increasingly being used to explore the dynamic responses of living systems in biochemical research. The complexity of the metabolome is outstanding, requiring the use of complementary analytical platforms and methods for its quantitative or qualitative profiling. In alignment with the selected analytical approach and the study aim, sample collection and preparation are critical steps that must be carefully selected and optimized to generate high-quality metabolomic data. This book showcases some of the most recent developments in the field of sample preparation for metabolomics studies. Novel technologies presented include electromembrane extraction of polar metabolites from plasma samples and guidelines for the preparation of biospecimens for the analysis with high-resolution μ magic-angle spinning nuclear magnetic resonance (HR-μMAS NMR). In the following chapters, the spotlight is on sample preparation approaches that have been optimized for diverse bioanalytical applications, including the analysis of cell lines, bacteria, single spheroids, extracellular vesicles, human milk, plant natural products and forest trees.
Metabolomics In Practice
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Author : Michael Lämmerhofer
language : en
Publisher: John Wiley & Sons
Release Date : 2013-02-14
Metabolomics In Practice written by Michael Lämmerhofer and has been published by John Wiley & Sons this book supported file pdf, txt, epub, kindle and other format this book has been release on 2013-02-14 with Science categories.
Unlike other handbooks in this emerging field, this guide focuses on the challenges and critical parameters in running a metabolomics study, including such often-neglected issues as sample preparation, choice of separation and detection method, recording and evaluating data as well as method validation. By systematically covering the entire workflow, from sample preparation to data processing, the insight and advice offered here helps to clear the hurdles in setting up and running a successful analysis, resulting in high-quality data from every experiment. Based on more than a decade of practical experience in developing, optimizing and validating metabolomics approaches as a routine technology in the academic and industrial research laboratory, the lessons taught here are highly relevant for all systems-level approaches, whether in systems biology, biotechnology, toxicology or pharmaceutical sciences. From the Contents: * Sampling and Sample Preparation in Microbial Metabolomics * Tandem Mass Spectrometry Hyphenated with HPLC and UHPLC for Targeted Metabolomics * GC-MS, LC-MS, CE-MS and Ultrahigh Resolution MS (FTICR-MS) in Metabolomics * NMR-based metabolomics analysis * Potential of Microfluidics and Single Cell Analysis in Metabolomics * Data Processing in Metabolomics * Validation and Measurement Uncertainty in Metabolomic Studies * Metabolomics and its Role in the Study of Mammalian Systems and in Plant Sciences * Metabolomics in Biotechnology and Nutritional Metabolomics and more.
Advanced Mass Spectrometry Based Analytical Separation Techniques For Probing The Polar Metabolome
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Author : Rawi Ramautar
language : en
Publisher: Royal Society of Chemistry
Release Date : 2021-07-13
Advanced Mass Spectrometry Based Analytical Separation Techniques For Probing The Polar Metabolome written by Rawi Ramautar and has been published by Royal Society of Chemistry this book supported file pdf, txt, epub, kindle and other format this book has been release on 2021-07-13 with Science categories.
The efficient analysis of polar and charged metabolites in biological samples remains a huge challenge in the field of metabolomics. Over the past years, novel mass spectrometry-based analytical tools have been developed to enable the sensitive and efficient profiling of polar ionogenic metabolites in various biological samples. This book gives the reader a comprehensive overview of these recent technological developments. Topics covered include the use of chemical labelling strategies for allowing the analysis of polar metabolites using reversed-phase liquid chromatography–mass spectrometry (RPLC-MS) and the latest methodological developments in RPLC-MS, hydrophilic interaction liquid chromatography (HILIC)-MS and ion-pair LC-MS approaches. Attention is also paid to developments in nano-LC-MS and capillary electrophoresis–mass spectrometry methods specifically for profiling polar metabolites in small volume biological samples. The utility of ion-mobility MS and NMR spectroscopy will also be outlined. Sample preparation is the key part in the analytical workflow employed for metabolomics. Therefore, ample emphasis will be given on recent solid-phase extraction and solid-phase micro-extraction methods. Finally, analytical techniques for chiral metabolic profiling will also be considered. Discussing the state-of-the-art of the proposed topics in one single book for probing the polar metabolome, using relevant examples, is unique and needed in the metabolomics field. This book has relevance and appeal to an international audience of analytical and biomedical researchers in industry and academia.
Chemical Derivatization In Gas Chromatography
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Author : J. Drozd
language : en
Publisher: Elsevier
Release Date : 1986-07-01
Chemical Derivatization In Gas Chromatography written by J. Drozd and has been published by Elsevier this book supported file pdf, txt, epub, kindle and other format this book has been release on 1986-07-01 with Science categories.
Chemical Derivatization in Gas Chromatography