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      Ion-Pairing Chromatography and Amine Derivatization Provide Complementary Approaches for the Targeted LC-MS Analysis of the Polar Metabolome

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          Abstract

          Liquid chromatography coupled to mass spectrometry is a key metabolomics/metabonomics technology. Reversed-phase liquid chromatography (RPLC) is very widely used as a separation step, but typically has poor retention of highly polar metabolites. Here, we evaluated the combination of two alternative methods for improving retention of polar metabolites based on 6-aminoquinoloyl- N-hydroxysuccinidimyl carbamate derivatization for amine groups, and ion-pairing chromatography (IPC) using tributylamine as an ion-pairing agent to retain acids. We compared both of these methods to RPLC and also to each other, for targeted analysis using a triple-quadrupole mass spectrometer, applied to a library of ca. 500 polar metabolites. IPC and derivatization were complementary in terms of their coverage: combined, they improved the proportion of metabolites with good retention to 91%, compared to just 39% for RPLC alone. The combined method was assessed by analyzing a set of liver extracts from aged male and female mice that had been treated with the polyphenol compound ampelopsin. Not only were a number of significantly changed metabolites detected, but also it could be shown that there was a clear interaction between ampelopsin treatment and sex, in that the direction of metabolite change was opposite for males and females.

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          Most cited references43

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          Proposed minimum reporting standards for chemical analysis Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI).

          There is a general consensus that supports the need for standardized reporting of metadata or information describing large-scale metabolomics and other functional genomics data sets. Reporting of standard metadata provides a biological and empirical context for the data, facilitates experimental replication, and enables the re-interrogation and comparison of data by others. Accordingly, the Metabolomics Standards Initiative is building a general consensus concerning the minimum reporting standards for metabolomics experiments of which the Chemical Analysis Working Group (CAWG) is a member of this community effort. This article proposes the minimum reporting standards related to the chemical analysis aspects of metabolomics experiments including: sample preparation, experimental analysis, quality control, metabolite identification, and data pre-processing. These minimum standards currently focus mostly upon mass spectrometry and nuclear magnetic resonance spectroscopy due to the popularity of these techniques in metabolomics. However, additional input concerning other techniques is welcomed and can be provided via the CAWG on-line discussion forum at http://msi-workgroups.sourceforge.net/ or http://Msi-workgroups-feedback@lists.sourceforge.net. Further, community input related to this document can also be provided via this electronic forum.
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            A positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue.

            The revival of interest in cancer cell metabolism in recent years has prompted the need for quantitative analytical platforms for studying metabolites from in vivo sources. We implemented a quantitative polar metabolomics profiling platform using selected reaction monitoring with a 5500 QTRAP hybrid triple quadrupole mass spectrometer that covers all major metabolic pathways. The platform uses hydrophilic interaction liquid chromatography with positive/negative ion switching to analyze 258 metabolites (289 Q1/Q3 transitions) from a single 15-min liquid chromatography-mass spectrometry acquisition with a 3-ms dwell time and a 1.55-s duty cycle time. Previous platforms use more than one experiment to profile this number of metabolites from different ionization modes. The platform is compatible with polar metabolites from any biological source, including fresh tissues, cancer cells, bodily fluids and formalin-fixed paraffin-embedded tumor tissue. Relative quantification can be achieved without using internal standards, and integrated peak areas based on total ion current can be used for statistical analyses and pathway analyses across biological sample conditions. The procedure takes ∼12 h from metabolite extraction to peak integration for a data set containing 15 total samples (∼6 h for a single sample).
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              Precision high-throughput proton NMR spectroscopy of human urine, serum, and plasma for large-scale metabolic phenotyping.

              Proton nuclear magnetic resonance (NMR)-based metabolic phenotyping of urine and blood plasma/serum samples provides important prognostic and diagnostic information and permits monitoring of disease progression in an objective manner. Much effort has been made in recent years to develop NMR instrumentation and technology to allow the acquisition of data in an effective, reproducible, and high-throughput approach that allows the study of general population samples from epidemiological collections for biomarkers of disease risk. The challenge remains to develop highly reproducible methods and standardized protocols that minimize technical or experimental bias, allowing realistic interlaboratory comparisons of subtle biomarker information. Here we present a detailed set of updated protocols that carefully consider major experimental conditions, including sample preparation, spectrometer parameters, NMR pulse sequences, throughput, reproducibility, quality control, and resolution. These results provide an experimental platform that facilitates NMR spectroscopy usage across different large cohorts of biofluid samples, enabling integration of global metabolic profiling that is a prerequisite for personalized healthcare.
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                Author and article information

                Journal
                J Proteome Res
                J Proteome Res
                pr
                jprobs
                Journal of Proteome Research
                American Chemical Society
                1535-3893
                1535-3907
                10 May 2022
                03 June 2022
                : 21
                : 6
                : 1428-1437
                Affiliations
                []Department of Metabolism, Digestion and Reproduction, Imperial College London , South Kensington, London SW7 2AZ, U.K.
                []AnalytiCon Discovery GmbH , Hermannswerder Haus 17, 14473 Potsdam, Germany
                [§ ]Center for Behavioral Sciences and Mental Health, Istituto Superiore di Sanità, Viale Regina Elena , 00161 Rome, Italy
                []Department of Psychology, Sapienza University of Rome , via dei Marsi 78, 00185 Rome, Italy
                Author notes
                Author information
                https://orcid.org/0000-0002-7982-4904
                https://orcid.org/0000-0002-8558-7394
                https://orcid.org/0000-0002-7433-8632
                https://orcid.org/0000-0002-1164-8465
                Article
                10.1021/acs.jproteome.2c00030
                9171846
                35536659
                80462ace-1f5e-4be2-8f13-3dfa02319643
                © 2022 The Authors. Published by American Chemical Society

                Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained ( https://creativecommons.org/licenses/by/4.0/).

                History
                : 17 January 2022
                Funding
                Funded by: H2020 Health, doi 10.13039/100010677;
                Award ID: 633589
                Funded by: Natural Environment Research Council, doi 10.13039/501100000270;
                Award ID: NE/S000240/1
                Funded by: Biotechnology and Biological Sciences Research Council, doi 10.13039/501100000268;
                Award ID: NA
                Categories
                Article
                Custom metadata
                pr2c00030
                pr2c00030

                Molecular biology
                metabolomics,metabonomics,ion-pairing,ampelopsin,healthy aging,uplc-ms,nmr spectroscopy,statistical heterospectroscopy

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