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Systematic Isolation and Structure Elucidation of Urinary Metabolites Optimized for the Analytical-Scale Molecular Profiling Laboratory
[Image: see text] Annotation and identification of metabolite biomarkers is critical for their biological interpretation in metabolic phenotyping studies, presenting a significant bottleneck in the successful implementation of untargeted metabolomics. Here, a systematic multistep protocol was develo...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical
Society
2019
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6666900/ https://www.ncbi.nlm.nih.gov/pubmed/31188566 http://dx.doi.org/10.1021/acs.analchem.9b00241 |
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author | Whiley, Luke Chekmeneva, Elena Berry, David J Jiménez, Beatriz Yuen, Ada H. Y. Salam, Ash Hussain, Humma Witt, Matthias Takats, Zoltan Nicholson, Jeremy Lewis, Matthew R. |
author_facet | Whiley, Luke Chekmeneva, Elena Berry, David J Jiménez, Beatriz Yuen, Ada H. Y. Salam, Ash Hussain, Humma Witt, Matthias Takats, Zoltan Nicholson, Jeremy Lewis, Matthew R. |
author_sort | Whiley, Luke |
collection | PubMed |
description | [Image: see text] Annotation and identification of metabolite biomarkers is critical for their biological interpretation in metabolic phenotyping studies, presenting a significant bottleneck in the successful implementation of untargeted metabolomics. Here, a systematic multistep protocol was developed for the purification and de novo structural elucidation of urinary metabolites. The protocol is most suited for instances where structure elucidation and metabolite annotation are critical for the downstream biological interpretation of metabolic phenotyping studies. First, a bulk urine pool was desalted using ion-exchange resins enabling large-scale fractionation using precise iterations of analytical scale chromatography. Primary urine fractions were collected and assembled into a “fraction bank” suitable for long-term laboratory storage. Secondary and tertiary fractionations exploited differences in selectivity across a range of reversed-phase chemistries, achieving the purification of metabolites of interest yielding an amount of material suitable for chemical characterization. To exemplify the application of the systematic workflow in a diverse set of cases, four metabolites with a range of physicochemical properties were selected and purified from urine and subjected to chemical formula and structure elucidation by respective magnetic resonance mass spectrometry (MRMS) and NMR analyses. Their structures were fully assigned as tetrahydropentoxyline, indole-3-acetic-acid-O-glucuronide, p-cresol glucuronide, and pregnanediol-3-glucuronide. Unused effluent was collected, dried, and returned to the fraction bank, demonstrating the viability of the system for repeat use in metabolite annotation with a high degree of efficiency. |
format | Online Article Text |
id | pubmed-6666900 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | American
Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-66669002019-07-31 Systematic Isolation and Structure Elucidation of Urinary Metabolites Optimized for the Analytical-Scale Molecular Profiling Laboratory Whiley, Luke Chekmeneva, Elena Berry, David J Jiménez, Beatriz Yuen, Ada H. Y. Salam, Ash Hussain, Humma Witt, Matthias Takats, Zoltan Nicholson, Jeremy Lewis, Matthew R. Anal Chem [Image: see text] Annotation and identification of metabolite biomarkers is critical for their biological interpretation in metabolic phenotyping studies, presenting a significant bottleneck in the successful implementation of untargeted metabolomics. Here, a systematic multistep protocol was developed for the purification and de novo structural elucidation of urinary metabolites. The protocol is most suited for instances where structure elucidation and metabolite annotation are critical for the downstream biological interpretation of metabolic phenotyping studies. First, a bulk urine pool was desalted using ion-exchange resins enabling large-scale fractionation using precise iterations of analytical scale chromatography. Primary urine fractions were collected and assembled into a “fraction bank” suitable for long-term laboratory storage. Secondary and tertiary fractionations exploited differences in selectivity across a range of reversed-phase chemistries, achieving the purification of metabolites of interest yielding an amount of material suitable for chemical characterization. To exemplify the application of the systematic workflow in a diverse set of cases, four metabolites with a range of physicochemical properties were selected and purified from urine and subjected to chemical formula and structure elucidation by respective magnetic resonance mass spectrometry (MRMS) and NMR analyses. Their structures were fully assigned as tetrahydropentoxyline, indole-3-acetic-acid-O-glucuronide, p-cresol glucuronide, and pregnanediol-3-glucuronide. Unused effluent was collected, dried, and returned to the fraction bank, demonstrating the viability of the system for repeat use in metabolite annotation with a high degree of efficiency. American Chemical Society 2019-06-12 2019-07-16 /pmc/articles/PMC6666900/ /pubmed/31188566 http://dx.doi.org/10.1021/acs.analchem.9b00241 Text en Copyright © 2019 American Chemical Society This is an open access article published under a Creative Commons Attribution (CC-BY) License (http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html) , which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. |
spellingShingle | Whiley, Luke Chekmeneva, Elena Berry, David J Jiménez, Beatriz Yuen, Ada H. Y. Salam, Ash Hussain, Humma Witt, Matthias Takats, Zoltan Nicholson, Jeremy Lewis, Matthew R. Systematic Isolation and Structure Elucidation of Urinary Metabolites Optimized for the Analytical-Scale Molecular Profiling Laboratory |
title | Systematic Isolation and Structure Elucidation of
Urinary Metabolites Optimized for the Analytical-Scale Molecular Profiling
Laboratory |
title_full | Systematic Isolation and Structure Elucidation of
Urinary Metabolites Optimized for the Analytical-Scale Molecular Profiling
Laboratory |
title_fullStr | Systematic Isolation and Structure Elucidation of
Urinary Metabolites Optimized for the Analytical-Scale Molecular Profiling
Laboratory |
title_full_unstemmed | Systematic Isolation and Structure Elucidation of
Urinary Metabolites Optimized for the Analytical-Scale Molecular Profiling
Laboratory |
title_short | Systematic Isolation and Structure Elucidation of
Urinary Metabolites Optimized for the Analytical-Scale Molecular Profiling
Laboratory |
title_sort | systematic isolation and structure elucidation of
urinary metabolites optimized for the analytical-scale molecular profiling
laboratory |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6666900/ https://www.ncbi.nlm.nih.gov/pubmed/31188566 http://dx.doi.org/10.1021/acs.analchem.9b00241 |
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