Development of a plasma pseudotargeted metabolomics method based on ultra-high-performance liquid chromatography–mass spectrometry
Untargeted methods are typically used in the detection and discovery of small organic compounds in metabolomics research, and ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) is one of the most commonly used platforms for untargeted metabolomics. Although t...
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| Veröffentlicht in: | Nature protocols 2020-08, Vol.15 (8), p.2519-2537 |
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| creator | Zheng, Fujian Zhao, Xinjie Zeng, Zhongda Wang, Lichao Lv, Wangjie Wang, Qingqing Xu, Guowang |
| description | Untargeted methods are typically used in the detection and discovery of small organic compounds in metabolomics research, and ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) is one of the most commonly used platforms for untargeted metabolomics. Although they are non-biased and have high coverage, untargeted approaches suffer from unsatisfying repeatability and a requirement for complex data processing. Targeted metabolomics based on triple-quadrupole mass spectrometry (TQMS) could be a complementary tool because of its high sensitivity, high specificity and excellent quantification ability. However, it is usually applicable to known compounds: compounds whose identities are known and/or are expected to be present in the analyzed samples. Pseudotargeted metabolomics merges the advantages of untargeted and targeted metabolomics and can act as an alternative to the untargeted method. Here, we describe a detailed protocol of pseudotargeted metabolomics using UHPLC-TQMS. An in-depth, untargeted metabolomics experiment involving multiple UHPLC-HRMS runs with MS at different collision energies (both positive and negative) is performed using a mixture obtained using small amounts of the analyzed samples. XCMS, CAMERA and Multiple Reaction Monitoring (MRM)-Ion Pair Finder are used to find and annotate peaks and choose transitions that will be used to analyze the real samples. A set of internal standards is used to correct for variations in retention time. High coverage and high-performance quantitative analysis can be realized. The entire protocol takes ~5 d to complete and enables the simultaneously semiquantitative analysis of 800–1,300 metabolites.
In pseudotargeted metabolomics, transitions used for multiple-reaction monitoring are chosen from mass spectrometry data obtained using mixtures of real samples. Semiquantitative information is obtained without knowing the identity of the compounds. |
| doi_str_mv | 10.1038/s41596-020-0341-5 |
| format | Article |
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In pseudotargeted metabolomics, transitions used for multiple-reaction monitoring are chosen from mass spectrometry data obtained using mixtures of real samples. 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Academic</collection><jtitle>Nature protocols</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Zheng, Fujian</au><au>Zhao, Xinjie</au><au>Zeng, Zhongda</au><au>Wang, Lichao</au><au>Lv, Wangjie</au><au>Wang, Qingqing</au><au>Xu, Guowang</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of a plasma pseudotargeted metabolomics method based on ultra-high-performance liquid chromatography–mass spectrometry</atitle><jtitle>Nature protocols</jtitle><stitle>Nat Protoc</stitle><addtitle>Nat Protoc</addtitle><date>2020-08-01</date><risdate>2020</risdate><volume>15</volume><issue>8</issue><spage>2519</spage><epage>2537</epage><pages>2519-2537</pages><issn>1754-2189</issn><eissn>1750-2799</eissn><abstract>Untargeted methods are typically used in the detection and discovery of small organic compounds in metabolomics research, and ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) is one of the most commonly used platforms for untargeted metabolomics. Although they are non-biased and have high coverage, untargeted approaches suffer from unsatisfying repeatability and a requirement for complex data processing. Targeted metabolomics based on triple-quadrupole mass spectrometry (TQMS) could be a complementary tool because of its high sensitivity, high specificity and excellent quantification ability. However, it is usually applicable to known compounds: compounds whose identities are known and/or are expected to be present in the analyzed samples. Pseudotargeted metabolomics merges the advantages of untargeted and targeted metabolomics and can act as an alternative to the untargeted method. Here, we describe a detailed protocol of pseudotargeted metabolomics using UHPLC-TQMS. An in-depth, untargeted metabolomics experiment involving multiple UHPLC-HRMS runs with MS at different collision energies (both positive and negative) is performed using a mixture obtained using small amounts of the analyzed samples. XCMS, CAMERA and Multiple Reaction Monitoring (MRM)-Ion Pair Finder are used to find and annotate peaks and choose transitions that will be used to analyze the real samples. A set of internal standards is used to correct for variations in retention time. High coverage and high-performance quantitative analysis can be realized. The entire protocol takes ~5 d to complete and enables the simultaneously semiquantitative analysis of 800–1,300 metabolites.
In pseudotargeted metabolomics, transitions used for multiple-reaction monitoring are chosen from mass spectrometry data obtained using mixtures of real samples. Semiquantitative information is obtained without knowing the identity of the compounds.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>32581297</pmid><doi>10.1038/s41596-020-0341-5</doi><tpages>19</tpages><orcidid>https://orcid.org/0000-0003-4298-3554</orcidid></addata></record> |
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| subjects | 631/1647/2196 631/45/320 Analytical Chemistry Biological Techniques Biomedical and Life Sciences Chromatography Chromatography, High Pressure Liquid Computational Biology/Bioinformatics Data processing High performance liquid chromatography Ions Life Sciences Liquid chromatography Mass Spectrometry Mass spectroscopy Metabolites Metabolomics Metabolomics - methods Methods Microarrays Monitoring Organic Chemistry Organic compounds Plasma - metabolism Protocol Quadrupoles Retention time Scientific imaging Spectroscopy Time Factors |
| title | Development of a plasma pseudotargeted metabolomics method based on ultra-high-performance liquid chromatography–mass spectrometry |
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