Metabolomic method: UPLC-q-ToF polar and non-polar metabolites in the healthy rat cerebellum using an in-vial dual extraction
Unbiased metabolomic analysis of biological samples is a powerful and increasingly commonly utilised tool, especially for the analysis of bio-fluids to identify candidate biomarkers. To date however only a small number of metabolomic studies have been applied to studying the metabolite composition o...
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| Veröffentlicht in: | PloS one 2015-04, Vol.10 (4), p.e0122883-e0122883 |
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| creator | Ebshiana, Amera A Snowden, Stuart G Thambisetty, Madhav Parsons, Richard Hye, Abdul Legido-Quigley, Cristina |
| description | Unbiased metabolomic analysis of biological samples is a powerful and increasingly commonly utilised tool, especially for the analysis of bio-fluids to identify candidate biomarkers. To date however only a small number of metabolomic studies have been applied to studying the metabolite composition of tissue samples, this is due, in part to a number of technical challenges including scarcity of material and difficulty in extracting metabolites. The aim of this study was to develop a method for maximising the biological information obtained from small tissue samples by optimising sample preparation, LC-MS analysis and metabolite identification. Here we describe an in-vial dual extraction (IVDE) method, with reversed phase and hydrophilic liquid interaction chromatography (HILIC) which reproducibly measured over 4,000 metabolite features from as little as 3mg of brain tissue. The aqueous phase was analysed in positive and negative modes following HILIC separation in which 2,838 metabolite features were consistently measured including amino acids, sugars and purine bases. The non-aqueous phase was also analysed in positive and negative modes following reversed phase separation gradients respectively from which 1,183 metabolite features were consistently measured representing metabolites such as phosphatidylcholines, sphingolipids and triacylglycerides. The described metabolomics method includes a database for 200 metabolites, retention time, mass and relative intensity, and presents the basal metabolite composition for brain tissue in the healthy rat cerebellum. |
| doi_str_mv | 10.1371/journal.pone.0122883 |
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The non-aqueous phase was also analysed in positive and negative modes following reversed phase separation gradients respectively from which 1,183 metabolite features were consistently measured representing metabolites such as phosphatidylcholines, sphingolipids and triacylglycerides. The described metabolomics method includes a database for 200 metabolites, retention time, mass and relative intensity, and presents the basal metabolite composition for brain tissue in the healthy rat cerebellum.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0122883</identifier><identifier>PMID: 25853858</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Amino acids ; Animals ; Biological properties ; Biological samples ; Biomarkers ; Biomarkers - metabolism ; Brain ; Care and treatment ; Cerebellum ; Cerebellum - chemistry ; Cerebellum - metabolism ; Chromatography ; Chromatography, Liquid ; Complications and side effects ; Down syndrome ; Health aspects ; Metabolites ; Metabolome ; Metabolomics ; Methods ; Optimization ; Phase separation ; Rats ; Retention time ; Sample preparation ; Spectrometry, Mass, Electrospray Ionization ; Sphingolipids ; Sugar</subject><ispartof>PloS one, 2015-04, Vol.10 (4), p.e0122883-e0122883</ispartof><rights>COPYRIGHT 2015 Public Library of Science</rights><rights>This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. 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To date however only a small number of metabolomic studies have been applied to studying the metabolite composition of tissue samples, this is due, in part to a number of technical challenges including scarcity of material and difficulty in extracting metabolites. The aim of this study was to develop a method for maximising the biological information obtained from small tissue samples by optimising sample preparation, LC-MS analysis and metabolite identification. Here we describe an in-vial dual extraction (IVDE) method, with reversed phase and hydrophilic liquid interaction chromatography (HILIC) which reproducibly measured over 4,000 metabolite features from as little as 3mg of brain tissue. The aqueous phase was analysed in positive and negative modes following HILIC separation in which 2,838 metabolite features were consistently measured including amino acids, sugars and purine bases. 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| issn | 1932-6203 1932-6203 |
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| subjects | Amino acids Animals Biological properties Biological samples Biomarkers Biomarkers - metabolism Brain Care and treatment Cerebellum Cerebellum - chemistry Cerebellum - metabolism Chromatography Chromatography, Liquid Complications and side effects Down syndrome Health aspects Metabolites Metabolome Metabolomics Methods Optimization Phase separation Rats Retention time Sample preparation Spectrometry, Mass, Electrospray Ionization Sphingolipids Sugar |
| title | Metabolomic method: UPLC-q-ToF polar and non-polar metabolites in the healthy rat cerebellum using an in-vial dual extraction |
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