Tissue-Specific Sample Dilution: An Important Parameter to Optimise Prior to Untargeted LC-MS Metabolomics

When developing a sample preparation protocol for LC−MS untargeted metabolomics of a new sample matrix unfamiliar to the laboratory, selection of a suitable injection concentration is rarely described. Here we developed a simple workflow to address this issue prior to untargeted LC&#87...

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Main Authors: Zhanxuan E. Wu, Marlena C. Kruger, Garth J.S. Cooper, Sally D. Poppitt, Karl Fraser
Format: Article
Language:English
Published: MDPI AG 2019-06-01
Series:Metabolites
Subjects:
Online Access:https://www.mdpi.com/2218-1989/9/7/124
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spelling doaj-9480b68a0f7d49e4bc0a1acac058712f2020-11-25T01:34:26ZengMDPI AGMetabolites2218-19892019-06-019712410.3390/metabo9070124metabo9070124Tissue-Specific Sample Dilution: An Important Parameter to Optimise Prior to Untargeted LC-MS MetabolomicsZhanxuan E. Wu0Marlena C. Kruger1Garth J.S. Cooper2Sally D. Poppitt3Karl Fraser4Food Nutrition & Health, Food and Bio-based Products, AgResearch Limited, Palmerston North 4442, New ZealandSchool of Health Sciences, Massey University, Palmerston North 4442, New ZealandCentre for Advanced Discovery and Experimental Therapeutics, Division of Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9NT, UKHigh-Value Nutrition National Science Challenge, Auckland 1142, New ZealandFood Nutrition & Health, Food and Bio-based Products, AgResearch Limited, Palmerston North 4442, New ZealandWhen developing a sample preparation protocol for LC−MS untargeted metabolomics of a new sample matrix unfamiliar to the laboratory, selection of a suitable injection concentration is rarely described. Here we developed a simple workflow to address this issue prior to untargeted LC−MS metabolomics using pig adipose tissue and liver tissue. Bi-phasic extraction was performed to enable simultaneous optimisation of parameters for analysis of both lipids and polar extracts. A series of diluted pooled samples were analysed by LC−MS and used to evaluate signal linearity. Suitable injected concentrations were determined based on both the number of reproducible features and linear features. With our laboratory settings, the optimum concentrations of tissue mass to reconstitution solvent of liver and adipose tissue lipid fractions were found to be 125 mg/mL and 7.81 mg/mL respectively, producing 2811 (ESI+) and 4326 (ESI−) linear features from liver, 698 (ESI+) and 498 (ESI−) linear features from adipose tissue. For analysis of the polar fraction of both tissues, 250 mg/mL was suitable, producing 403 (ESI+) and 235 (ESI−) linear features from liver, 114 (ESI+) and 108 (ESI−) linear features from adipose tissue. Incorrect reconstitution volumes resulted in either severe overloading or poor linearity in our lipid data, while too dilute polar fractions resulted in a low number of reproducible features (<50) compared to hundreds of reproducible features from the optimum concentration used. Our study highlights on multiple matrices and multiple extract and chromatography types, the critical importance of determining a suitable injected concentration prior to untargeted LC−MS metabolomics, with the described workflow applicable to any matrix and LC−MS system.https://www.mdpi.com/2218-1989/9/7/124LC–MS untargeted metabolomicslipidomicstissue metabolite profilingsample preparation for metabolomics
collection DOAJ
language English
format Article
sources DOAJ
author Zhanxuan E. Wu
Marlena C. Kruger
Garth J.S. Cooper
Sally D. Poppitt
Karl Fraser
spellingShingle Zhanxuan E. Wu
Marlena C. Kruger
Garth J.S. Cooper
Sally D. Poppitt
Karl Fraser
Tissue-Specific Sample Dilution: An Important Parameter to Optimise Prior to Untargeted LC-MS Metabolomics
Metabolites
LC–MS untargeted metabolomics
lipidomics
tissue metabolite profiling
sample preparation for metabolomics
author_facet Zhanxuan E. Wu
Marlena C. Kruger
Garth J.S. Cooper
Sally D. Poppitt
Karl Fraser
author_sort Zhanxuan E. Wu
title Tissue-Specific Sample Dilution: An Important Parameter to Optimise Prior to Untargeted LC-MS Metabolomics
title_short Tissue-Specific Sample Dilution: An Important Parameter to Optimise Prior to Untargeted LC-MS Metabolomics
title_full Tissue-Specific Sample Dilution: An Important Parameter to Optimise Prior to Untargeted LC-MS Metabolomics
title_fullStr Tissue-Specific Sample Dilution: An Important Parameter to Optimise Prior to Untargeted LC-MS Metabolomics
title_full_unstemmed Tissue-Specific Sample Dilution: An Important Parameter to Optimise Prior to Untargeted LC-MS Metabolomics
title_sort tissue-specific sample dilution: an important parameter to optimise prior to untargeted lc-ms metabolomics
publisher MDPI AG
series Metabolites
issn 2218-1989
publishDate 2019-06-01
description When developing a sample preparation protocol for LC−MS untargeted metabolomics of a new sample matrix unfamiliar to the laboratory, selection of a suitable injection concentration is rarely described. Here we developed a simple workflow to address this issue prior to untargeted LC−MS metabolomics using pig adipose tissue and liver tissue. Bi-phasic extraction was performed to enable simultaneous optimisation of parameters for analysis of both lipids and polar extracts. A series of diluted pooled samples were analysed by LC−MS and used to evaluate signal linearity. Suitable injected concentrations were determined based on both the number of reproducible features and linear features. With our laboratory settings, the optimum concentrations of tissue mass to reconstitution solvent of liver and adipose tissue lipid fractions were found to be 125 mg/mL and 7.81 mg/mL respectively, producing 2811 (ESI+) and 4326 (ESI−) linear features from liver, 698 (ESI+) and 498 (ESI−) linear features from adipose tissue. For analysis of the polar fraction of both tissues, 250 mg/mL was suitable, producing 403 (ESI+) and 235 (ESI−) linear features from liver, 114 (ESI+) and 108 (ESI−) linear features from adipose tissue. Incorrect reconstitution volumes resulted in either severe overloading or poor linearity in our lipid data, while too dilute polar fractions resulted in a low number of reproducible features (<50) compared to hundreds of reproducible features from the optimum concentration used. Our study highlights on multiple matrices and multiple extract and chromatography types, the critical importance of determining a suitable injected concentration prior to untargeted LC−MS metabolomics, with the described workflow applicable to any matrix and LC−MS system.
topic LC–MS untargeted metabolomics
lipidomics
tissue metabolite profiling
sample preparation for metabolomics
url https://www.mdpi.com/2218-1989/9/7/124
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