Systematic evaluation of extraction methods for multiplatform-based metabotyping: application to the Fasciola hepatica metabolome

Jasmina Saric, Elizabeth J Want, Urs Duthaler, Matthew Lewis, Jennifer Keiser, John P Shockcor, Gordon A Ross, Jeremy K Nicholson, Elaine Holmes, Marina F M Tavares, Jasmina Saric, Elizabeth J Want, Urs Duthaler, Matthew Lewis, Jennifer Keiser, John P Shockcor, Gordon A Ross, Jeremy K Nicholson, Elaine Holmes, Marina F M Tavares

Abstract

Combining data from multiple analytical platforms is essential for comprehensive study of the molecular phenotype (metabotype) of a given biological sample. The metabolite profiles generated are intrinsically dependent on the analytical platforms, each requiring optimization of instrumental parameters, separation conditions, and sample extraction to deliver maximal biological information. An in-depth evaluation of extraction protocols for characterizing the metabolome of the hepatobiliary fluke Fasciola hepatica , using ultra performance liquid chromatography and capillary electrophoresis coupled with mass spectroscopy is presented. The spectrometric methods were characterized by performance, and metrics of merit were established, including precision, mass accuracy, selectivity, sensitivity, and platform stability. Although a core group of molecules was common to all methods, each platform contributed a unique set, whereby 142 metabolites out of 14,724 features were identified. A mixture design revealed that the chloroform:methanol:water proportion of 15:59:26 was globally the best composition for metabolite extraction across UPLC-MS and CE-MS platforms accommodating different columns and ionization modes. Despite the general assumption of the necessity of platform-adapted protocols for achieving effective metabotype characterization, we show that an appropriately designed single extraction procedure is able to fit the requirements of all technologies. This may constitute a paradigm shift in developing efficient protocols for high-throughput metabolite profiling with more-general analytical applicability.

Figures

Figure 1
Figure 1
Typical mass chromatograms and electropherograms of 80% methanol extracts of F. hepatica flukes acquired at ESI+ mode in (A) C18 column, (B) HILIC column, and (C) CE capillary. Key: GPC, glycerophosphocholine; the remaining terms have the nomenclature α-phosphatidylcholine (L)_n (which represents a mixture of α-phosphatidylcholines (L)_n numbered 1 to 23 by order of elution in the RPLC-MS method (refs (18) and (33)); tentative identification based on m/z searches at http://www.lipidmaps.org/data/structure/LMSDSearch.php): L19, 1-hexadecanoyl-2-octadecadienoyl-sn-glycero-3-phosphocholine; L20, 1-nonanoyl-2-tricosanoyl-sn-glycero-3-phosphocholine; L21, 1-hexadecanoyl-2-octadecenoyl-sn-glycero-3-phosphocholine; and L23, 1-octadecanoyl-2-octadecenoyl-sn-glycero-3-phosphocholine.
Figure 2
Figure 2
Venn diagram depicting the 142 metabolites identified across all analytical platforms. The metabolites are color-coded according to the method by which they were identified: red (five methods), brown (four methods), green (three methods), blue (two methods) and black (one method). Common metabolites to all five methods: (71) glutathione, (81) hypoxanthine, (83) inosine, (84) isoleucine, (85) leucine, (90) methionine, (104) phenylalanine, (130) tryptophan, and (132) tyrosine. (For a complete list of identified metabolites, see the Supporting Information (Table S-2).)
Figure 3
Figure 3
Optimization of extraction of a pooled F. hepatica sample using a mixture design approach and RPLC-MS analysis in ESI+ mode. Analyses were performed only on the aqueous extracts. Extractions were performed in (a) 80% methanol, (b) 20% methanol, (c) 90:10 chloroform:20% methanol, (d) 65% methanol, and (e) 15:15:70 chloroform:20% methanol:80% methanol.
Figure 4
Figure 4
Representation of responses as a function of extraction composition for RPLC-MS at ESI+ mode. Responses are computed as the peak area of (A) phenylalanine and (B) α-phosphatidylcholine (L)_23 (1-octadecanoyl-2-octadecenoyl-sn-glycero-3-phosphocholine) corrected by the biomass weight.

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