Abstract
The degree of precision in measuring accurate masses in LC MS/MS-based metabolomics experiments is a determinant in the successful identification of the metabolites present in the original extract. Using the methods described here, complex broccoli extracts containing hundreds of small-molecule compounds (mass range 100–1,400 Da) can be profiled at resolutions up to 100,000 (full width half maximum, FWHM), useful for accurate and sensitive relative quantification experiments. Using external instrument calibration, analyte masses can be measured with high (sub-ppm to a maximum of 2 ppm) accuracy, leading to compound identifications based on elemental composition analysis. Unambiguous identification of four analytes (citric acid, chlorogenic acid, phenylalanine, and UDP-d-glucose) is used to validate the performance of the different MS/MS fragmentation regimes. Identifications are carried out either via resonance excitation collision induced dissociation (CID) or via higher energy collision dissociation (HCD) experiments, and validated by infrared multiphoton dissociation (IRMPD) fragmentation of standards. Such results, obtained on both hybrid and non-hybrid systems from metabolite profiling and identification experiments, provide evidence that the strategies selected can be successfully applied to other LC-MS based projects for plant metabolomic studies.
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Acknowledgements
The authors would like to thank Michael Athanas from VAST Scientific, Robert Mistrik from HighChem, Helen Jenkins and Robert Hall as partners in the META-PHOR project, for advice and useful discussions. This work was in part supported by the EU FP VI project META-PHOR (FOOD-CT-2006-036220).
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Oppermann, M., Damoc, N.E., Crone, C., Moehring, T., Muenster, H., Hornshaw, M. (2011). High Precision Measurement and Fragmentation Analysis for Metabolite Identification. In: Hardy, N., Hall, R. (eds) Plant Metabolomics. Methods in Molecular Biology, vol 860. Humana Press. https://doi.org/10.1007/978-1-61779-594-7_10
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DOI: https://doi.org/10.1007/978-1-61779-594-7_10
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