Abstract
In metabolomic studies the identification of biomarkers is a key step but represents a serious bottleneck since the de novo identification of natural products is a lengthy process. A strategy for the dereplication and peak annotation of plant biomarkers is presented based on high resolution mass spectra acquired on a quadrupole-time-of-flight mass spectrometry coupled to ultra-high pressure liquid chromatography and chemotaxonomy information. A rational approach for the targeted LC-MS micro-isolation of biomarkers followed by de novo identification by NMR at the microgram scale is described, based on gradient transfer from the analytical scale and chromatographic modelling. The methodology is illustrated by the identification of various stress biomarkers of the plant wound response using Arabidopsis thaliana as a model.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Guy C, Kopka J, Moritz T (2008) Plant metabolomics coming of age. Physiol Plant 132:113–116
Fiehn O, Kopka J, Dörmann P et al (2000) Metabolite profiling for plant functional genomics. Nat Biotechnol 18:1157–1161
Zhou B, Xiao JF, Tuli L et al (2012) LC-MS-based metabolomics. Mol Biosystems 8:470–481
Leiss KA, Choi YH, Verpoorte R et al (2011) An overview of NMR-based metabolomics to identify secondary plant compounds involved in host plant resistance. Phytochem Rev 10:205–216
Wolfender JL, Glauser G, Boccard J et al (2009) MS-based plant metabolomic approaches for biomarker discovery. Nat Prod Commun 4:1417–1430
Dunn WB (2008) Current trends and future requirements for the mass spectrometric investigation of microbial, mammalian and plant metabolomes. Phys Biol 5:011001
Boccard J, Veuthey JL, Rudaz S (2010) Knowledge discovery in metabolomics: an overview of MS data handling. J Sep Sci 33:290–304
Glauser G, Guillarme D, Grata E et al (2008) Optimized liquid chromatography-mass spectrometry approach for the isolation of minor stress biomarkers in plant extracts and their identification by capillary nuclear magnetic resonance. J Chromatogr A 1180:90–98
Heinisch S, Lesellier E, Podevin C et al (1997) Computerized optimization of RP-HPLC separation with nonaqueous or partially aqueous mobile phases. Chromatographia 44:529–537
Guillarme D, Nguyen DTT, Rudaz S et al (2008) Method transfer for fast liquid chromatography in pharmaceutical analysis: application to short columns packed with small particle. Part II: gradient experiments. Eur J Pharm Biopharm 68:430–440
Kind T, Fiehn O (2007) Seven Golden Rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry. BMC Bioinformatics 8:105–124
Grata E, Boccard J, Guillarme D et al (2008) UPLC-TOF-MS for plant metabolomics: a sequential approach for wound marker analysis in Arabidopsis thaliana. J Chromatogr B 871:261–270
Huang N, Siegel MM, Kruppa GH et al (1999) Automation of a Fourier transform ion cyclotron resonance mass spectrometer for acquisition, analysis, and E-mailing of high-resolution exact-mass electrospray ionization mass spectral data. J Am Soc Mass Spectrom 10:1166–1173
Nielsen KF, Mansson M, Rank C et al (2011) Dereplication of microbial natural products by LC-DAD-TOFMS. J Nat Prod 74:2338–2348
Markham KR (1982) Techniques of flavonoid identification. Academic press, London
Molinski TF (2010) NMR of natural products at the ‘nanomole-scale’. Nat Prod Rep 27:321–329
Wolfender JL, Queiroz EF, Hostettmann K (2005) Phytochemistry in the microgram domain—a LC-NMR perspective. Magn Reson Chem 43:697–709
Olson DL, Norcross JA, O’Neil-Johnson M et al (2004) Microflow NMR: concepts and capabilities. Anal Chem 76:2966–2974
Glauser G, Boccard J, Rudaz S et al (2010) Mass spectrometry-based metabolomics oriented by correlation analysis for wound-induced molecule discovery: identification of a novel jasmonate glucoside. Phytochem Anal 21:95–101
Guillarme D, Nguyen DTT, Rudaz S et al (2007) Method transfer for fast liquid chromatography in pharmaceutical analysis: application to short columns packed with small particle. Part I: isocratic separation. Eur J Pharm Biopharm 66:475–482
Guillarme D, Grata E, Glauser G et al (2009) Some solutions to obtain very efficient separations in isocratic and gradient modes using UHPLC technology. J Chromatogr A1216:3232–3243
Funari C, Eugster P, Martel S et al. (2012) High resolution Ultra High Pressure Liquid Chromatography Time-of-Flight Mass Spectrometry dereplication strategy for the metabolite profiling of Brazilian Lippia species. J Chromatogr A 1259:167–178
Waridel P, Wolfender JL, Ndjoko K et al (2001) Evaluation of quadrupole time-of-flight tandem mass spectrometry and ion-trap multiple-stage mass spectrometry for the differentiation of C-glycosidic flavonoid isomers. J Chromatogr A 926:29–41
van der Hooft JJJ, Vervoort J, Bino RJ et al (2011) Polyphenol identification based on systematic and robust high-resolution accurate mass spectrometry fragmentation. Anal Chem 83:409–416
Acknowledgments
This work was supported by Swiss National Science Foundation grants no. 205320_135190 and CRSII3_127187. G.G. and J.L.W. are also grateful to the National Center of Competence in Research (NCCR) Plant Survival and to the Swiss Plant Science Web (SPSW).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Eugster, P.J., Glauser, G., Wolfender, JL. (2013). Strategies in Biomarker Discovery. Peak Annotation by MS and Targeted LC-MS Micro-Fractionation for De Novo Structure Identification by Micro-NMR. In: Roessner, U., Dias, D. (eds) Metabolomics Tools for Natural Product Discovery. Methods in Molecular Biology, vol 1055. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-577-4_19
Download citation
DOI: https://doi.org/10.1007/978-1-62703-577-4_19
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-576-7
Online ISBN: 978-1-62703-577-4
eBook Packages: Springer Protocols