Strategies in biomarker discovery. Peak annotation by MS and targeted LC-MS micro-fractionation for de novo structure identification by micro-NMR.

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.