Development and Validation of a Liquid Chromatography-Electrospray Ionization-Time-of-Flight Mass Spectrometry Method for Induced Changes in Nicotiana attenuata Leaves during Simulated Herbivory

A liquid chromatography electrospray ionization time-of-flight mass spectrometry (HPLC/ESI-TOF-MS) procedure was developed to characterize changes induced in Nicotiana attenuata leaves 1 h and 5 days after wounding and application of Manduca sexta elicitors. The constancy of the measurement conditions was first confirmed for 22 selected analytes spanning the entire chromatogram. Using the Profile Analysis software, we extracted 367 buckets, which were analyzed by principal component analysis and two-factorial ANOVA. One hundred seventy-three buckets were found to be statistically regulated, 128 due to time effects, and 85 due to treatment effects. In vivo (15)N-isotope labeling was used to facilitate the annotation and the interpretation of the fragmentation pattern of nitrogen-containing metabolites, and a correlation analysis was performed to test mathematical relationships existing among potential in-source fragments. Additionally, tandem MS measurements of the most regulated ions are presented. Altogether, this study defines a framework for the mining and annotation of major herbivory-elicited changes in Nicotiana attenuate.