Untargeted metabolomics provides a comprehensive platform for identifying 
metabolites whose levels are altered between two or more populations. By using 
liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS), 
hundreds to thousands of peaks with a unique m/z ratio and retention time are 
routinely detected from most biological samples in an untargeted profiling 
experiment. Each peak, termed a metabolomic feature, can be characterized on the 
basis of its accurate mass, retention time and tandem mass spectral 
fragmentation pattern. Here a seven-step protocol is suggested for such a 
characterization by using the METLIN metabolite database. The protocol starts 
from untargeted metabolomic LC-Q-TOF-MS data that have been analyzed with the 
bioinformatics program XCMS, and it describes a strategy for selecting 
interesting features as well as performing subsequent targeted tandem MS. The 
seven steps described will require 2-4 h to complete per feature, depending on 
the compound.