Bar Coding MS² Spectra for Metabolite Identification

Metabolite identifications are most frequently achieved in untargeted metabolomics by matching precursor mass and full, high-resolution MS² spectra to metabolite databases and standards. Here we considered an alternative approach for establishing metabolite identifications that does not rely on full, high-resolution MS² spectra. First, we select mass-to-charge regions containing the most informative metabolite fragments and designate them as bins. We then translate each metabolite fragmentation pattern into a binary code by assigning 1's to bins containing fragments and 0's to bins without fragments. With 20 bins, this binary-code system is capable of distinguishing 96% of the compounds in the METLIN MS² library. A major advantage of the approach is that it extends untargeted metabolomics to low-resolution triple quadrupole (QqQ) instruments, which are typically less expensive and more robust than other types of mass spectrometers. We demonstrate a method of acquiring MS² data in which the third quadrupole of a QqQ instrument cycles over 20 wide isolation windows (coinciding with the location and width of our bins) for each precursor mass selected by the first quadrupole. Operating the QqQ instrument in this mode yields diagnostic bar codes for each precursor mass that can be matched to the bar codes of metabolite standards. Furthermore, our data suggest that using low-resolution bar codes enables QqQ instruments to make MS²-based identifications in untargeted metabolomics with a specificity and sensitivity that is competitive to high-resolution time-of-flight technologies.