MS² and LC libraries for untargeted metabolomics: Enhancing method development and identification confidence

As part of the “omics” technologies in the life sciences, metabolomics is becoming increasingly important. In untargeted metabolomics, unambiguous metabolite identification and the inevitable coverage bias that comes with the selection of analytical conditions present major challenges. Reliable compound annotation is essential for translating metabolomics data into meaningful biological information. Here, we developed a fast and transferable method for generating in-house MS² libraries to improve metabolite identification. Using the new method we established an in-house MS² library that includes over 4,000 fragmentation spectra of 506 standard compounds for 6 different normalized collision energies (NCEs). Additionally, we generated a comprehensive liquid chromatography (LC) library by testing 57 different LC-MS conditions for 294 compounds. We used the library information to develop an untargeted metabolomics screen with maximum coverage of the metabolome that was successfully tested in a study of 360 human serum samples. The current work demonstrates a workflow for LC–MS/MS-based metabolomics, with enhanced metabolite identification confidence and the possibility to select suitable analysis conditions according to the specific research interest.