| Summary: | The utility of metabolomics is well documented; however, its full scientific promise has not yet been realized due to multiple technical challenges. These grand challenges include accurate chemical identification of all observable metabolites and the limiting depth-of-coverage of current metabolomics methods. Here, we report a combinatorial solution to aid in both grand challenges using UHPLC-trapped ion mobility spectrometry coupled to tandem mass spectrometry (UHPLC-TIMS-TOF-MS). TIMS offers additional depth-of-coverage through increased peak capacities realized with the multi-dimensional UHPLC-TIMS separations. Metabolite identification confidence is simultaneously enhanced by incorporating orthogonal collision cross section (CCS) data matching. To facilitate metabolite identifications, we created a CCS library of 146 plant natural products. This library was generated using TIMS with N<sub>2</sub> drift gas to record the <sup>TIMS</sup>CCS<sub>N2</sub> of plant natural products with a high degree of reproducibility; i.e., average RSD = 0.10%. The robustness of <sup>TIMS</sup>CCS<sub>N2</sub> data matching was tested using authentic standards spiked into complex plant extracts, and the precision of CCS measurements were determined to be independent of matrix affects. The utility of the UHPLC-TIMS-TOF-MS/MS in metabolomics was then demonstrated using extracts from the model legume <i>Medicago truncatula</i> and metabolites were confidently identified based on retention time, accurate mass, molecular formula, and CCS.
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