Strategy for Nontargeted Metabolomic Annotation and Quantitation Using a High-Resolution Spectral-Stitching Nanoelectrospray Direct-Infusion Mass Spectrometry with Data-Independent Acquisition.

Direct-infusion nanoelectrospray ionization high-resolution mass spectrometry (DI-nESI-HRMS) is an alternative approach to chromatography-MS-based techniques for nontargeted metabolomics, offering a high sample throughout. However, its annotation accuracy of analytes is still full of challenges. In this study, we proposed a strategy for the annotation and quantitation of nontargeted metabolomic data using a spectral-stitching DI-nESI-HRMS with data-independent acquisition. The metabolite annotation strategy included the isotopic distribution, MS/MS spectrum similarity, and precursor and product ion correlation as well as matching of the extracted metabolite features along with the targeted metabolite precursors. Two groups of mixed standard solutions containing 40 and 79 metabolites were, respectively, used to establish the metabolite annotation strategy and validate its reliability. The results showed that the detected standards could be well annotated at top three explanations and total qualitative percentages were 100% (40 of 40) for the standard solution and 94.9% (74 of 78) for the standards spiked into the serum matrix. The intensity of the precursor ions was used for quantitation except for isomers, which were quantified by the intensities of the characteristic product ions if available. Finally, the strategy was applied to study serum metabolomics in diabetes, and the results demonstrated that it is promising for a large-scale cohort metabolomic study.