Evaluation of Ion Mobility Spectrometry for Improving Constitutional Assignment in Natural Product Mixtures br

The comprehensive chemical characterization of biological samples remains a central challenge in the field of natural products. Conventional workflows using liquid chromatography(LC)-coupled high-resolution tandem mass spectrometry (MS/MS or MS2) allow the detection of relevant small molecules while providing diagnostic fragment ions for their structural assignment. Still, many natural product extracts are of a molecular complexity that challenges the resolving power of modern LC-MS2pipelines.In this study, we examined the effect of integrating ion mobilityspectrometry (IMS) to our LC-MS2platform for the character-ization of natural product mixtures. IMS provides an additional axisof separation in the gas phase as well as experimental collisioncross-sectional (CCS) values. We analyzed a mixture of 20commercial standards at 2 concentration ranges, either solubilized in solvent or spiked into an actinobacterial extract. Data wereacquired in positive ion mode using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) MS2fragmentation approaches and assessed for both chemical coverage and spectral quality. IMS-DIA identified the largest number of standards in the spiked extract at the lower concentration of standards (17), followed by IMS-DDA (10), DDA (8), and DIA (6). Inaddition, we examined how these data sets performed in the Global Natural Products Social Molecular Networking (GNPS)platform. Overall, integrating IMS increased both metabolite detection and the quality of MS2spectra, particularly for samples analyzed in DIA mode