Integrating Ion Mobility–Resolved Tandem Mass Spectrometry and Microcrystal Electron Diffraction for Structural Elucidation

We analyzed a synthetic mixture at the sub-µg scale using an integrated strategy that combined high-resolution mass spec-trometry (HRMS), ion mobility spectrometry (IMS), tandem mass spectrometry (MS/MS), collision cross-section (CCS) and fragmentation pathway modelling, together with microcrystal electron diffraction (MicroED). We demonstrate that ion mobility-resolved MS/MS (IMS-HRMS-MS/MS) experiments can generate detailed structural information, which, when pro-cessed with accurate fragmentation chemistry and CCS predictors applied to plausible reaction products, can be used for robust structural elucidation for material of sub-µg quantity. The structural insights obtained from IMS-HRMS-MS/MS are comparable in quality to those from MicroED performed also at the sub-µg scale, and the two techniques provide complementary information. A pipeline is proposed that integrates IMS-HRMS-MS/MS and MicroED, leveraging the strengths of each technique in a synergistic manner