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Antimicrobial resistance is a major threat to human health as resistant pathogens spread globally, and the development of new antimicrobials is slow. Since many antimicrobials function by targeting cell wall and membrane components, high-throughput lipidomics for bacterial phenotyping is of high interest for researchers to unveil lipid-mediated pathways when dealing with a large number of lab-selected or clinical strains. However, current practice for lipidomic analysis requires the cultivation of bacteria on a large scale, which does not replicate the growth conditions for high-throughput bioassays that are normally carried out in 96-well plates, such as susceptibility tests, growth curve measurements, and biofilm quantitation. Analysis of bacteria grown under the same condition as other bioassays would better inform the differences in susceptibility and other biological metrics. In this work, a high-throughput method for cultivation and lipidomic analysis of antimicrobial-resistant bacteria was developed for standard 96-well plates exemplified by methicillin-resistant Staphylococcus aureus (MRSA). By combining a 30-mm liquid chromatography (LC) column with ion mobility (IM) separation, elution time could be dramatically shortened to 3.6 min for a single LC run without losing major lipid features. Peak capacity was largely rescued by the addition of the IM dimension. Through multi-linear calibration, the deviation of retention time can be limited to within 5%, making database-based automatic lipid identification feasible. This high-throughput method was further validated by characterizing the lipidomic phenotypes of antimicrobial-resistant mutants derived from the MRSA strain, W308, grown in a 96-well plate. [ABSTRACT FROM AUTHOR]

Ion mobility-mass spectrometry (IM-MS) has gained increased applications in the characterization and identification of drugs and drug metabolites, largely owning to the complementary separation of analyte ions based on their gas-phase size and shape in the IM dimension in addition to their mass-to-charge ratios. In this review, we discuss recent advances in such applications. We first introduce various types of IM techniques, focusing on those that allow the measurement of collision cross section (CCS), the physical property of an ion that reflects its gas-phase size and shape. Next, we discuss the IM-MS landscape of the large chemical space of drugs and multimodal distributions of certain drugs in IM separation due to the presence of protomers. We then review drug metabolism reactions and discuss the application of IM-MS in separation and identification of isomeric drug metabolites. Subsequently, we discuss various approaches to generate theoretical and predicted CCS data, including theory-based calculation methods and data-driven prediction models, and currently available resources on these approaches. Finally, current limitations and future directions of application of IM-MS are discussed. [Display omitted] • IM-MS landscape of the large chemical space of drugs. • Multimodal distribution of protomers of drugs in IM separation. • Characterization and identification of isomeric drug metabolites. • Theoretical approaches for the calculation and prediction of collision cross sections. [ABSTRACT FROM AUTHOR]