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Introduction: Ion mobility-mass spectrometry (IM-MS) is an emerging technique in the -omics fields that has broad potential applicability to the clinical lab. As a rapid, gas-phase structure-based separation technique, IM-MS offers promise in isomer separations and can be easily combined with existing LC-MS methods (i.e., LC-IM-MS). Several experimental conditions, including analyte cation adducts and drift composition further provide a means to tune separations for global and/or targeted applications., Objectives: The primary objective of this study was to demonstrate the utility of IM-MS under a range of experimental conditions for detection of glucocorticoids, and specifically for the separation of several isomeric pairs., Methods: LC-IM-MS was used to characterize 16 glucocorticoids including three isomer pairs: cortisone/prednisolone, betamethasone/dexamethasone, and flunisolide/triamcinolone acetonide. Collision cross section (CCS) values were measured for all common adducts (e.g., protonated and sodiated) using both step-field and single-field methods. Alternative alkali, alkaline earth, and transition metals were introduced, such that their adducts could also be measured. Finally, four different drift gases (helium, nitrogen, argon, and carbon dioxide) were compared for their relative separation capability., Results: LC-IM-MS offered a robust, multidimensional separation technique that allowed for the 16 glucocorticoids to be analyzed and separated in three-dimensions (retention time, CCS, and m / z ). Despite the relatively modest resolution of isomer pairs under standard conditions (i.e., nitrogen drift gas, sodiated ions, etc.), improvements were observed for alkaline earth and transition metals (notable barium adducts) and in carbon dioxide drift gas., Conclusion: In summary, LC-IM-MS offers potential as a clinical method due to its ease of coupling with traditional LC-MS methods and its promise for tuning separations to better resolve targeted and/or global isomers in complex biological samples., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could affect the work described in this article., (© 2022 THE AUTHORS.)

Globin-coupled sensors (GCS) usually consist of three domains: a sensor/globin, a linker, and a transmitter domain. The globin domain (GD), activated by ligand binding and/or redox change, induces an intramolecular signal transduction resulting in a response of the transmitter domain. Depending on the nature of the transmitter domain, GCSs can have different activities and functions, including adenylate and di-guanylate cyclase, histidine kinase activity, aerotaxis and/or oxygen sensing function. The gram-negative delta-proteobacterium Geobacter sulfurreducens expresses a protein with a GD covalently linked to a four transmembrane domain, classified, by sequence similarity, as GCS ( Gs GCS). While its GD is fully characterized, not so its transmembrane domain, which is rarely found in the globin superfamily. In the present work, Gs GCS was characterized spectroscopically and by native ion mobility-mass spectrometry in combination with cryo-electron microscopy. Although lacking high resolution, the oligomeric state and the electron density map were valuable for further rational modeling of the full-length Gs GCS structure. This model demonstrates that Gs GCS forms a transmembrane domain-driven tetramer with minimal contact between the GDs and with the heme groups oriented outward. This organization makes an intramolecular signal transduction less likely. Our results, including the auto-oxidation rate and redox potential, suggest a potential role for Gs GCS as redox sensor or in a membrane-bound e - /H + transfer. As such, Gs GCS might act as a player in connecting energy production to the oxidation of organic compounds and metal reduction. Database searches indicate that GDs linked to a four or seven helices transmembrane domain occur more frequently than expected., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)