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Mass Spectrometry Combinations for Structural Characterization of Sulfated-Steroid Metabolites

  • Research Article
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Journal of The American Society for Mass Spectrometry

Abstract

Steroid conjugates, which often occur as metabolites, are challenging to characterize. One application is female-mouse urine, where steroid conjugates serve as important ligands for the pheromone-sensing neurons. Although the two with the highest abundance in mouse urine were previously characterized with mass spectrometry (MS) and NMR to be sulfated steroids, many more exist but remain structurally unresolved. Given that their physical and chemical properties are similar, they are likely to have a sulfated steroid ring structure. Because these compounds occur in trace amounts in mouse urine and elsewhere, their characterization by NMR will be difficult. Thus, MS methods become the primary approach for determining structure. Here, we show that a combination of MS tools is effective for determining the structures of sulfated steroids. Using 4-pregnene analogs, we explored high-resolving power MS (HR-MS) to determine chemical formulae; HD exchange MS (HDX-MS) to determine number of active, exchangeable hydrogens (e.g., OH groups); methoxyamine hydrochloride (MOX) derivatization MS, or reactive desorption electrospray ionization with hydroxylamine to determine the number of carbonyl groups; and tandem MS (MSn), high-resolution tandem MS (HRMS/MS), and GC-MS to obtain structural details of the steroid ring. From the fragmentation studies, we deduced three major fragmentation rules for this class of sulfated steroids. We also show that a combined MS approach is effective for determining structure of steroid metabolites, with important implications for targeted metabolomics in general and for the study of mouse social communication in particular.

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Acknowledgments

The authors thank Dr. Hao Chen for providing us with electrosonic spray ionization and hel** set up the DESI experiments, Jan Crowley for hel** with the GC-MS experiments, and Fong-Fu Hsu for assistance in accurate-mass, product-ion spectra. This research was supported by the National Institute of General Medical Sciences (8 P41 GM103422-35 to MLG) and the National Institute on Deafness and Other Communication Disorders (R01 DC005964 to TEH), both of the National Institutes of Health.

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Authors and Affiliations

  1. Center for Biomedical and Bioorganic Mass Spectrometry, Department of Chemistry, Washington University in St. Louis, St. Louis, MO, 63130, USA

    Yuetian Yan, Don L. Rempel & Michael L. Gross

  2. Department of Anatomy and Neurobiology, Washington University School of Medicine, Washington University in St. Louis, St. Louis, MO, 63130, USA

    Timothy E. Holy

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  1. Yuetian Yan
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  2. Don L. Rempel
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Correspondence to Michael L. Gross.

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Yan, Y., Rempel, D.L., Holy, T.E. et al. Mass Spectrometry Combinations for Structural Characterization of Sulfated-Steroid Metabolites. J. Am. Soc. Mass Spectrom. 25, 869–879 (2014). https://doi.org/10.1007/s13361-014-0836-9

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