MSACL 2023 Abstract

Introduction: Metabolites produced by the gut microbiome play a crucial and diverse role on host physiology, which are detectable in a wide range of biological samples including feces, plasma, urine, and cerebrospinal fluid. Microbiota dysbiosis has been associated with the development of diseases, however, the metabolic link has yet to be detected. The detailed and targeted analysis of these metabolites is important for the discovery of biomarkers and unknown bioactive molecules.[1]

Methods: Mass spectrometric metabolomics is the method of choice for identification and quantification of these metabolites. Advanced methods at the interface of chemistry and biology coupled with metabolomics analysis are required but still limited. We have therefore developed a unique and multifunctional chemoselective probe with synthetic 13C/12C isotopically labelled analogues that allows for comparative and quantitative analysis of metabolites in human samples at low concentrations.[2] We have termed this unique method quantitative Quantitative Sensitive CHEmoselective MetAbolomics (quant-SCHEMA). Coupled to magnetic beads, this method allows the straightforward chemoselective extraction of metabolites from human samples.[2-6] The captured metabolites are released under mild conditions through the cleavage of the newly developed bioorthogonal cleavage site p-nitrocinnamyloxycarbonyl (Noc). This isolation procedure of specific classes of metabolites from sample matrices led to significantly increased mass spectrometric sensitivity by sixth orders of magnitude and facilitates the detection of metabolites at femtomole quantities.

Results: Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Due to the combination of natural and isotope-labeled conjugates before LC-MS analysis, this new method does not require any normalization procedure including internal standards or normalization of different sample types. This is necessary in general metabolomics analysis to reduce technical errors. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This method also facilitates detection of 21 metabolites that were not previously detected and can now even be quantified.It enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.

Conclusion: The chemoselective probe was applied for analysis of amine- or carbonyl-containing metabolites in human fecal, urine and plasma samples. We have discovered several metabolites previously unreported and performed metabolic profiling in these sample types and confirmation of the presence of medically relevant gut microbiota-derived metabolites.

Reference:
[1] M.S. Donia, M.A. Fischbach, Science, 2015, 349, 1254766.
[2] W. Lin, L.P. Conway, M. Vujasinovic, J.-M. Löhr, D. Globisch, Angew. Chem. Int. Ed. 2021, 60, 23232-23240.
[3] N. Garg, L. P. Conway, C. Ballet, M. S. P. Correia, F. Olsson, M. Vujasinovic, J. M. Löhr, D. Globisch, Angew. Chem., Int. Ed., 2018, 57, 13805–13809
[4] L. P. Conway, N. Garg, W. Lin, M. Vujasinovic, J.-M. Löhr, D. Globisch, Chem. Comm. 2019, 55, 9080–9083.
[5] W. Lin, L. P. Conway, A. Block, G. Sommi, M. Vujasinovic, J.-M. Löhr, D. Globisch, Analyst, 2020, 145, 3822–3831.
[6] W. Lin, Z. Yang, A. Kaur, A. Block, M. Vujasinovic, J.-M. Löhr, D. Globisch, RSC Chem. Biol. 2021, 2, 1479-1483.