Abstract
Chemical probes that target classes of proteins based on shared functional properties have emerged as powerful tools for proteomics. The metabolome rivals, if not surpasses, the proteome in terms of size and complexity, suggesting that efforts to profile metabolites would also benefit from targeted technologies. Here we apply the principle of chemoselective probes to the metabolome, creating a general strategy to tag, enrich and profile large classes of small molecules from biological systems. Key to success was incorporation of a protease-cleavage step to release captured metabolites in a format compatible with liquid chromatography–mass spectrometry (LC-MS) analysis. This technology, termed metabolite enrichment by tagging and proteolytic release (METPR), is applicable to small molecules of any physicochemical class, including polar, labile and low-mass (<100 Da) compounds. We applied METPR to profile changes in the thiol metabolome of human cancer cells treated with the antioxidant N-acetyl-L-cysteine.
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Acknowledgements
We thank H.P. Benton and A. Nordström for assistance with Q-TOF experiments. We also thank G. Simon for help in generating figures, and members of the Cravatt laboratory for helpful discussion and critical review of the manuscript. This work was supported by the American Cancer Society (PF-06-009-01-CDD, to E.E.C.), the National Institutes of Health (CA087660) and the Skaggs Institute for Chemical Biology.
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Supplementary information
Supplementary Fig. 1
Synthesis of reactive group-functionalized resins. (PDF 124 kb)
Supplementary Fig. 2
Protein precipitation as assessed by SDS-PAGE (10% SDS) visualized with silver stain. (PDF 129 kb)
Supplementary Fig. 3
Profiling metabolites with varied physicochemical properties by METPR. (PDF 154 kb)
Supplementary Fig. 4
METPR analysis of small molecule standards in human urine. (PDF 103 kb)
Supplementary Fig. 5
MS2 analysis of tagged small-molecules generated in METPR experiments. (PDF 191 kb)
Supplementary Table 1
Table of detected masses from NAC treatment. (PDF 43 kb)
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Carlson, E., Cravatt, B. Chemoselective probes for metabolite enrichment and profiling. Nat Methods 4, 429–435 (2007). https://doi.org/10.1038/nmeth1038
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DOI: https://doi.org/10.1038/nmeth1038
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