| الوصف: |
Protein prenylation is an essential post-translational modification that plays a key role in facilitating protein localization. Aberrations in protein prenylation have been indicated in multiple disease pathologies including progeria, some forms of cancer, and Alzheimer's disease. While there are single-cell methods to study prenylation, these methods cannot simultaneously assess prenylation and other cellular changes in the complex cell environment. Here, we report a novel method to monitor, at the single-cell level, prenylation and expression of autophagy markers. An isoprenoid analogue containing a terminal alkyne, substrate of prenylation enzymes, was metabolically incorporated into cells in culture. Treatment with a terbium reporter containing an azide functional group, followed by copper-catalyzed azide-alkyne cycloaddition, covalently attached terbium ions to prenylated proteins within cells. In addition, simultaneous treatment with a holmium-containing analogue of the reporter, without an azide functional group, was used to correct for non-specific retention at the single-cell level. This procedure was compatible with other mass cytometric sample preparation steps that use metal-tagged antibodies. We demonstrate that this method reports changes in levels of prenylation in competitive and inhibitor assays, while tracking autophagy molecular markers with metal-tagged antibodies. The method reported here makes it possible to track prenylation along with other molecular pathways in single cells of complex systems, which is essential to elucidate the role of this post-translational modification in disease, cell response to pharmacological treatments, and aging. |
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