Protein prenylation is an essential post-translational modification in which an isoprenoid is attached to proteins near their C-terminus. The modification is crucial for correct localization and function of many key proteins in the cell. Alterations in protein prenylation have been observed in multiple age-related diseases indicating it may play an important role in the aging process. To investigate prenylation in the context of cellular aging, we developed a novel method to track overall levels of prenylation at the single-cell level with Mass Cytometry. Mass cytometry enables the use of up to 40 parameters to analyze proteins, cell cycle stages, mRNA and other cell processes simultaneously in single cells. The new method is the first to use a small molecule probe and reporter to track prenylation with mass cytometry. A synthetic isoprenoid analogue containing a terminal alkyne is metabolically incorporated after addition to the culture media. The cells are then fixed and permeabilized before incubating with an azide functionalized reporter containing a chelated terbium ion. The isoprenoid analogue and terbium reporter react through copper catalyzed azide-alkyne cycloaddition. The whole cells are then introduced into the mass cytometer where the terbium intensity can be related back to the cellular level of prenylation. A correction method was developed and applied using a holmium-containing reporter lacking the azide functional group to account for non-specific binding of the terbium reporter. This prenylation analysis method has been successfully applied in L6 rat myoblast cell models of reduced autophagy function to characterize changes in prenylation related to decline in autophagy function with age. In the future, this prenylation measurement tool will be combined with antibody panels for prenylated proteins and other vital cellular pathways to aid in elucidating pharmacological targets for aging and age-related diseases.

Determining changes in prenylation through mass cytometry single-cell analysis

ACS Spring Meeting 2021

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