2021 GLRM 306

Single-cell Analysis of Prenylation in Mammalian Cells via Mass Cytometry

Zoe Maxwell¹, maxwe235@umn.edu, Heather M. Brown¹, Kiall F. Suazo¹, Elyse Krautkramer¹, Kevin Liu¹, Mark D. Distefano³, Edgar A. Arriaga². (1) Chemistry, University of Minnesota Twin Cities, Minneapolis, Minnesota, United States (3) Univ of Minnesota, Minneapolis, Minnesota, United States

Mass cytometry has become an important single-cell analysis tool due to its ability to measure over 40 parameters simultaneously through the detection of metal isotopes by ICP-MS. Traditional reporters for mass cytometry are antibodies linked with polymers that chelate metal isotopes often from the lanthanide series. Only using antibody reporters in experiments limits what targets can be detected in the cell. To expand the questions that can be answered using mass cytometry, there is a need to develop additional reporters for important cellular components. In this work, a novel probe and reporter method has been established to track changes in prenylation with mass cytometry. Prenylation is an essential post-translational modification which is the enzyme mediated attachment of an isoprenoid to proteins to facilitate correct localization and signaling. Disruption of prenylation has been indicate in multiple diseases such as progeria and Alzheimer’s. To track changes in prenylation, a farnesyl diphosphate analog containing a terminal alkyne is incorporated into cells during culture. After harvest, fixation and permeabilization the cells then undergo a copper catalyzed click reaction to attach a reporter containing a terminal azide and chelating a terbium ion. The intensity of terbium after the cells go through the mass cytometer can then be related back to level of prenylation in each cell. A non-specific binding reporter similar in structure to the terbium reporter has also been included in this method to track non-specific binding in the cells. To demonstrate applicability, the method has been successfully applied in two cell culture models of reduced autophagy function. In the future, this novel method can be used to investigate changes in prenylation levels in cellular models of disease, aging and after pharmaceutical treatments. Development of this technique has expanded the available cellular targets that can be investigated by mass cytometry and opened the door to expansion of similar reporters for other post-translational modifications or functional measurements.