Abstract
In spite of recent advances in proteomics, quantitative analyses of protein–protein interactions (PPIs) or post-translational modifications (PTMs) in rare cell populations remain challenging. This is in particular true for analyses of rare immune and/or stem cell populations that are directly isolated from humans or animal models, and which are often characterized by multiple surface markers. To overcome these limitations, here we have developed proximity ligation imaging cytometry (PLIC), a protocol for proteomic analysis of rare cells. Specifically, by employing PLIC on medullary thymic epithelial cells (mTECs), which serve as a paradigm for a rare immune population, we demonstrate that PLIC overcomes the inherent limitations of conventional proteomic approaches and enables a high-resolution detection and quantification of PPIs and PTMs at a single cell level.
Highlights
In spite of recent advances in proteomics, quantitative analyses of protein–protein interactions (PPIs) or post-translational modifications (PTMs) in rare cell populations remain challenging
The proximity ligation assay (PLA) assay enables a highly sensitive and robust analysis of protein associations, one of its key limitations is that it is much less suitable for proteomic analysis of rare cell populations and/or populations defined by expression of multiple surface markers, which are the typical hallmarks of many immune cell subsets
We have previously shown that Sirt[1] is able to deacetylate autoimmune regulator (Aire) on multiple lysine residues in transfected HEK-293 cells[24], experimental evidence validating that Aire is deacetylated by Sirt[1] in medullary thymic epithelial cells (mTECs) is still missing, due to the inherent limitations of conventional proteomic tools for analysis of rare cell populations
Summary
In spite of recent advances in proteomics, quantitative analyses of protein–protein interactions (PPIs) or post-translational modifications (PTMs) in rare cell populations remain challenging. In spite of the recent progress in our understanding of how Aire regulates expression of its target genes (reviewed in Anderson and Su10), most of the molecular insights into its modus operandi come from in vitro proteomic studies[11,12,13,14,15,16] Validation of these findings under physiological conditions, including analyses of Aire’s interacting partners or PTMs in bona fide mTECs, using currently available proteomic approaches remains technically not feasible. To overcome these limitations, we sought to develop an analytical approach, which would enable a quantitative analysis of PPIs and/or PTMs in rare cell populations that are often defined by the expression of multiple surface markers, such as mTECs, in an accurate, quantitative, and reproducible manner. Our study uses mTECs as an experimental paradigm, we demonstrate that PLIC is suitable for proteomic analysis of other populations of the immune system for which standard proteomic approaches have been technically challenging
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