Quantitative proteomics to predict functional consequences of ubiquitylation events during T cell stimulation

Abstract

Abstract The post-translational attachment of ubiquitin to cellular proteins can alter protein function and half-life, thereby profoundly impacting cell signaling and fate decisions. New proteomics-based techniques have been developed that have the potential to reveal relationships between ubiquitylation and protein fate. Here, we assess the feasibility and scalability of using quantitative proteomic approaches to identify ubiquitylated proteins in primary T cells in a high throughput manner. Furthermore, we sought to identify TCR-dependent changes in protein ubiquitylation. We utilized immunoprecipitation of ubiquitin remnant peptides (peptides with modified lysine residues) and quantitative mass spectrometry to quantify changes in protein-specific ubiquitylation in CD4+ T cells stimulated via the TCR compared to resting T cells. Our analysis identifies thousands of modified lysines in primary T cells, and reveals dynamic changes in ubiquitylation of hundred of proteins, including key signaling transducers, during TCR stimulation. We then performed whole cell proteomic analysis to quantify changes in relative protein abundance during T cell activation. By comparing changes in ubiquitylation with changes in protein abundance in stimulated CD4+ T cells, these data can be used as a resource to predict the consequences of TCR-induced protein ubiquitylation. This work establishes ubiquitin remnant profiling and whole proteome analysis as important techniques for global analysis of ubiquitylation in primary T cells.