Abstract
Calcium ions play important roles in nearly every biological process, yet whole-proteome analysis of calcium effectors has been hindered by lack of high-throughput, unbiased, and quantitative methods to identify proteins-calcium engagement. To address this, we adapted protein thermostability assays in the budding yeast, human cells, and mouse mitochondria. Based on calcium-dependent thermostability, we identified 2884 putative calcium-regulated proteins across human, mouse, and yeast proteomes. These data revealed calcium engagement of novel signaling hubs and cellular processes, including metabolic enzymes and the spliceosome. Cross-species comparison of calcium-protein engagement and mutagenesis experiments identified residue-specific cation engagement, even within well-known EF-hand domains. Additionally, we found that the dienoyl-CoA reductase DECR1 binds calcium at physiologically-relevant concentrations with substrate-specific affinity, suggesting direct calcium regulation of mitochondrial fatty acid oxidation. These unbiased, proteomic analyses of calcium effectors establish a key resource to dissect cation engagement and its mechanistic effects across multiple species and diverse biological processes.