Optimization of calmodulin-affinity chromatography for brain and organelles

Abstract

Calmodulin (CaM) is a Ca²⁺-binding signaling protein that binds to and activates many target proteins, known as calmodulin-binding proteins (CaM-BPs). They are involved in multiple cellular processes. Despite the diversity and importance of CaM-BPs, many remain to be identified and characterized. We performed extensive optimization of a CaM-affinity capture method, using commercial CaM-chromatographic material. We identify both the Ca²⁺-dependent and -independent CaM binding proteomes in both mouse brain and in rat brain neuronal organelles, synaptosomes, and compared cytosolic with membrane associated targets. Fractionation of peptides, derived from on-resin tryptic digestion, using hydrophilic interaction liquid chromatography (HILIC) was combined with reversed-phase liquid chromatography tandem mass spectrometry (LC-MS/MS) to improve identification of low abundance CaM-BPs in a reproducible and sensitive manner. Various detergents were tested for the most efficient membrane protein solubilization for pull-down of membrane-associated CaM-BPs. We identified 3529 putative mouse brain CaM-BPs, of which 1629 were integral membrane or membrane-associated. Among them, 170 proteins were known CaM-BPs or previously reported as potential CaM-BPs while 696 contained predicted CaM binding motifs. In synaptosomes we identified 2698 CaM-BPs and 2783 unique phosphopeptides derived from 984 of the potential synaptosomal CaM-BPs. Overall, our improved workflow provides unmatched sensitivity for the identification of the CaM binding proteome and its associated phosphoproteome and this now enables sensitive analysis of organelle-specific CaM-BPs.