Optimization of calmodulin-affinity chromatography for brain and organelles

Publication date: Available online 23 July 2015 Source:EuPA Open Proteomics Author(s): Katarzyna Kulej, Simone Sidoli, Giuseppe Palmisano, Alistair V.G. Edwards, Phillip J. Robinson, Martin R. Larsen 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 3,529 putative mouse brain CaM-BPs, of which 1,629 were integral membrane or membrane-associated. Among them, 170 proteins were known CaM-BPs or previously reported as potential CaM-BPs while 696 contained pre...

Source: EuPA Open Proteomics - July 23, 2015 Category: Bioinformatics Source Type: research