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Abstract
The profiling of subproteomes from complex mixtures on the basis of small molecule interactions shared by members of protein families or small molecule interaction domains present in a subset of proteins is an increasingly important approach in functional proteomics. Capture CompoundTM Mass Spectrometry (CCMS) is a novel technology to address this issue. CCs are trifunctional molecules that accomplish the reversible binding of target protein families to a selectivity group (small molecule), covalent capturing of the bound proteins by photoactivated cross-linking through a reactivity group, and pullout of the small molecule-protein complexes through a sorting function, e.g. biotin. Here we present the design, synthesis, and application of a new Capture Compound to target and identify cAMP-binding proteins in complex protein mixtures. Starting with modest amounts of total protein mixture (65–500 μg), we demonstrate that the cAMP-CCs can be used to isolate bona fide cAMP-binding proteins from lysates of Escherichia coli, mammalian HepG2 cells, and subcellular fractions of mammalian brain, respectively. The identified proteins captured by the cAMP-CCs range from soluble cAMP-binding proteins, such as the catabolite gene activator protein from E. coli and regulatory subunits of protein kinase A from mammalian systems, to cAMP-activated potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channels from neuronal membranes and specifically synaptosomal fractions from rat brain. The latter group of proteins has never been identified before in any small molecule protein interaction and mass spectrometry-based proteomics study. Given the modest amount of protein input required, we expect that CCMS using the cAMP-CCs provides a unique tool for profiling cAMP-binding proteins from proteome samples of limited abundance, such as tissue biopsies.
Highlights
The profiling of subproteomes from complex mixtures on the basis of small molecule interactions shared by members of protein families or small molecule interaction domains present in a subset of proteins is an increasingly important approach in functional proteomics
We report the applicability of the Capture CompoundTM Mass Spectrometry (CCMS) approach for the capturing of cAMP-binding hyperpolarization-activated cyclic nucleotide-gated (HCN) channel proteins from rat brain synaptosome preparations as well
Chemistry and Stability Characteristics of cAMP-Capture CompoundTM (CC)— Capture Compounds are trifunctional small organic molecules that can be used for equilibrium binding of target proteins to the selectivity group, subsequent covalent capture of the target protein via a photoactivatable reactivity group, and pullout of the Capture Compound-protein conjugates via a sorting function, e.g. biotin
Summary
All standard chemical reagents and solvents were obtained from commercial suppliers and were used without further purification. For on-bead experiments, 25 l of cAMP-CC (100 M) were incubated with 50 l of Dynabeadsா MyOneTM Streptavidin C1 (Invitrogen Dynal) in a 0.2-ml PCR tube with flat cap (Thermo Scientific) at room temperature for 30 min. For off-bead experiments, 10 l of cAMP-CCs (100 M) were mixed with 500 g of cell lysate and 20 l of 5ϫ concentrated capture buffer in a 0.2-ml PCR tube with flat cap (Thermo Scientific). The capture experiments in the rat brain synaptosomal fractions preparations (for preparation, see below) were performed to the capturing in E. coli and HepG2 lysate with slight modifications with respect to protein concentration and WB: ϳ65 g of protein were applied in capture experiments, and 0.1% n-dodecyl-maltoside (Glycon, Luckenwalde, Germany) was added to the capture and the wash buffer
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