The cAMP Capture Compound Mass Spectrometry as a Novel Tool for Targeting cAMP-binding Proteins: FROM PROTEIN KINASE A TO POTASSIUM/SODIUM HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATED CHANNELS

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 Compound^TM 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.cAMP is an important biological second messenger molecule involved in many biological processes, such as adaptation of bacteria to low glucose growing conditions, chemotaxis in slime molds, and various signal transduction processes in metazoa downstream of the activation of hormone receptors (1). The concentration level of cAMP in biological systems is tightly controlled by the activity of adenylyl cyclases that catalyze the formation of cAMP and by the activity of phosphodiesterases, which catalyze the degradation of cAMP. Given the importance of signaling cascades downstream of hormone or neurotransmitter receptors that involve increased formation or degradation of cAMP, the identification and profiling of cAMP effector proteins can be expected to be an essential contribution to elucidate the molecular basis of physiological as well as pathophysiological signaling events.Bona fide effectors of cAMP are proteins that contain a cyclic nucleotide binding domain (CNBD).1 This motif represents a protein domain initially defined and characterized by the crystal structure of the major known cAMP-binding protein from Escherichia coli, the catabolite gene activator protein (2). This domain is present in all known mammalian cAMP-binding proteins as well. Three major classes of proteins exist that contain CNBDs. The first group contains protein kinase A subunits, namely regulatory subunits of protein kinase A isozymes (3), as well as the cGMP-dependent protein kinases (4). A group of Rap guanine nucleotide exchange factors (Epac proteins) that contain CNBDs (5) comprises the second group. Both groups contain key proteins involved in signaling cascades. A number of ion channels that can be directly regulated by cAMP contain CNBDs, such as the cyclic nucleotide-gated channels (6), make up the third group. In particular, potassium/sodium hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a crucial role in the pacemaking of heart and brain activity (7). A relatively small number of further proteins that contain CNBDs, such as phosphodiesterase isoforms and a sodium-hydrogen exchange transporter, can be retrieved from searches in databases such as Swiss-Prot.Among the methodological repertoire applied in functional proteomics, small molecule affinity-based techniques seem to be ideal for the task of profiling the cAMP binding proteome subset. Established strategies make use of cAMP affinity beads. These beads comprise cAMP derivatives covalently attached to the polymer backbone via an aminoalkyl linker. The linker may vary in length of the alkyl chain and in the attachment position at the nucleobase (8, 9). This approach, however, suffers from the relatively large amount of protein input required to obtain significant data, precluding e.g. the profiling of the target proteins in samples of limited abundance. Furthermore, it has not been demonstrated yet that affinity-based enrichment of cAMP-binding proteins is suitable for cAMP-binding membrane proteins that are known to be difficult to access. On the other hand, soluble cAMP- and cGMP-binding proteins along with their interaction partners were robustly identified with this methodology. Another approach described in the literature used a cyclic guanosine monophosphate analogue immobilized on a Biacore chip to isolate cGMP- and cAMP-binding proteins from a cell lysate, estimate the quantity of the material, and elute proteins for proteolysis and identification by LC-MS/MS. In addition, for single purified proteins, binding constants can be measured (10). The applicability of this approach to transmembrane cGMP/cAMP-binding proteins, however, has yet to be determined.Here we describe the synthesis and application of a trifunctional Capture Compound^TM (CC) (see Fig. 1A) as a novel approach for the functional isolation of cAMP-binding proteins from complex protein mixtures using low amounts of protein input. In contrast to current pulldown approaches, the CC enables the covalent linkage to the target proteins by a photoactivatable reactivity group in addition to the reversible binding of target proteins by the selectivity group. The Capture Compound-protein conjugate can be isolated from the complex protein mixture via the sorting function (a biotin moiety) of the Capture Compound by means of streptavidin-coated magnetic beads (see Fig. 1, B and C) (11). The cAMP-binding protein-selective Capture Compound described here was successfully applied to the isolation of cAMP-binding proteins from E. coli lysate and cultured eukaryotic HepG2 cells, respectively. Furthermore, we report the applicability of the CCMS approach for the capturing of cAMP-binding HCN channel proteins from rat brain synaptosome preparations as well. To our knowledge, this has not yet been achieved by any cAMP affinity bead approach. In addition, the ion channels, which by antibody- and in situ hybridization-based techniques have been shown to be located in neuronal tissues at synaptic sites (12, 13), have also escaped detection in many detailed proteomics profiling studies conducted to establish the protein complements of synaptic structures (see e.g. Refs. 14–17). Our data suggest that the cAMP-CC approach is uniquely efficient and sensitive for the identification and profiling of cAMP-binding proteins in complex protein mixtures.Open in a separate windowFig. 1.A, schematic design of a CC. Three functionalities are coupled to a core. The selectivity function (red), e.g. modified cAMP, for target recognition; the reactivity function (orange), e.g. diazirines, for covalent cross-linking; and the sorting function (yellow), e.g. biotin, for pullout of captured proteins; and a variable linker (green) that can modify the hydrophilicity of the system are shown. B, structure of 8-AHA-cAMP-CC (7c), which represents one of several cAMP-CCs that are available. C, flow chart of the CCMS technology.