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 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 microg), 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.
Luo
Yan
Y
GmbH, Volmerstrasse 5, 12489 Berlin, Germany.
Blex
Christian
C
Baessler
Olivia
O
Glinski
Mirko
M
Dreger
Mathias
M
Sefkow
Michael
M
Köster
Hubert
H
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002457:Cell Extracts; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005454:Fluorescence Polarization; D056945:Hep G2 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats; D016622:Silver Staining; D012964:Sodium / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism
D002457:Cell Extracts; D054815:Cyclic Nucleotide-Gated Cation Channels; D012964:Sodium; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases