title: Profiling of methyltransferases and other S-Adenosyl-L-homocysteine-binding proteins by Capture Compound mass spectrometry.
abstract: There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions. We describe a generic approach based on trifunctional Capture Compounds, in which the initial equilibrium-driven interaction between a small molecule probe and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function of the Capture Compound and the surface of the target protein(s). Subsequently, Capture Compound - protein conjugates are isolated from complex biological mixtures via the sorting function of the Capture Compound. Here, we describe the application of a trifunctional Capture Compound that carries the methyltransferase product inhibitor S-Adenosyl-L -homocysteine as the selectivity function for the isolation of methyltransferases from a complex lysate of Escherichia coli DH5α cells. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free S-Adenosyl-L -homocysteine.
authors: Caprotec Bioanalytics GmbH, Berlin, Germany.
authors: Thomas
authors: T
authors: Lenz
authors: Peter
authors: P
authors: Poot
authors: Elmar
authors: E
authors: Weinhold
authors: Mathias
authors: M
authors: Dreger
mesh_terms: D001331:Automation; D002853:Chromatography, Liquid; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D013058:Mass Spectrometry / Q000379:methods*; D008780:Methyltransferases / Q000378:metabolism*; D036103:Nanotechnology; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D012435:S-Adenosylhomocysteine / Q000378:metabolism*; D016622:Silver Staining; D014357:Trypsin / Q000378:metabolism
chemical_list: D010455:Peptides; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; D014357:Trypsin