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In JoVE (1)
- Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)
Other Publications (2)
Articles by Peter Poot in JoVE
Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)
Thomas Lenz1, Peter Poot2, Olivia Gräbner1, Mirko Glinski1, Elmar Weinhold2, Mathias Dreger1, Hubert Köster1
1Department of Biochemistry / Analytics, caprotec bioanalytics GmbH, 2Institute of Organic Chemistry, RWTH Aachen University
Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.
Other articles by Peter Poot on PubMed
Synthesis of S-adenosyl-L-homocysteine Capture Compounds for Selective Photoinduced Isolation of Methyltransferases
Chembiochem : a European Journal of Chemical Biology. Jan, 2010 | Pubmed ID: 20049756
Understanding the interplay of different cellular proteins and their substrates is of major interest in the postgenomic era. For this purpose, selective isolation and identification of proteins from complex biological samples is necessary and targeted isolation of enzyme families is a challenging task. Over the last years, methods like activity-based protein profiling (ABPP) and capture compound mass spectrometry (CCMS) have been developed to reduce the complexity of the proteome by means of protein function in contrast to standard approaches, which utilize differences in physical properties for protein separation. To isolate and identify the subproteome consisting of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (methylome), we developed and synthesized trifunctional capture compounds containing the chemically stable cofactor product S-adenosyl-L-homocysteine (SAH or AdoHcy) as selectivity function. SAH analogues with amino linkers at the N6 or C8 positions were synthesized and attached to scaffolds containing different photocrosslinking groups for covalent protein modification and biotin for affinity isolation. The utility of these SAH capture compounds for selective photoinduced protein isolation is demonstrated for various methyltransferases (MTases) acting on DNA, RNA and proteins as well as with Escherichia coli cell lysate. In addition, they can be used to determine dissociation constants for MTase-cofactor complexes.
Profiling of Methyltransferases and Other S-Adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry
Methods in Molecular Biology (Clifton, N.J.). 2012 | Pubmed ID: 22065221
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.