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Chemical Proteomics

Volume 803 of the series Methods in Molecular Biology pp 97-125

Date:

Profiling of Methyltransferases and Other S-Adenosyl-l-Homocysteine-Binding Proteins by Capture Compound Mass Spectrometry

  • Thomas LenzAffiliated withcaprotec bioanalytics GmbH
  • , Peter PootAffiliated withInstitute of Organic Chemistry, RWTH University
  • , Elmar WeinholdAffiliated withInstitute of Organic Chemistry, RWTH University
  • , Mathias DregerAffiliated withcaprotec bioanalytics GmbH Email author 

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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.

Key words

Capture Compound Photo-crosslink Small molecule–protein interaction Methyltransferase S-Adenosyl-l-homocysteine SAH- AdoHcy S-Adenosyl-l-methionine SAM AdoMet Functional proteo­mics LC-MS/MS