A method for connecting solution-phase enzyme activity assays with immobilized format analysis by mass spectrometry.
This paper reports an enzyme activity assay that combines the assets of both homogeneous and solid-phase formats. In this method, enzyme reactions are carried out in solution using substrates that are tagged with an immobilization reagent that allows the substrates to be selectively immobilized to self-assembled monolayers (SAMs), for direct analysis by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). As a model enzyme reaction, this work examined the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to an arginine side chain of a peptide substrate by the enzyme protein arginine methyltransferase 1 (RMT1). A cysteine-terminated peptide substrate was methylated by RMT1 in solution and then applied to a maleimide-presenting SAM to give selective immobilization of the peptide. Time-dependent analysis of methylation using MALDI-TOFMS clearly showed that both the presence and relative amount of the two reaction products-the mono- and dimethylated peptides-can be conveniently evaluated. This assay strategy is rapid, takes advantage of solution-phase assay conditions, avoids the use of labels and complicated purification steps, and is applicable to multianalyte analyses.
Min
Dal-Hee
DH
Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 5735 South Ellis Avenue, Chicago, Illinois 60637, USA.
Yeo
Woon-Seok
WS
Mrksich
Milan
M
D004800:Enzymes, Immobilized / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D044367:Phase Transition; D011484:Protein-Arginine N-Methyltransferases / Q000737:chemistry*; D012436:S-Adenosylmethionine / Q000378:metabolism; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors
D004800:Enzymes, Immobilized; D012996:Solutions; D012436:S-Adenosylmethionine; D011484:Protein-Arginine N-Methyltransferases