An Assay Based on SAMDI Mass Spectrometry for Profiling Protein Interaction Domains

Patrick T. O'Kane, Milan Mrksich*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


This paper describes an assay that can profile the binding of a protein to ligands and can rank the affinities of a library of ligands. The method is based on the enhanced rate of an enzyme-mediated reaction that follows from colocalization of the enzyme and substrate by a protein-ligand interaction. This assay uses a self-assembled monolayer that presents a candidate peptide ligand for a receptor and a peptide substrate for an enzyme. The receptor is prepared as a fusion to the relevant enzyme so that binding of the receptor to the immobilized ligand brings the enzyme to the surface, where it can more rapidly modify its substrate. The extent of conversion of the substrate to product is therefore a measure of the average time the ligand-receptor complex is present and is quantified using the SAMDI mass spectrometry technique. The approach is used to profile the binding of chromodomain proteins to methylated lysine peptides derived from the histone 3 protein. The relative affinities for the peptide ligands found in this work agreed with results from prior studies. Additionally, this work revealed cross-talk interactions whereby phosphorylation of certain residues impaired binding of chromodomains to the peptide ligands. The method presented here, which we term protein interaction by SAMDI (PI-SAMDI), has the advantages that it is applicable to low-affinity interactions because the complexes are not observed directly, but rather leave a "covalent record" of the interaction that is measured with mass spectrometry and because it is compatible with laboratory automation for high-throughput analysis.

Original languageEnglish (US)
Pages (from-to)10320-10327
Number of pages8
JournalJournal of the American Chemical Society
Issue number30
StatePublished - Aug 2 2017

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


Dive into the research topics of 'An Assay Based on SAMDI Mass Spectrometry for Profiling Protein Interaction Domains'. Together they form a unique fingerprint.

Cite this