Label-free detection of protein-protein interactions on biochips.
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Gold↗
Biomedical subjects
Dal-Hee Min
Publications and source records attributed to Dal-Hee Min.
Profiling kinase activities by using a peptide chip and mass spectrometry.
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Gold↗
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.
Enzymes, Immobilized↗
Chemical screening by mass spectrometry to identify inhibitors of anthrax lethal factor.
Mass spectrometry (MS) analysis is applicable to a broad range of biological analytes and has the important advantage that it does not require analytes to be labeled. A drawback of MS methods, however, is the need for chromatographic steps to prepare the analyte, precluding MS from being used in chemical screening and rapid analysis. Here, we report that surfaces that are chemically tailored for characterization by matrix-assisted laser-desorption ionization time-of-flight MS eliminate the need for sample processing and make this technique adaptable to parallel screening experiments. The tailored substrates are based on self-assembled monolayers that present ligands that interact with target proteins and enzymes. We apply this method to screen a chemical library against protease activity of anthrax lethal factor, and report a compound that inhibits lethal factor activity with a K(i) of 1.1 microM and blocks the cleavage of MEK1 in 293 cells.
Antigens, Bacterial↗
Peptide arrays: towards routine implementation.
Peptide arrays have attracted wide interest as tools for discovering biochemical interactions. Because many protein binding and enzyme activities are directed towards peptides, the preparation of arrays having hundreds to thousands of immobilized peptides offers an unprecedented opportunity for identifying interactions of proteins. This short opinion reviews recent progress in the preparation of peptide arrays and their use in the characterization of biomolecular interactions and the discovery of new reagents for biological researches. This body of work establishes the feasibility of this technology and suggests that it will find much wider use in research groups.
Biotechnology↗
Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands.
This paper describes a method for the selective and covalent immobilization of proteins to surfaces with control over the density and orientation of the protein. The strategy is based on binding of the serine esterase cutinase to a self-assembled monolayer presenting a phosphonate ligand and the subsequent displacement reaction that covalently binds the ligand to the enzyme active site. Surface plasmon resonance (SPR) spectroscopy showed that cutinase binds irreversibly to a monolayer presenting the capture ligand at a density of 1% mixed among tri(ethylene glycol) groups. The covalent immobilization is specific for cutinase, and the glycol-terminated monolayer effectively prevents unwanted nonspecific adsorption of proteins. To demonstrate that the method could be used to immobilize proteins of interest, a cutinase-calmodulin fusion protein was constructed and immobilized to the monolayer. SPR showed that calcineurin selectively associated with the immobilized calmodulin. This capture ligand immobilization method combines the advantages that the immobilization reaction is highly selective for the intended protein, the tether is covalent and, hence, stable, and the method avoids the need for synthetic modification and rigorous purification of proteins before immobilization. These characteristics make the method well suited to a range of applications and, in particular, for constructing protein microarrays.
Binding Sites↗