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Analysis of protein tyrosine phosphatase interactions with microarrayed phosphopeptide substrates using imaging mass spectrometry
- McKee, Christopher J., Hines, Harry B., Ulrich, Robert G.
- Analytical biochemistry 2013 v.442 no.1 pp. 62-67
- desorption, enzyme activity, gold, image analysis, ionization, mass spectrometry, microarray technology, protein-protein interactions, protein-tyrosine-phosphatase, recombinant proteins, surface plasmon resonance, tyrosine
- Microarrays of peptide and recombinant protein libraries are routinely used for high-throughput studies of protein–protein interactions and enzymatic activities. Imaging mass spectrometry (IMS) is currently applied as a method to localize analytes on thin tissue sections and other surfaces. Here, we have applied IMS as a label-free means to analyze protein–peptide interactions in a microarray-based phosphatase assay. This IMS strategy visualizes the entire microarray in one composite image by collecting a predefined raster of matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry spectra over the surface of the chip. Examining the bacterial tyrosine phosphatase YopH, we used IMS as a label-free means to visualize enzyme binding and activity with a microarrayed phosphopeptide library printed on chips coated with either gold or indium–tin oxide. Furthermore, we demonstrate that microarray-based IMS can be coupled with surface plasmon resonance imaging to add kinetic analyses to measured binding interactions. The method described here is within the capabilities of many modern MALDI–TOF instruments and has general utility for the label-free analysis of microarray assays.