title: Analysis of protein tyrosine phosphatase interactions with microarrayed phosphopeptide substrates using imaging mass spectrometry.
abstract: 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.
authors.affiliation: U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA. Electronic address: mckee.473@buckeyemail.osu.edu.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: McKee
authors.forename: Harry B
authors.initials: HB
authors.lastname: Hines
authors.forename: Robert G
authors.initials: RG
authors.lastname: Ulrich
mesh_terms: D057166:High-Throughput Screening Assays; D013058:Mass Spectrometry; D010748:Phosphopeptides / Q000737:chemistry*; D040081:Protein Array Analysis; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D015010:Yersinia pestis / Q000201:enzymology
chemical_list: D010748:Phosphopeptides; D011994:Recombinant Proteins; D017027:Protein Tyrosine Phosphatases
keywords: Imaging mass spectrometry; MALDI; Peptide microarray; Phosphatase assay; SPRi; YopH
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