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Parallel determination of multiple protein metabolite interactions using cell extract, protein microarrays and mass spectrometric detection

Authors

  • Victor N. Morozov,

    Corresponding author
    1. Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia
    • Beckman Institute for Biomedical Research, 28835 Single Oak Drive, Temecula, CA 92590, USA.
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  • Tamara Ya. Morozova,

    1. Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia
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  • Kenneth L. Johnson,

    1. Biomedical Mass Spectrometry and Functional Proteomics Facility and Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905, USA
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  • Stephen Naylor

    1. Biomedical Mass Spectrometry and Functional Proteomics Facility and Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905, USA
    2. Department of Molecular Pharmacology and Experimental Therapeutics and Clinical Pharmacology Unit and Division of Biomedical Engineering, Mayo Clinic and Foundation, Rochester, MN 55905, USA
    Current affiliation:
    1. Beyond Genomics, 40 Bear Hill Road, Waltham, MA 02451, USA.
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Abstract

Analysis of interactive networks between proteins and other molecular constituents is of paramount importance to delineate complex cellular processes. In order to facilitate this process, new technologies that allow rapid, high-throughput parallel screening, as well as identification of constituents, are necessary. A particularly powerful combination in this regard could be the use of multiprotein microarrays coupled with mass spectrometry (MS). In the initial step of the method development we applied MS to single-protein microarrays. We demonstrated that even a simplified version of the method allows rapid parallel label-free assay of specific protein interactions with multiple metabolites derived from complex artificial and natural mixtures. The microarrays fabricated by the electrospray deposition technique and cross-linked in glutaraldehyde vapor were brought into contact with droplets of solution containing either a natural extract of baker's yeast cells or an artificial cocktail of metabolites. After washing, the microarrays were placed into 75% methanol to denature proteins and release specifically bound metabolites. The eluates were then analyzed by electrospray ionization mass spectrometry (ESI-MS) to simultaneously detect all the metabolites bound. Such a procedure applied to ten different proteins demonstrated that 50–400 ng of cross-linked protein is enough to obtain ion intensities from metabolites that are well distinguishable above noise. The compatibility of microplates and different microarray designs with MS detection is discussed. Copyright © 2003 John Wiley & Sons, Ltd.

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