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A nanostructure-initiator mass spectrometry-based enzyme activity assay

Academic Article
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Overview

authors

  • Northen, T. R.
  • Lee, Jiing-Dwan
  • Hoang, L.
  • Raymond, J.
  • Hwang, D. R.
  • Yannone, S. M.
  • Wong, Chi-Huey
  • Siuzdak, Gary

publication date

  • March 2008

journal

  • Proceedings of the National Academy of Sciences of the United States of America  Journal

abstract

  • We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorous-phase interactions. This "soft" immobilization allows efficient desorption/ionization while also enabling the use of surface-washing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is sensitive to subpicogram levels of enzyme, detects both addition and cleavage reactions (sialyltransferase and galactosidase), is applicable over a wide range of pHs and temperatures, and can measure activity directly from crude cell lysates. The ability of the Nimzyme assay to analyze complex mixtures is illustrated by identifying and directly characterizing beta-1,4-galactosidase activity from a thermophilic microbial community lysate. The optimal enzyme temperature and pH were found to be 65 degrees C and 5.5, respectively, and the activity was inhibited by both phenylethyl-beta-d-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced gamma-proteobacterium. In general, this assay provides an efficient method for detection and characterization of enzymatic activities in complex biological mixtures prior to sequencing or cloning efforts. More generally, this approach may have important applications for screening both enzymatic and inhibitor libraries, constructing and screening glycan microarrays, and complementing fluorous-phase organic synthesis.

subject areas

  • Bacteria
  • Biological Assay
  • Enzyme Inhibitors
  • Humans
  • Hydrogen-Ion Concentration
  • Mass Spectrometry
  • Microchip Analytical Procedures
  • Nanostructures
  • Sialyltransferases
  • Substrate Specificity
  • Temperature
  • beta-Galactosidase
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Research

keywords

  • carbohydrate
  • fluorous phase
  • microbial community
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Identity

PubMed Central ID

  • PMC2268803

International Standard Serial Number (ISSN)

  • 0027-8424

Digital Object Identifier (DOI)

  • 10.1073/pnas.0712332105

PubMed ID

  • 18319341
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Additional Document Info

start page

  • 3678

end page

  • 3683

volume

  • 105

issue

  • 10

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