A nanostructure-initiator mass spectrometry-based enzyme activity assay

Trent R. Northen, Jinq Chyi Lee, Linh Hoang, Jason Raymond, Der Ren Hwang, Steven M. Yannone, Chi Huey Wong, Gary Siuzdak

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106 Scopus citations

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 fluorousphase 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 β-1,4-galactosidase activity from a thermophilic microbial community lysate. The optimal enzyme temperature and pH were found to be 65°C and 5.5, respectively, and the activity was inhibited by both phenylethyl-β-D-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced γ-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.

Original languageEnglish (US)
Pages (from-to)3678-3683
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number10
DOIs
StatePublished - Mar 25 2008

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Keywords

  • Carbohydrate
  • Fluorous phase
  • Microbial community

ASJC Scopus subject areas

  • General

Cite this

Northen, T. R., Lee, J. C., Hoang, L., Raymond, J., Hwang, D. R., Yannone, S. M., Wong, C. H., & Siuzdak, G. (2008). A nanostructure-initiator mass spectrometry-based enzyme activity assay. Proceedings of the National Academy of Sciences of the United States of America, 105(10), 3678-3683. https://doi.org/10.1073/pnas.0712332105