A NIMS enzyme assay

Schematic of the Nimzyme assay. (a) Substrates are immobilized in the fluorous phase of the NIMS surface. (b) The surface is incubated with a crude cell lysate. (c) After washing, the surface is analyzed by NIMS. Scale bar = 2 µm. (Adapted with permission. Copyright 2008 National Academy of Sciences, U.S.A.)
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Gary Siuzdak, Chi-Huey Wong, and colleagues at Scripps Research Institute, the University of California Merced, and Lawrence Berkeley National Laboratory report a method to study the enzymatic activity of complex biological samples by using nanostructure initiator MS (NIMS). NIMS is a laser desorption/ionization technique in which analytes are adsorbed from liquid-coated nanostructured surfaces.

The researchers call their new technique the Nimzyme assay; in it, enzyme substrates are attached via a fluorinated tag to the fluorous phase, a unique material that is immiscible with aqueous and organic phases. A crude cell lysate is flooded onto the surface, allowed to react, and then washed away before NIMS analysis.

The researchers used the Nimzyme assay to look at two enzymatic reactions: hydrolysis of lactose by β-1,4-galactosidase and modification of the same substrate by α-2,3-sialyltransferase. They first analyzed crude E. coli lysates and found that they could obtain relatively clean mass spectra after reaction of their immobilized substrates with the lysate.

The scientists also used their Nimzyme assay to analyze the crude lysate of a thermophilic microbial community obtained from a hot spring in Yellowstone National Park. They could determine the optimal pH and temperature of the galactosidase reaction.

The researchers note that their assay has sensitivity comparable to more traditional fluorescent enzyme assays and that their technique can be extended to study a multitude of other enzymatic activities. (Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 3678–3683)