A nanostructure-initiator mass spectrometry-based enzyme activity assay.
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.
Northen
Trent R
TR
Department of Molecular Biology and Scripps Center for Mass Spectrometry and Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Lee
Jinq-Chyi
JC
Hoang
Linh
L
Raymond
Jason
J
Hwang
Der-Ren
DR
Yannone
Steven M
SM
Wong
Chi-Huey
CH
Siuzdak
Gary
G
D001419:Bacteria / Q000187:drug effects / Q000201:enzymology; D001681:Biological Assay / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D013058:Mass Spectrometry / Q000379:methods*; D046208:Microchip Analytical Procedures; D049329:Nanostructures; D012799:Sialyltransferases / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D001616:beta-Galactosidase / Q000378:metabolism*
D004791:Enzyme Inhibitors; D012799:Sialyltransferases; D001616:beta-Galactosidase