A nanostructure-initiator mass spectrometry-based enzyme activity assay

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 surfacewashing 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 ␤-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-␤-D-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced ␥-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.fluorous phase ͉ microbial community ͉ carbohydrate

From Soil to Structure, a Novel Dimeric β-Glucosidase Belonging to Glycoside Hydrolase Family 3 Isolated from Compost Using Metagenomic Analysis

“…The hydrolysis of cellobiose, cellotetraose, and maltose by the wild-type ␤-glucosidase and the three mutants was further qualitatively analyzed by nanostructure-initiator mass spectrometry (10)-based enzyme activity assays (Nimzyme) (11,12). The principle of Nimzyme has been described previously (11,12).…”

“…The principle of Nimzyme has been described previously (11,12). In brief, hydrolysis reactions were measured at 50°C in 25 mM MES buffer (pH 6.5).…”

Chemistry meets nutrition: Toward a systems biological description of human metabolism

“…However, many obstacles to metabolite identification and quantification still persist [21][22][23]. For example, the complexity and inhomogeneity of crude cell and tissue lysates impede detection of reaction products, surmounting these often demands additional chromatographic steps that lower sensitivity and throughput of the analysis.…”

Ionic matrix for matrix-enhanced surface-assisted laser desorption ionization mass spectrometry imaging (ME-SALDI-MSI)

“…While the loss of matrix under vacuum does not necessarily impose a problem in MALDI profiling experiments where most measurements occur in seconds, it cannot be ignored during MS imaging experiments because of extended interrogation time and the preferred solvent-free approach for matrix deposition [7]. For example, for a commercial MALDI-TOF instrument equipped with a 20-Hz N 2 laser, approximately 720 pixels can be imaged in 1 h when 100 laser shots are averaged per pixel, which translates to ϳ55.6 h of instrument time to examine a 1-cm 2 tissue section with spatial resolution of 50 m, not counting the time spent for translational stage movement. Even with a high pulse rate laser installed, the interrogation time for most imaging experiments is sufficiently long to experience the impacts of matrix loss, most notably the inevitable change in ionization efficiency across tissue that skews the actual spatial distribution of chemical composition in the sample imaged.…”

“…S urface-assisted laser desorption ionization mass spectrometry (SALDI-MS) has been used as a complementary tool to matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in small molecule profiling [1,2]. Its applications in tissue imaging have been demonstrated by various groups where porous surfaces or inorganic nanoparticles have been successfully utilized as the energy mediating reagents [3][4][5].…”