Use of Nanostructure-Initiator Mass Spectrometry to Deduce Selectivity of Reaction in Glycoside Hydrolases.
Ontology highlight
ABSTRACT:
SUBMITTER: Deng K
PROVIDER: S-EPMC4621489 | biostudies-literature | 2015
REPOSITORIES: biostudies-literature
Publications
Use of Nanostructure-Initiator Mass Spectrometry to Deduce Selectivity of Reaction in Glycoside Hydrolases.
Deng Kai K Takasuka Taichi E TE Bianchetti Christopher M CM Bergeman Lai F LF Adams Paul D PD Northen Trent R TR Fox Brian G BG
Frontiers in bioengineering and biotechnology 20151027
Chemically synthesized nanostructure-initiator mass spectrometry (NIMS) probes derivatized with tetrasaccharides were used to study the reactivity of representative Clostridium thermocellum β-glucosidase, endoglucanases, and cellobiohydrolase. Diagnostic patterns for reactions of these different classes of enzymes were observed. Results show sequential removal of glucose by the β-glucosidase and a progressive increase in specificity of reaction from endoglucanases to cellobiohydrolase. Time-depe ...[more]
Similar Datasets
Project description: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.
| S-EPMC2268803 | biostudies-literature
Project description:In a variety of neurological diseases, pathological progression is cell type and region specific. Previous reports suggest that mass spectrometry imaging has the potential to differentiate between brain regions enriched in specific cell types. Here, we utilized a matrix-free surface mass spectrometry approach, nanostructure initiator mass spectrometry (NIMS), to show that spatial distributions of multiple lipids can be used as a 'fingerprint' to discriminate between neuronal- and glial- enriched brain regions. In addition, glial cells from different brain regions can be distinguished based on unique lipid profiles. NIMS images were generated from sagittal brain sections and were matched with immunostained serial sections to define glial cell enriched areas. Tandem mass spectrometry (LC-MS/MS QTOF) on whole brain extracts was used to identify 18 phospholipids. Multivariate statistical analysis (Nonnegative Matrix Factorization) enhanced differentiation of brain regions and cell populations compared to single ion imaging methods. This analysis resolved brain regions that are difficult to distinguish using conventional stains but are known to have distinct physiological functions. This method accurately distinguished the frontal (or somatomotor) and dorsal (or retrosplenial) regions of the cortex from each other and from the pons region.
| S-EPMC3698601 | biostudies-literature
Project description:Harnessing the biotechnological potential of the large number of proteins available in sequence databases requires scalable methods for functional characterization. Here we propose a workflow to address this challenge by combining phylogenomic guided DNA synthesis with high-throughput mass spectrometry and apply it to the systematic characterization of GH1 ?-glucosidases, a family of enzymes necessary for biomass hydrolysis, an important step in the conversion of lignocellulosic feedstocks to fuels and chemicals. We synthesized and expressed 175 GH1s, selected from over 2000 candidate sequences to cover maximum sequence diversity. These enzymes were functionally characterized over a range of temperatures and pHs using nanostructure-initiator mass spectrometry (NIMS), generating over 10,000 data points. When combined with HPLC-based sugar profiling, we observed GH1 enzymes active over a broad temperature range and toward many different ?-linked disaccharides. For some GH1s we also observed activity toward laminarin, a more complex oligosaccharide present as a major component of macroalgae. An area of particular interest was the identification of GH1 enzymes compatible with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), a next-generation biomass pretreatment technology. We thus searched for GH1 enzymes active at 70 °C and 20% (v/v) [C2mim][OAc] over the course of a 24-h saccharification reaction. Using our unbiased approach, we identified multiple enzymes of different phylogentic origin with such activities. Our approach of characterizing sequence diversity through targeted gene synthesis coupled to high-throughput screening technologies is a broadly applicable paradigm for a wide range of biological problems.
| S-EPMC4168791 | biostudies-literature
Project description:BackgroundTissue imaging of treatment-induced metabolic changes is useful for optimizing cancer therapies, but commonly used methods require trade-offs between assay sensitivity and spatial resolution. Nanostructure-Initiator Mass Spectrometry imaging (NIMS) permits quantitative co-localization of drugs and treatment response biomarkers in cells and tissues with relatively high resolution. The present feasibility studies use NIMS to monitor phosphorylation of 3'-deoxy-3'-fluorothymidine (FLT) to FLT-MP in lymphoma cells and solid tumors as an indicator of drug exposure and pharmacodynamic responses.MethodsNIMS analytical sensitivity and spatial resolution were examined in cultured Burkitt's lymphoma cells treated briefly with Rapamycin or FLT. Sample aliquots were dispersed on NIMS surfaces for single cell imaging and metabolic profiling, or extracted in parallel for LC-MS/MS analysis. Docetaxel-induced changes in FLT metabolism were also monitored in tissues and tissue extracts from mice bearing drug-sensitive tumor xenografts. To correct for variations in FLT disposition, the ratio of FLT-MP to FLT was used as a measure of TK1 thymidine kinase activity in NIMS images. TK1 and tumor-specific luciferase were measured in adjacent tissue sections using immuno-fluorescence microscopy.ResultsNIMS and LC-MS/MS yielded consistent results. FLT, FLT-MP, and Rapamycin were readily detected at the single cell level using NIMS. Rapid changes in endogenous metabolism were detected in drug-treated cells, and rapid accumulation of FLT-MP was seen in most, but not all imaged cells. FLT-MP accumulation in xenograft tumors was shown to be sensitive to Docetaxel treatment, and TK1 immunoreactivity co-localized with tumor-specific antigens in xenograft tumors, supporting a role for xenograft-derived TK1 activity in tumor FLT metabolism.ConclusionsNIMS is suitable for monitoring drug exposure and metabolite biotransformation with essentially single cell resolution, and provides new spatial and functional dimensions to studies of cancer metabolism without the need for radiotracers or tissue extraction. These findings should prove useful for in vitro and pre-clinical studies of cancer metabolism, and aid the optimization of metabolism-based cancer therapies and diagnostics.
| S-EPMC3834492 | biostudies-literature
Project description:Background:Producing valuable fuels and chemicals from lignin is a key factor for making lignocellulosic biomass economically feasible; however, significant roadblocks exist due to our lack of detailed understanding of how lignin is enzymatically depolymerized and of the range of possible lignin fragments that can be produced. Development of suitable enzymatic assays for characterization of putative lignin active enzymes is an important step towards improving our understanding of the catalytic activities of relevant enzymes. Previously, we have successfully built an assay platform based on glycan substrates containing a charged perfluorinated tag and nanostructure-initiator mass spectrometry to study carbohydrate active enzymes, especially various glycosyl hydrolyses. Here, we extend this approach to develop a reliable and rapid assay to study lignin-modifying enzymes. Results:Two ?-aryl ether bond containing model lignin dimer substrates, designed to be suitable for studying the activities of lignin-modifying enzymes (LMEs) by nanostructure-initiator mass spectrometry (NIMS), were successful synthesized. Small-angle neutron scattering experiments showed that these substrates form micelles in solution. Two LMEs, laccase from the polypore mushroom Trametes versicolor, and manganese peroxidase (MnP) from white rot fungus Nematoloma frowardii, were tested for catalytic activity against the two model substrates. We show that the reaction of laccase and MnP with phenolic substrate yields products that arise from the cleavage of the carbon-carbon single bond between the ?-carbon and the adjacent aryl carbon, consistent with the mechanism for producing phenoxy radical as reaction intermediates. Reactions of the nonphenolic substrate with laccase, on the other hand, adopt a different pathway by producing an ?-oxidation product; as well as the cleavage of the ?-aryl ether bond. No cleavage of the carbon-carbon bond between the ?-carbon and the aryl carbon was observed. To facilitate understanding of reaction kinetics, the reaction time course for laccase activity on the phenolic substrate (I) was generated by the simultaneous measurement of all products at different time points of the reaction. Withdrawal of only a small sample aliquot (0.2 ?L at each time point) ensured minimum perturbation of the reaction. The time course can help us to understand the enzyme kinetics. Conclusions:A new assay procedure has been developed for studying lignin-modifying enzymes by nanostructure-initiator mass spectrometry. Enzyme assays of a laccase and a MnP on phenolic and nonphenolic ?-aryl ether substrates revealed different primary reaction pathways due to the availability of the phenoxy radical intermediates. Our assay provides a wealth of information on bond cleavage events not available using conventional colorimetric assays and can easily be carried out in microliter volumes and the quantitative analysis of product formation and kinetics is rapidly achieved by NIMS. This is the first time that NIMS technology was applied to study the activities of lignin-modifying enzymes. Unlike other previous works, our use of amphiphilic guaiacylglycerol ?-O-4 substrate (I) enables the formation of micelles. This approach helps avoid the re-polymerization of the resulting monomeric product. As a result, our assay can clearly demonstrate the degradation pathways of phenolic guaiacylglycerol ?-O-4 type of molecules with laccase and MnP.
| S-EPMC6158898 | biostudies-other
Project description:Lignocellulosic biomass is composed of three major biopolymers: cellulose, hemicellulose and lignin. Analytical tools capable of quickly detecting both glycan and lignin deconstruction are needed to support the development and characterization of efficient enzymes/enzyme cocktails. Previously we have described nanostructure-initiator mass spectrometry-based assays for the analysis of glycosyl hydrolase and most recently an assay for lignin modifying enzymes. Here we integrate these two assays into a single multiplexed assay against both classes of enzymes and use it to characterize crude commercial enzyme mixtures. Application of our multiplexed platform based on nanostructure-initiator mass spectrometry enabled us to characterize crude mixtures of laccase enzymes from fungi Agaricus bisporus (Ab) and Myceliopthora thermophila (Mt) revealing activity on both carbohydrate and aromatic substrates. Using time-series analysis we determined that crude laccase from Ab has the higher GH activity and that laccase from Mt has the higher activity against our lignin model compound. Inhibitor studies showed a significant reduction in Mt GH activity under low oxygen conditions and increased activities in the presence of vanillin (common GH inhibitor). Ultimately, this assay can help to discover mixtures of enzymes that could be incorporated into biomass pretreatments to deconstruct diverse components of lignocellulosic biomass.
| S-EPMC8175421 | biostudies-literature
Project description:Nanostructure imaging mass spectrometry (NIMS) with fluorinated gold nanoparticles (f-AuNPs) is a nanoparticle assisted laser desorption/ionization approach that requires low laser energy and has demonstrated high sensitivity. Here we describe NIMS with f-AuNPs for the comprehensive analysis of metabolites in biological tissues. F-AuNPs assist in desorption/ionization by laser-induced release of the fluorocarbon chains with minimal background noise. Since the energy barrier required to release the fluorocarbons from the AuNPs is minimal, the energy of the laser is maintained in the low μJ/pulse range, thus limiting metabolite in-source fragmentation. Electron microscopy analysis of tissue samples after f-AuNP NIMS shows a distinct "raising" of the surface as compared to matrix assisted laser desorption ionization ablation, indicative of a gentle desorption mechanism aiding in the generation of intact molecular ions. Moreover, the use of perfluorohexane to distribute the f-AuNPs on the tissue creates a hydrophobic environment minimizing metabolite solubilization and spatial dislocation. The transfer of the energy from the incident laser to the analytes through the release of the fluorocarbon chains similarly enhances the desorption/ionization of metabolites of different chemical nature, resulting in heterogeneous metabolome coverage. We performed the approach in a comparative study of the colon of mice exposed to three different diets. F-AuNP NIMS allows the direct detection of carbohydrates, lipids, bile acids, sulfur metabolites, amino acids, nucleotide precursors as well as other small molecules of varied biological origins. Ultimately, the diversified molecular coverage obtained provides a broad picture of a tissue's metabolic organization.
| S-EPMC10283077 | biostudies-literature
Project description:Across many environments microbial glycoside hydrolases support the enzymatic processing of carbohydrates, a critical function in many ecosystems. Little is known about how the microbial composition of a community and the potential for carbohydrate processing relate to each other. Here, using 1,934 metagenomic datasets, we linked changes in community composition to variation of potential for carbohydrate processing across environments. We were able to show that each ecosystem-type displays a specific potential for carbohydrate utilization. Most of this potential was associated with just 77 bacterial genera. The GH content in bacterial genera is best described by their taxonomic affiliation. Across metagenomes, fluctuations of the microbial community structure and GH potential for carbohydrate utilization were correlated. Our analysis reveals that both deterministic and stochastic processes contribute to the assembly of complex microbial communities.
| S-EPMC5218504 | biostudies-literature
Project description:Few aerobic hyperthermophiles degrade polysaccharides. We describe the genome-enabled enrichment and isolation of an aerobic hyperthermophile, Fervidibacter sacchari, which was originally ascribed to candidate phylum Fervidibacteria. F. sacchari uses polysaccharides and monosaccharides as sole carbon sources from 65-87.5 °C, and its genome encodes 117 glycoside hydrolases (GHs) spanning 49 GH families, including 31 homologs of understudied GH109, GH177, and GH179 domains. Here, we analyzed the transcriptomes of F. sacchari cells grown on eight different sole carbon and energy sources (beta-glucan, chondroitin sulfate, corn stover, gellan gum, locust bean gum, starch, xanthan gum, and xyloglucan) to link glycoside hydrolase substrate to function, as well as identify potential regulatory mechanisms. These data will provide preliminary characterization of novel carbohydrate-active enzymes at high temperatures.
2023-12-31 | GSE249938 | GEO
Project description:During the last decades, the impact of hyperthermophiles and their enzymes has been intensively investigated for implementation in various high-temperature biotechnological processes. Biocatalysts of hyperthermophiles have proven to show extremely high thermo-activities and thermo-stabilities and are identified as suitable candidates for numerous industrial processes with harsh conditions, including the process of an efficient plant biomass pretreatment and conversion. Already-characterized archaea-originated glycoside hydrolases (GHs) have shown highly impressive features and numerous enzyme characterizations indicated that these biocatalysts show maximum activities at a higher temperature range compared to bacterial ones. However, compared to bacterial biomass-degrading enzymes, the number of characterized archaeal ones remains low. To discover new promising archaeal GH candidates, it is necessary to study in detail the microbiology and enzymology of extremely high-temperature habitats, ranging from terrestrial to marine hydrothermal systems. State-of-the art technologies such as sequencing of genomes and metagenomes and automated binning of genomes out of metagenomes, combined with classical microbiological culture-dependent approaches, have been successfully performed to detect novel promising biomass-degrading hyperthermozymes. In this review, we will focus on the detection, characterization and similarities of archaeal GHs and their unique characteristics. The potential of hyperthermozymes and their impact on high-temperature industrial applications have not yet been exhausted.
| S-EPMC7469102 | biostudies-literature