Nanostructure‐Initiator Mass Spectrometry (NIMS) for the Analysis of Enzyme Activities

Wolfgang Reindl1, Kai Deng2, Xiaoliang Cheng1, Anup K. Singh2, Blake A. Simmons2, Paul D. Adams3, Trent R. Northen1

1 Department of Bioenergy/GTL & Structural Biology, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, 2 Biotechnology and Bioengineering & Biomass Science and Conversion Technology Departments, Sandia National Laboratories, Livermore, California, 3 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110221
Online Posting Date:  June, 2012
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Abstract

Enzymes are essential to innumerable vital processes in all fields of life. Additionally, enzymatic activities are utilized in a multitude of biological, medical, and technical applications. Using cellulases and other glycoside hydrolases for the conversion of lignocellulosic biomass into biofuel has become an active area of research for applied enzymology. Development of high‐throughput, high‐specificity enzyme assays for glycoside hydrolases is critical to identify, characterize, and optimize such activities for efficient biomass degradation. Here, we present a detailed protocol describing our recently reported nanostructure‐initiator mass spectrometry (NIMS)‐based technique for the rapid analysis of glycoside hydrolase activities. NIMS enzyme activity (Nimzyme) assays can be used to characterize already known, or identify novel glycoside hydrolases. Importantly, several enzymatic activities can be tested in parallel in a multiplexed arrangement and under a broad range of assay conditions. Curr. Protoc. Chem. Biol. 4:123‐142 © 2012 by John Wiley & Sons, Inc.

Keywords: nanostructure‐initiator mass spectrometry; NIMS; Nimzyme; enzyme assay; glycoside hydrolase; lignocellulosic biofuel

     
 
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Table of Contents

  • Introduction
  • Strategic Planing
  • Basic Protocol 1: Determination of an Enzyme Activity Profile using a Single Substrate Nimzyme Assay
  • Basic Protocol 2: Parallel Analysis of Multiple Enzyme Activities using a Multiplexed Nimzyme Assay
  • Basic Protocol 3: Direct Identification of Multiple Enzyme Activities in Crude Environmental Samples
  • Support Protocol 1: Synthesis of Nimzyme Substrates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determination of an Enzyme Activity Profile using a Single Substrate Nimzyme Assay

  Materials
  • Enzyme: β‐glucosidase (NS50010) from the Biomass kit from Novozymes; enzyme stock solution: protein density 1.2 g/ml with 250 cellobiase Units (CBU) per g
  • Buffers: 50 mM sodium acetate (pH 4.8), McIlvaine's citrate/phosphate buffer (pH 2.6 and 6.0; see recipe), 50 mM Tris⋅Cl (pH 9.0); all reagents are grade p.a. from Sigma
  • Nimzyme substrate stock solution: 2 mM CB‐F17 with perfluorinated tag (Fig. A) in H 2O (see recipe)
  • Ice‐cold methanol, HPLC grade (Sigma)
  • Standard heat block or PCR machine (e.g., PTC‐225 Tetrad PCR System from MJ Research)
  • NIMS chip (see Strategic Planning) and modified MALDI plate (see Critical Parameters), diamond scribe, metal ruler
  • Lint‐ or fiber‐free laboratory tissue
  • MALDI mass spectrometer (e.g., 4800 MALDI TOF/TOF mass analyzer from AB Sciex) and software

Basic Protocol 2: Parallel Analysis of Multiple Enzyme Activities using a Multiplexed Nimzyme Assay

  Materials
  • Enzymes (both enzymes are part of the Biomass kit from Novozymes):
    • β‐glucosidase (NS50010), enzyme stock solution: protein density 1.2 g/ml with 250 cellobiase Units (CBU) per gram
    • β‐glucanase/xylanase (NS22002), enzyme stock solution: protein density 1.2 g/ml with 45 fungal β‐glucanase units (FBG) and ∼470 Farvet xylan units (FXU) per gram
  • Buffers: 50 mM sodium acetate (pH 4.8), McIlvaine's citrate/phosphate buffer (pH 6.0) (see recipes); all reagents are grade p.a. from Sigma
  • Substrate stock solutions: 2 mM CB‐F17 with perfluorinated tag (Fig. A) in H 2O and 2 mM XB‐F17 with perfluorinated tag (Fig. B) in H 2O (see recipe)
  • Ice‐cold methanol, HPLC grade (Sigma)
  • Standard heat block or PCR machine (e.g., PTC‐225 Tetrad PCR System from MJ Research)
  • NIMS chip (see Strategic Planning) and modified MALDI plate (see Critical Parameters), diamond scribe, metal ruler
  • Lint‐ and fiber‐free laboratory tissue
  • MALDI mass spectrometer (e.g., 4800 MALDI TOF/TOF mass analyzer from AB Sciex) and software

Basic Protocol 3: Direct Identification of Multiple Enzyme Activities in Crude Environmental Samples

  Materials
  • Environmental sample: soil from a standard garden compost
  • Medium and buffer: M9 minimal medium with trace elements (M9TE) and 20 g/liter switchgrass (see recipe), 50 mM sodium acetate (pH 5.0); all reagents are grade p.a. from Sigma
  • Substrate stock solutions: 2 mM CB‐F17 with perfluorinated tag (Fig. A) in H 2O, 2 mM XB‐F17 with perfluorinated tag (Fig. B) in H 2O (see recipe)
  • Ice‐cold methanol, HPLC grade (Sigma)
  • Hammer
  • 60°C shaking incubator
  • Centrifuge
  • Standard heat block or PCR machine (e.g., PTC‐225 Tetrad PCR System from MJ Research) for incubation
  • NIMS chip (see Strategic Planning) and modified MALDI plate (see Critical Parameters), diamond scribe, metal ruler
  • Lint‐ and fiber‐free laboratory tissue
  • MALDI mass spectrometer (e.g., 4800 MALDI TOF/TOF mass analyzer from AB Sciex) and software

Support Protocol 1: Synthesis of Nimzyme Substrates

  Materials
  • Acidic ceric ammonium molybdate
  • Ninhydrate
  • Triethylamine
  • Asymmetric dimethyl‐arginine HCl salt (Compound 1)
  • Compound 2 (see Lee et al., )
  • Dimethylformamide (DMF)
  • Nitrogen
  • Deuterated H 2O (D 2O)
  • Compound 4 (see Chang et al., )
  • Compound 5
  • Anhydrous methylene chloride
  • 3 Å molecular sieves
  • Trimethylsilyl trifluoromethanesulfonate (TMSOTf)
  • Sodium methoxide (NaOMe)
  • Methanol (MeOH)
  • DOWEX 50WX2‐200 (H+)
  • Chloroform
  • Tetrahydrofuran (THF)
  • 10% Pd/C
  • N,N′‐Diisopropylcarbodiimide
  • 1‐hydroxybenzotriazole
  • Compound 10 (see Vrsanska et al., )
  • Thin‐layer chromatography (TLC) plates (e.g., EM silica gel 60 F254 plates from Sigma)
  • NMR spectrometer (e.g., AVB‐400 or AV‐600 from Bruker)
  • Ice bath
  • Rotary evaporator (e.g., Buchi R‐200 rotary evaporator from Buchi)
  • Whatman filter paper
  • Plastic funnel
  • Flash column chromatography system (e.g., CombiFlash Rf from Teledyne ISCO)
NOTE: All chemicals are available from Sigma. They are all reagent grade and are used without further purification.
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Figures

Videos

Literature Cited

Literature Cited
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