Metallomics Using Inductively Coupled Plasma Mass Spectrometry

Brian J. Vaccaro1, Angeli L. Menon1, W. Andrew Lancaster1, Michael W. W. Adams1

1 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch120031
Online Posting Date:  September, 2012
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Abstract

Inductively Coupled Plasma Mass Spectrometry (ICP‐MS) is a highly sensitive elemental analysis technique that has been widely applied in many fields. Here we describe applications using a broad‐scale approach to examine metal usage in biology. These protocols address questions such as: Which elements from the surrounding environment are taken up into the cells of a given organism? How does this vary between different organisms? Which metals are “bound” and which are “free”? With which type(s) of proteins are the “bound” metals associated? This allows for investigations into several branches of bioinorganic chemistry including uptake, toxicity, detoxification, bioremediation, and the discovery of new uses for elements. In the protocols presented here, there is an emphasis on metals, and, more narrowly, on transition metals because these comprise the majority of tightly protein‐bound, low‐abundance elements. Nonmetals, metalloids, main‐group metals, and f‐block metals are also analyzed and investigated. The sample preparation procedure requires acid lability for detection, which likely eliminates certain nonmetals, such as selenium. However, one advantage of the protocols described is that they are readily adapted to measure any element of interest. Curr. Protoc. Chem. Biol. 4:249‐274 © 2012 by John Wiley & Sons, Inc.

Keywords: metalloproteome; metallome; ICP‐MS; metalloprotein; metalloenzyme; bioinorganic chemistry; metallobiochemistry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: ICP‐MS Analysis of Soluble Protein Samples
  • Basic Protocol 2: Preparation of Washed Cytoplasmic Extract for the Identification of Soluble Metal‐Macromolecular Complexes (≥3 kDa)
  • Basic Protocol 3: Anaerobic Purification of Metalloproteins Using ICP‐MS and Conventional Protein Chromatography
  • Support Protocol 1: Acid Washing Plasticware for ICP‐MS Sample Handling
  • Support Protocol 2: Acid Washing Glassware for Anaerobic Cytoplasmic Washes and Purification of Metalloproteins
  • Support Protocol 3: Preparing Anaerobic Solutions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: ICP‐MS Analysis of Soluble Protein Samples

  Materials
  • Tuning solution for ICP‐MS 7500cs (Agilent PN: 5185‐5959)
  • Internal standard: IV‐ICPMS‐71D (Inorganic Ventures, http://www.inorganicventures.com/)
  • Glass‐distilled deionized (gdd) H 2O
  • Trace‐metal free nitric acid(2% and 5% v/v solutions; Sigma‐Aldrich, cat. no. 7697‐37‐2)
  • External calibration standards (Inorganic Ventures, http://www.inorganicventures.com/):
    • IV‐ICPMS‐71A
    • CCS‐5
    • CMS‐2
  • Protein sample (Basic Protocols protocol 22 and protocol 33)
  • Agilent 7500ce ICP‐MS with micro‐mist nebulizer
  • Cetac ASX‐520 autosampler (http://www.cetac.com)
  • 15‐ml conical tubes (e.g., BD Falcon), acid washed ( protocol 4)
  • 37°C shaking incubator
  • Beckman Allegra 6R centrifuge and Beckman GH‐3.8 rotor
NOTE: The protein and salt concentrations in the sample, as well as the buffer used, will vary depending on the type of chromatography performed and the level of separation required. The pH of samples generated by Basic Protocols protocol 22 and protocol 33 is typically between 5 and 9. Acidfication in predigestion will lower the pH, and additional acid can be added if the pH is usually high and a less dilute solution is required.

Basic Protocol 2: Preparation of Washed Cytoplasmic Extract for the Identification of Soluble Metal‐Macromolecular Complexes (≥3 kDa)

  Materials
  • Cell lysis/wash buffer: 50 mM Tris⋅Cl, pH 8.0, anaerobic (see recipe)
  • DNase I (Sigma‐Aldrich, cat. no. DN25‐1G)
  • 3 g wet weight of freshly prepared cell pellet or flash frozen P. furiosus cells (stored at −80°C); these cells are not currently commercially available, but large‐scale fermentations can be conducted or done on a contract basis [e.g., The Bioexpression and Fermentation (BFF) facility at the University of Georgia (http://bff.uga.edu/)]
  • Bradford Protein Assay Kit (BioRad, cat no. 500‐0002)
  • Anaerobic glove chamber (Type B vinyl anaerobic chamber; Coy Laboratory Products, Inc., http://www.coylab.com/)
  • 5‐ 10‐, and 25‐ml (or 30‐ml) glass serum bottles with rubber stoppers
  • 50‐ml beakers with magnetic stir bars
  • Magnetic stirrer (in anaerobic chamber)
  • Polycarbonate centrifuge tubes with cap assemblies (10.4 ml capacity; Beckman, cat. no. 355603)
  • Ultracentrifuge (Beckman Optima L‐90K)
  • Fixed angle rotor for ultracentrifuge (Beckman 70.1 Ti: 12 × 10.4 ml capacity)
  • Amicon Ultra‐4 centrifugal filter devices (3K nominal molecular weight limit; Millipore)
  • Tabletop Eppendorf centrifuge 5430 (in anaerobic chamber)
  • Eppendorf fixed angle rotor F‐35‐6‐30 with adaptors (holds 6 × 15 ml centrifugal filter devices)
  • 1.5‐ml sterile polypropylene (pp) screw cap tubes (Phenix Research Products, cat. no. SCS‐015FS, http://www.phenixresearch.com/)
NOTE: All steps are carried out at room temperature unless otherwise stated, although samples from microbes that grow at much lower temperature may require processing at 4°C to preserve the integrity of their proteins. The protocol described here is for 3 g wet weight of cells, but can be scaled up if desired.NOTE: All clean glassware and stir bars used for the experiment should be acid washed as described in protocol 5. Cleaned centrifuge tubes/lid assemblies should be soaked and rinsed multiple times in glass‐distilled deionized (gdd) water or equivalent. Chemicals should be of the highest purity possible. All solutions should be made in gdd water and stored in acid‐washed glass containers.NOTE: All manipulations should be done under strictly anaerobic conditions i.e., using a vacuum manifold and/or in an anaerobic chamber maintained with 5% hydrogen gas with the balance composed of an inert gas (we use argon). The gas mix containing the H 2 gas is circulated through a palladium‐coated alumina catalyst and removes O 2 by forming water. Generally, O 2 levels equilibrate to 0 to 5 parts per million (ppm). For more details refer to the Coy Laboratory Products Catalog (http://www.coylab.com/). Transfer all glassware, stir bars, rubber stoppers, tip boxes, pipets, centrifuge tubes with caps (open), and centrifugal filtration devices with caps (open) into the anaerobic chamber the evening before starting the experiment. This will remove any diffused oxygen in the plastic/glass/pipet tips/rubber stoppers, so that everything is oxygen‐free by the time the sample is introduced into the chamber.

Basic Protocol 3: Anaerobic Purification of Metalloproteins Using ICP‐MS and Conventional Protein Chromatography

  Materials
  • Buffer A: 50 mM Tris⋅Cl, pH 8.0, anaerobic (see recipe for cell/lysis wash buffer in Reagents and Solutions; see protocol 6 for anaerobic preparation)
  • Glass distilled deionized (gdd) H 2O
  • 300 g wet weight of freshly prepared cell pellet or flash frozen P. furiosus cells (stored at −80°C); these cells are not currently commercially available, but large‐scale fermentations can be conducted or done on a contract basis [e.g., The Bioexpression and Fermentation (BFF) facility at the University of Georgia (http://bff.uga.edu/)]
  • DNase I (Sigma‐Aldrich, cat. no. DN25‐1G)
  • Buffer B: 50 mM Tris⋅Cl, pH 8.0/2 M NaCl, anaerobic (see protocol 6 for anaerobic preparation)
  • Argon source
  • Bradford Protein Assay Kit (e.g., BioRad, cat. no. 500‐0002V)
  • Buffer C: 50 mM Tris⋅Cl, pH 8.0/300 mM NaCl, anaerobic (see protocol 6 for anaerobic preparation)
  • Buffer D: 5 mM potassium phosphate, pH 8.0, anaerobic (see protocol 6 for anaerobic preparation)
  • Buffer E: 500 mM potassium phosphate, pH 8.0, anaerobic (see protocol 6 for anaerobic preparation)
  • Buffer F: 50 mM potassium phosphate/2 M (NH 4) 2SO 4, pH 7.0, anaerobic (see protocol 6 for anaerobic preparation)
  • Buffer G: 50 mM potassium phosphate, pH 7.0, anaerobic (see protocol 6 for anaerobic preparation)
  • Buffer H: 50 mM Tris⋅Cl, pH 8.0, anaerobic (see protocol 6 for anaerobic preparation)
  • Buffer I: : 50 mM Tris⋅Cl, pH 8.0/1 M NaCl, anaerobic (see protocol 6 for anaerobic preparation)
  • Vacuum manifold (connected to mechanical vacuum pump and argon tank)
  • Stir bars
  • Stir plate
  • Anaerobic glove chamber (Type B vinyl anaerobic chamber; Coy Laboratory Products, Inc., http://www.coylab.com/)
  • Polycarbonate centrifuge tubes with cap assemblies (70‐ml capacity; Beckman, cat. no. 35562)
  • Ultracentrifuge (Beckman Optima L‐90K)
  • Fixed angle rotor for ultracentrifuge (Beckman 45 Ti: 6 × 94‐ml capacity)
  • Glassware (acid washed; see protocol 5) and accessories:
    • Two 15‐liter carboys that can hold a vacuum
    • Three 4 liter sidearm flasks
    • Two 1 liter sidearm flasks
    • Two 250 ml sidearm flasks
    • 5‐125 ml serum bottles/vials
    • Rubber stoppers to fit all flasks, bottles, and vials
    • Plastic/rubber hosing
    • Clamps for sealing off rubber hosing
    • Hose barb connectors
  • Syringes and needles
  • 1.1‐liter DEAE Sepharose Fast Flow (FF) column (GE Healthcare, cat. no. 17‐0709‐05, column—GE XK 50/60)
  • Advantec 450 ml stirred pressure filtration cell (Model # UHP‐76, http://www.advantecmfs.com/)
  • 76‐mm ultrafiltration membrane (3‐kDa nominal molecular weight limit; NMWL; Millipore, cat. no. PLBC07610)
  • Protein chromatography system (e.g., ÄKTA BASIC)
  • Superdex 75 26/60 (320 ml) size‐exclusion column (GE Healthcare)
  • 8‐ml hydroxyapatite column (BioRad CHT Type I, 20 µm particle ceramic hydroxyapatite, cat. no. 158‐2000, column—GE XK 16/20)
  • 1 ml phenyl Sepharose column (e.g., HiTrap Phenyl HP; GE Healthcare)
  • 1 ml Mono Q 5/50 GL column (GE Healthcare)
  • 5‐ml sample loop (GE Healthcare, cat. no. 18‐1140‐53)
  • Additional reagents and equipment for preparation of anaerobic solutions ( protocol 6), ICP‐MS ( protocol 1), and SDS‐PAGE (Gallagher, )
NOTE: All clean glassware and stir bars used for the experiment should be acid washed as described in protocol 5. All solutions should be made in gdd water and stored in acid‐washed glass containers.NOTE: All collection vessels (flasks, bottles, vials, etc.) for column flow throughs, washes, and fractions should be made anaerobic by taking them empty and open into an anaerobic glove chamber and sealing them in the glove chamber with a rubber stopper (see protocol 6, step 2) or septum before removal from the glove box and use. It is best to let permeable items, particularly septa, sit in the glove box at least overnight to allow absorbed oxygen to diffuse out; thus, it is convenient to keep a stock of septa anaerobic in the glove box. Vessels can alternatively be made anaerobic on a vacuum manifold by repeatedly exchanging vacuum and inert gas five to seven times.

Support Protocol 1: Acid Washing Plasticware for ICP‐MS Sample Handling

  Materials
  • 2% (v/v) nitric acid prepared from reagent‐grade nitric acid (Sigma‐Aldrich, cat. no. 438073) and gdd water
  • Glass distilled deionized (gdd) water
  • Plasticware to be used for ICP‐MS
  • Three high density polyethylene (HDPE) tanks (Norton)
  • Large laboratory tissues (Kimwipes)

Support Protocol 2: Acid Washing Glassware for Anaerobic Cytoplasmic Washes and Purification of Metalloproteins

  Materials
  • Reagent‐grade nitric acid (Sigma‐Aldrich, cat. no. 438073)
  • Glass‐distilled deionized (gdd) water or equivalent purity water
  • Long acid‐resistant polyethylene or latex gloves
  • Polypropylene tank with lid (Nalgene, 27 liter; Fisher Scientific, cat. no. 14‐831‐112)
  • Polypropylene tray (Nalgene, 15 liter; Fisher Scientific, cat. no. 13‐359‐20 B)
  • Stir bars
  • Magnetic stirrer
  • Clean glassware to be acid washed
  • Clean Teflon‐coated stir bars to be acid‐washed
  • pH paper

Support Protocol 3: Preparing Anaerobic Solutions

  Materials
  • Solution (e.g., buffer) for anaerobic treatment
  • Argon source
  • Sidearm Erlenmeyer flask
  • Stir bar
  • Rubber stoppers to fit container
  • Soft rubber septa for solution removal
  • Stir plate
  • Vacuum manifold (connected to mechanical vacuum pump and argon tank)
  • Plastic/rubber hosing
  • Clamps for sealing off rubber hosing
  • Hose barb connectors
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Figures

Videos

Literature Cited

Literature Cited
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