Metallomics Using Inductively Coupled Plasma Mass Spectrometry

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

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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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) H₂ O
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 H₂ gas is circulated through a palladium‐coated alumina catalyst and removes O₂ by forming water. Generally, O₂ 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) H₂ O
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 (NH₄ )₂ SO₄ , 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

Figure 1. Flowchart showing the preparation of the cytoplasmic (soluble) fraction and the series of buffer exchange steps that the soluble fraction undergoes to generate the 256‐fold washed cytoplasmic extract. The fractions generated at each step are analyzed by ICP‐MS.

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Figure 2. Anaerobic chromatography setup showing anaerobic buffer introduction, anaerobic sample collection, and the direction of buffer/sample and argon flow.

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Figure 3. Elution of soluble P. furiousus proteins from a DEAE anion‐exchange column showing molybdenum (‐x‐) and protein (⋅⋅ o ⋅⋅) concentrations. Box shows fractions pooled for PF1972 purification (adapted from Cvetkovic et al., ).

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Figure 4. Elution from second level size‐exclusion column of the fraction pool from Figure 3 showing molybdenum (‐x‐) and protein (⋅⋅ o ⋅⋅) concentrations. Box shows fractions pooled for PF1972 purification, (adapted from Cvetkovic et al., ).

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Figure 5. Elution from a third level hydroxyapatite column of fraction pool from Figure 4 showing molybdenum (‐x‐) and protein (⋅⋅ o ⋅⋅) concentrations. Box shows fractions pooled for PF1972 purification (adapted from Cvetkovic et al., ).

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Figure 6. Elution from a fourth level phenyl Sepharose column of fraction pool from Figure 5 showing molybdenum concentrations (‐x‐). Box shows fractions pooled for PF1972 purification (adapted from Cvetkovic et al., ).

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Figure 7. (A ) Elution from a fifth‐level Mono Q column of fraction pool from Figure 6, showing molybdenum concentrations (‐x‐). Box shows purified PF1972. (B ) SDS‐PAGE gel of purified PF1972 (adapted from Cvetkovic et al., ).

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Figure 8. The susceptibility of metals to removal from the cytoplasmic fraction by filtration (3 kDa cut‐off). In each plot, the bars represent (from left to right) the total amount of metal in the cytoplasm before (S100) and after washing (S100w), the metal in the flowthrough (FT), and in the three subsequent wash steps (w‐1, w‐2, and w‐3). The three bar graphs show representative examples of metal‐macromolecule interactions. (A ) unbound metal/weak interactions; (B ) tight interactions; and (C ) mixed (a mixture of both types of interactions).

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Videos

Literature Cited

Literature Cited
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Metallomics Using Inductively Coupled Plasma Mass Spectrometry

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited