Modification of Cysteine

Gregory A. Grant1

1 Washington University School of Medicine, Department of Medicine and Department of Developmental Biology, St. Louis, Missouri
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 15.1
DOI:  10.1002/cpps.22
Online Posting Date:  February, 2017
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Abstract

This unit describes a number of methods for modifying cysteine residues of proteins and peptides. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N‐ethylmaleimide (NEM) is presented, and alternate protocols describe similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and N‐(iodoethyl)‐trifluoroacetamide are also presented. Two procedures that are often used for subsequent sequence analysis of the protein, alkylation with 4‐vinylpyridine and acrylamide, are described, and a specialized procedure for 4‐vinylpyridine alkylation of protein that has been adsorbed onto a sequencing membrane is also presented. Reversible modification of cysteine residues by way of sulfitolysis is described, and a protocol for oxidation with performic acid for amino acid compositional analysis is also provided. Gentle oxidation of cysteine residues to disulfides by exposure to air is described. Support protocols are included for recrystallization of iodoacetic acid, colorimetric detection of free sulfhydryls, and desalting of modified samples. © 2017 by John Wiley & Sons, Inc.

Keywords: cysteine; disulfide; alkylation; dithiothreitol; TCEP; cysteine modification

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Alkylation of a Protein of Known Size and Composition with Haloacyl Reagents or N‐Ethylmaleimide
  • Alternate Protocol 1: Alkylation of a Protein of Unknown Size and Composition with Haloacyl Reagents or N‐Ethylmaleimide
  • Alternate Protocol 2: Alkylation of ≤50 μg Protein with Haloacyl Reagents or N‐Ethylmaleimide
  • Basic Protocol 2: Alkylation with 3‐Bromopropylamine
  • Basic Protocol 3: Alkylation with N‐(Iodoethyl)‐Trifluoroacetamide
  • Basic Protocol 4: Alkylation with 4‐Vinylpyridine
  • Basic Protocol 5: Alkylation with Acrylamide
  • Basic Protocol 6: Sulfitolysis
  • Basic Protocol 7: Oxidation with Performic Acid
  • Basic Protocol 8: Air Oxidation to a Disulfide
  • Basic Protocol 9: Alkylation of Cysteine in Protein Applied to Sequencer Membranes
  • Support Protocol 1: Recrystallization of Iodoacetic Acid
  • Support Protocol 2: Colorimetric Quantitation of Free Sulfhydryls
  • Support Protocol 3: Desalting the Sample After Cysteine Modification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Alkylation of a Protein of Known Size and Composition with Haloacyl Reagents or N‐Ethylmaleimide

  Materials
  • Protein, lyophilized
  • Tris/guanidine buffer, pH 8.0 (see recipe) or 0.1 M Tris·Cl, pH 8.0 ( appendix 2E)
  • 50 mM dithiothreitol (DTT; 7.7 mg/ml) or 50 mM TCEP HCl (14.3 mg/ml) in Tris buffer
  • Nitrogen
  • Alkylating agent (see recipe)
  • 2‐mercaptoethanol (2‐ME; 14.4 M). Also called β‐mercaptoethanol (β−ME or BME)
  • 0.5‐ to 2.5‐ml microcentrifuge tube
  • Additional reagents and equipment for desalting modified protein (see protocol 14)

Alternate Protocol 1: Alkylation of a Protein of Unknown Size and Composition with Haloacyl Reagents or N‐Ethylmaleimide

  Materials
  • Protein, lyophilized
  • 20 mM and 2 M dithiothreitol (DTT) in 0.5 M Tris·Cl (pH 8.2; appendix 2E)/6 M guanidine·HCl
  • 0.5 M 3‐bromopropylamine (see recipe)
  • Nitrogen
  • 0.5‐ to 2.5‐ml microcentrifuge tube
  • Additional reagents and equipment for desalting modified protein (see protocol 14)

Alternate Protocol 2: Alkylation of ≤50 μg Protein with Haloacyl Reagents or N‐Ethylmaleimide

  Materials
  • Protein, lyophilized
  • 0.2 M N‐ethylmorpholine acetate (pH 8.1)/6 M guanidine·HCl
  • 50 mM dithiothreitol (DTT; 7.7 mg/ml)
  • Nitrogen
  • N‐(iodoethyl)‐trifluoroacetamide
  • Methanol
  • 2 N acetic acid
  • 0.5‐ to 2.5‐ml microcentrifuge tube
  • Additional reagents and equipment for desalting modified protein (see protocol 14)

Basic Protocol 2: Alkylation with 3‐Bromopropylamine

  Materials
  • 1 M Tris·Cl (pH 8.5; appendix 2E)/4 mM EDTA
  • 8 M guanidine·HCl
  • Protein, lyophilized
  • 10% (v/v) 2‐mercaptoethanol (2‐ME)
  • Argon
  • 4‐vinylpyridine, fresh
  • 0.5‐ to 2.5‐ml microcentrifuge tube
  • Additional reagents and equipment for desalting modified protein (see protocol 14)

Basic Protocol 3: Alkylation with N‐(Iodoethyl)‐Trifluoroacetamide

  Materials
  • Protein, lyophilized
  • 0.3 M Tris·Cl, pH 8.3 ( appendix 2E)
  • 50 mM dithiothreitol (DTT; 7.7 mg/ml; optional). TCEP may be used as in protocol 1
  • 6 M acrylamide
  • 10% (v/v) 2‐mercaptoethanol (2‐ME)
  • 0.5‐ to 1.5‐ml microcentrifuge tube
  • Additional reagents and equipment for desalting modified protein (see protocol 14)
CAUTION: Acrylamide monomer is neurotoxic. A mask should be worn when weighing acrylamide powder. Gloves should be worn while handling the solution, and the solution should not be pipetted by mouth.

Basic Protocol 4: Alkylation with 4‐Vinylpyridine

  Materials
  • Protein, lyophilized
  • Sodium sulfite reagent buffer (see recipe)
  • 4 M urea/1 M Tris·Cl, pH 7.5
  • 2‐mercaptoethanol (2‐ME)
  • 0.5‐ to 2.5‐ml microcentrifuge tube
  • Additional reagents and equipment for desalting modified protein (see protocol 14)

Basic Protocol 5: Alkylation with Acrylamide

  Materials
  • Formic acid
  • 30% hydrogen peroxide
  • Protein, lyophilized and chilled
  • 48% (v/v) hydrobromic acid
  • Ice bath

Basic Protocol 6: Sulfitolysis

  Materials
  • Protein or peptide, lyophilized
  • 0.1 M ammonium bicarbonate
  • Additional reagents and equipment for reversed‐phase HPLC (unit 11.6; Mant and Hodges, ) and desalting oxidized protein (see protocol 14)

Basic Protocol 7: Oxidation with Performic Acid

  Materials
  • Protein
  • Reduction/alkylation cocktail (see recipe)
  • Sequencer reagents (Perkin‐Elmer or equivalent)
  • Biobrene coated glass‐fiber disk, precycled
  • PE‐ABD sequencer, cartridge (Perkin‐Elmer) or equivalent

Basic Protocol 8: Air Oxidation to a Disulfide

  Materials
  • Iodoacetic acid (IAA)
  • Diethyl ether
  • Petroleum ether, ice cold
  • Buchner funnel
  • Whatman No. 1 filter paper or equivalent
  • Amber bottle with Teflon‐lined lid

Basic Protocol 9: Alkylation of Cysteine in Protein Applied to Sequencer Membranes

  Materials
  • 0.1 M sodium phosphate, pH 8.0 ( appendix 2E)
  • Reagent solution: 4 mg/ml Ellman reagent/0.1 M sodium phosphate, pH 8.0
  • Cysteine standard stock solution (see recipe)
  • Unknown sample
  • 13 × 100‐mm clear test tubes
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Figures

Videos

Literature Cited

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