Determination of Disulfide‐Bond Linkages in Proteins

Hsin‐Yao Tang1, David W. Speicher1

1 The Wistar Institute, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 11.11
DOI:  10.1002/0471140864.ps1111s37
Online Posting Date:  September, 2004
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The formation of disulfide bonds in proteins is an important post‐translational modification that is critical for stabilizing the native structures of proteins. The disulfide linkages in a protein cannot be predicted from its amino acid sequence; therefore, determination of disulfide bond linkages in the protein will provide insights into its three‐dimensional structure and contribute to the understanding of its structural‐functional relationship. This unit details a series of protocols that have been applied successfully to locate disulfide bonds in proteins. The general strategy involves chemical or proteolytic cleavage of the protein followed by chromatographic separation of the resultant peptides. Disulfide‐containing peptides are identified by the alteration of mobility as a consequence of disulfide bond reduction, and are further characterized by mass spectrometry and/or N‐terminal sequencing. A partial reduction and alkylation strategy for mapping disulfide linkages in peptides with multiple disulfide bonds is also presented.

Keywords: disulfide bond determination; chemical and protease cleavage; HPLC; mass spectrometry; Edman sequencing

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Trypsin Cleavage and Disulfide‐Bond Mapping of Proteins
  • Alternate Protocol 1: Cyanogen Bromide Cleavage of Protein
  • Alternate Protocol 2: Partial Reduction and Alkylation of Disulfide Bonds
  • Support Protocol 1: Determination of Total Disulfide Bonds
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Trypsin Cleavage and Disulfide‐Bond Mapping of Proteins

  • Urea buffer (see recipe)
  • Protein sample, lyophilized or aqueous (i.e., in mildly acidic buffer without serine protease inhibitors and with known concentration)
  • Argon
  • Urea, solid (for aqueous proteins only)
  • 1M HCl
  • 1M NaOH
  • 50 mM sodium phosphate, pH 6.5 ( appendix 2E)
  • Reaction buffer: 3 M urea/50 mM sodium phosphate, pH 6.5
  • Trifluoroacetic acid (TFA)
  • Reducing solution, pH 8.0 (see recipe)
  • Solvent A: 0.1% TFA in H 2O
  • Solvent B: 0.1% TFA in 95% acetonitrile
  • 50% acetonitrile/0.1% TFA
  • Immobilized tosylphenylalanine chloromethylketone (TPCK)‐treated trypsin F7m column (MoBiTec)
  • 10‐ml syringe
  • HPLC system, with 2.1‐mm C18 reversed‐phase column (e.g., ZORBAX 300SB‐C18), UV detector, fraction collector, peak detector, and chart recorder
  • C18 ZipTip (Millipore)
  • Additional reagents and materials for RP‐HPLC (unit 11.6) and MALDI mass spectrometry analysis of peptides (unit 16.2), and tandem mass spectrometry (unit 16.10)

Alternate Protocol 1: Cyanogen Bromide Cleavage of Protein

  • Glacial and 5% acetic acid
  • Solution of purified protein with known sequence
  • 88% and 70% formic acid
  • CNBr
  • Acetonitrile
  • Desalting column (i.e., Bio‐Rad Econo‐Pac 10DG or equivalent)
  • Fraction collector
  • Glass vial with Teflon‐lined cap
  • Aluminum foil
  • Additional reagents and materials for desalting protein samples (unit 8.3) and SDS‐PAGE (unit 10.1)

Alternate Protocol 2: Partial Reduction and Alkylation of Disulfide Bonds

  • Reducing solution, pH 3.5 (see recipe)
  • RP‐HPLC‐purified peptide complex (see protocol 1)
  • 1 M alkylation solution (see recipe)
  • 0.3% and 10% TFA

Support Protocol 1: Determination of Total Disulfide Bonds

  • Protein solution
  • Guanidine buffer (see recipe)
  • Reducing solution, pH 6.5 (see recipe)
  • 80 mM alkylation solution (see recipe)
  • 0.3% TFA
  • 50% acetonitrile/0.1% TFA
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Literature Cited

Literature Cited
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