Enzymatic Digestion of Proteins in Gels for Mass Spectrometric Identification and Structural Analysis

Kathryn L. Stone1, Kenneth R. Williams1

1 W.M. Keck Foundation Biotechnology Resource Laboratory, Yale University School of Medicine, New Haven, Connecticut
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 11.3
DOI:  10.1002/0471140864.ps1103s38
Online Posting Date:  November, 2004
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Abstract

Enzymatic digestion of proteins is a key technique used in protein identification. By combining the digestion with mass spectrometric detection, proteins at very low femtomole levels, and in some cases subfemtomole levels, can be identified. Typically, one‐ or two‐dimensional SDS‐PAGE is used to isolate the proteins of interest, followed by staining with Coomassie blue, digestion‐compatible silver stain, or Sypro Ruby for detection. Two‐dimensional (2‐D) fluorescence difference gel electrophoresis (DIGE), which uses Cy3 and Cy5 dyes for detection, allows comparison of two different sample states in order to locate proteins that are up‐ or down‐regulated. In each case, an in‐gel digestion, usually tryptic, is used with mass spectrometry to identify these proteins of interest. For large numbers of gel spots, robotic digestion can save time and money.

Keywords: digestion; DIGE; protein identification; trypsin; SDS‐PAGE

     
 
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Table of Contents

  • Basic Protocol 1: Manual Digestion of Proteins in Gels
  • Alternate Protocol 1: Removal of Silver Prior to In‐Gel Digestion of Silver‐Stained Proteins
  • Alternate Protocol 2: Automated In‐Gel Trypsin Digestion Using the Amersham Biosciences Ettan TA Digester
  • Support Protocol 1: C18 ZipTip Clean‐Up/Desalting of In‐Gel Digests Prior To Nanospray Ms/Ms Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Manual Digestion of Proteins in Gels

  Materials
  • Protein sample separated on an SDS‐polyacrylamide gel (unit 10.1; include appropriate standard proteins on gel) or on a two‐dimensional isoelectric focusing/SDS polyacrylamide gel (unit 10.4) and stained with Coomassie brilliant blue (unit 10.5), silver (unit 10.5), or other protein stain
  • 0.1% (v/v) trifluoroacetic acid (TFA)/60% (v/v) acetonitrile
  • 50% (v/v) acetonitrile
  • 50% (v/v) acetonitrile in 50 mM NH 4HCO 3 (store up to 1 week at 4°C)
  • 50% (v/v) acetonitrile in 10 mM NH 4HCO 3 (store up to 1 week at 4°C)
  • 6.7 µl/ml trypsin working solution or endoproteinase Lys‐C working solution (see reciperecipes)
  • 10 mM NH 4HCO 3 (store up to 1 week at 4°C)
  • 20% (v/v) trifluoroacetic acid (TFA)/60% (v/v) acetonitrile
  • 0.1% (v/v) and 100% TFA
  • 5% acetonitrile (v/v) in 100 mM NH 4HCO 3 (store up to 1 week at 4°C)
  • 45 mM dithiothreitol (DTT) (store up to 1 week at 4°C)
  • 100 mM iodoacetamide (store up to 1 week at 4°C)
  • 0.05% (v/v) trifluoroacetic acid/5% (v/v) acetonitrile
  • HPLC buffer A: 0.06% (v/v) TFA
  • HPLC buffer B: 0.052% (v/v) TFA in 80% (v/v) acetonitrile
  • Fine forceps
  • Platform rocker (Thermolyne Vari‐Mix or equivalent)
  • 37°C incubator or heating block
  • HPLC system (Hewlett Packard 1090M or equivalent), equipped with 250‐µl injection loop
  • Vydac C18 1.0 × 250–mm, 300‐angstrom pore size, 5‐micron particle size reversed‐phase HPLC column (Separations Group) or equivalent
  • Fraction collector with peak separator (e.g., Isco Foxy fraction collector with a Model 2150 peak detector or equivalent)

Alternate Protocol 1: Removal of Silver Prior to In‐Gel Digestion of Silver‐Stained Proteins

  • 5% (v/v) acetic acid
  • 30 mM potassium ferricyanide (prepare fresh)
  • 100 mM sodium thiosulfate (prepare fresh)

Alternate Protocol 2: Automated In‐Gel Trypsin Digestion Using the Amersham Biosciences Ettan TA Digester

  • 75% (v/v) acetonitrile
  • 0.1 mg/ml trypsin stock solution (see recipe but prepare fresh)
  • Ettan Spot Picker (Amersham Biosciences; optional)
  • Ettan TA Digester (Amersham Biosciences)

Support Protocol 1: C18 ZipTip Clean‐Up/Desalting of In‐Gel Digests Prior To Nanospray Ms/Ms Analysis

  • 100% and 15% acetonitrile
  • 0.1% (v/v) TFA/50% (v/v) acetonitrile
  • 50% (v/v) acetonitrile/0.1% (v/v) formic acid
  • C18 ZipTips (P‐10 tip size; Millipore)
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Figures

Videos

Literature Cited

Literature Cited
   Chaudhuri, A., Polyakova, J., Zbrzezna, V., Williams, K.R., Gulati, S., and Pogo, O. 1993. Cloning of glycoprotein D cDNA, which encodes the major subunit of the Duffy blood group system and the receptor for the Plasmodium vivax malaria parasite. Proc. Natl. Acad. Sci. U.S.A. 90:10793‐10797.
   Gharahdaghi, F., Weinberg, C.R., Meagher, D.A., Imai, B.S., and Mische, S.M. 1999. Mass spectrometric identification of proteins from silver‐stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity. Electrophoresis 20:601‐605.
   Han, D., Eng, J., Zhou, H., and Aebersold, R. 2001. Quantitative profiling of differentiation‐induced microsomal proteins using isotope‐coded affinity tags and mass spectrometry. Nat. Biotechnol. 19:945‐951.
   Jahnen, W., Ward, L.D., Reid, G.E., Moritz, R.L., and Simpson, R.J. 1990. Internal amino acid sequencing of proteins by in situ cyanogen bromide cleavage in polyacrylamide gels. Biochem. Biophys. Res. Commun. 166:139‐145.
   O'Farrell, P.H. 1975. High resolution two‐dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007‐4021.
   Safer, B., Cohen, R.B., Garfinkel, S., and Thompson, J.A. 1988. DNA affinity labeling of adenovirus type 2 upstream promoter sequence‐binding factors identifies two distinct proteins. Mol. Cell. Biol. 8:105‐113.
   Scheler, C., Lamer, S., Pan, Z., Li, Xin‐Ping, Salnikow, J., and Jungblut, P. 1998. Peptide mass fingerprint sequence coverage from differently stained proteins on two‐dimensional electrophoresis patterns by matrix assisted laser desorption/ionization mass spectrometry. Electrophoresis 19:918‐927.
   Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. 1996. Mass spectrometric sequencing of proteins from silver‐stained polyacrylamide gels. Anal. Chem. 68:850‐858.
   Tonge, R., Shaw, J., Middleton, B., Rowlinson, R., Rayner, S., Young, J., Pognan, F., Hawkins E., Currie, I., and Davison, M. 2001. Validation and development of fluorescence two‐dimensional differential gel electrophoresis proteomics technology. Proteomics 1:377‐396.
   Williams, K.R. and Stone, K.L. 1997. Enzymatic cleavage and HPLC peptide mapping of proteins. Mol. Biotechnol. 8:155‐167.
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