Enzymatic Digestion of Proteins in Solution

Lise R. Riviere1, Paul Tempst2

1 OsteoArthritis Sciences, Inc., Cambridge, Massachusetts, 2 Memorial Sloan‐Kettering Cancer Center, New York, New York
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 11.1
DOI:  10.1002/0471140864.ps1101s00
Online Posting Date:  May, 2001
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Abstract

Analysis of protein covalent structure is less complex and more accurate when performed on peptides derived from the larger protein. In contrast to acid‐promoted total hydrolysis, peptides are typically generated by selective proteolysis, i.e., by specifically cleaving peptide bonds with endoproteases that have varying degrees of specificity. This unit presents a protocol that can be used to generate peptide fragments from intact, undenatured proteins. Fragments can be analyzed directly by mass spectrometry (MS) or, more often, are first separated by reversed‐phase HPLC (RP‐HPLC) and then analyzed by MS or automated sequencing. Most proteins are resistant to enzymatic proteolysis under nondenaturing conditions or are not soluble in aqueous solution. Digestion procedures performed in the presence of chaotropic agents and SDS are described, and support protocols provide instructions for preparing enzyme stocks and reducing and alkylating peptides prior to sequencing or HPLC analysis.

     
 
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Table of Contents

  • Basic Protocol 1: Digestion of Proteins Under Nondenaturing Conditions
  • Alternate Protocol 1: Digestion of Proteins in the Presence of Urea or Guanidine·HCl
  • Alternate Protocol 2: Digestion of Proteins in the Presence of SDS
  • Support Protocol 1: Preparing and Using Enzyme Stocks
  • Support Protocol 2: Reduction and S‐Alkylation of Peptides in Digest Mixtures
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Digestion of Proteins Under Nondenaturing Conditions

  Materials
  • 100 pmol to 5 nmol protein sample, as pellet or solution
  • 1 × and 10× digestion buffer (see Table 11.1.1)
  • 20% 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propanesulfonate (CHAPS), 20% octyl glucoside, or 20% Nonidet P‐40 (NP‐40; Calbiochem)
  • 100% acetonitrile
  • 1 µg/µl enzyme stock (see protocol 4; store at −20°C)
  • Trifluoroacetic acid (TFA; Pierce)
  • Solvent A: 0.1% (v/v) TFA in water
  • Solvent B: 0.09% (v/v) TFA in 70% (v/v) acetonitrile (Burdick & Jackson)
  • Sonicator bath (Branson)
  • Phast Gel system (Pharmacia), optional
  • HPLC system, with C18 or C4 reversed‐phase column, 4.6‐mm or 2.1‐mm (e.g., Vydac); UV detector; chart recorder
  • Additional reagents and materials for SDS‐PAGE (unit 10.1), gel staining (unit 10.5), and HPLC analysis of peptides (unit 11.6)
    Table 1.1.1   Materials   Conditions for Endoprotease Activity   Conditions for Endoprotease Activity

    Condition Enzyme
    T C KC DN EC S H P E PA
    Nondenaturing
    Buffer 0.1 M AB 0.1 M AB 0.1 M AB 0.1 M AB 0.05 M AB 0.1 M AB 0.1 M AB/1 mM CaCl 2 0.1% TFA 0.2 M TC 0.1 M AB/1 mM EDTA
    Time 2 hr 2 hr 2 hr 5 hr 5 hr 1 hr 2 hr 1 hr 18 hr 5 hr
    Low/high pH
    Acid +
    NH 4OH 0.1 M 1 M
    Chaotropes
    Urea 4 M 2 M 8 M 2 M 2 M c 8 M c 8 M c 2 M 8 M c
     Buffer 0.1 M AB/+5 mM CaCl 2 0.1 M AB 0.2 M TC 0.1 M AB 0.05 M AB 0.1 M TC 0.1 M AB/+5 mM Ca 0.1 M TC/+5 mM Ca ND 0.2 M TC 0.1 M AB/1 mM EDTA
     Time 15 hr 5 hr 5 hr 5 hr 5 hr 1 hr 2 hr 18 hr 5 hr
    Gu⋅HCl 2 M 2 M 1 M c 1 M c 2 M c 2 M 2 M c 2 M
     Buffer 0.2 M AB 0.2 M TC 0.1 M AB 0.2 M TC 0.05 M AB 0.1 M AB 12 mM HCl 0.2 M TC 0.1 M AB/1 mM EDTA
     Time 24 hr 5 hr 18 hr 18 hr 2 hr 2 hr 18 hr 18 hr
    Detergents d
    SDS <0.1% 0.1% c 1% <0.1% 0.1% c 1% 0.1% c ND 0.1%
    CHAPS 2% 1% 2% 2% 2% c 2% c 2% ND 1% 1%
    OG and NP‐40 2% 1% 2% 2% 2% c 2% c 2% ND 1% 2% c
    Organic solvents d
    MeCN 40% 30% 40% 40% 20% c 40% 40% 20% c 40% 40%
    IPA 40% 20% 40% 40% 20% c 40% c 40% c 20% c 20% 40%
    Reducing agent
    2‐ME 0.5% 0.1% 0.5% ND ND ND ND ND

     aBuffers listed here are 1× stocks; it may be necessary to prepare 10× stocks for certain parts of the protocols. Listed here are the highest concentrations of additives that still permit adequate proteolysis.
     bAbbreviations: Enzymes: C, chymotrypsin; DN, endoproteinase Asp‐N; E, elastase; EC, endoproteinase Glu‐C; H, thermolysin; KC, endoproteinase Lys‐C; P, pepsin; PA, papain; S, subtilisin; T, trypsin. Other: —, does not work; +, works at pH 2.0; AB, ammonium bicarbonate; Gu⋅HCl, guanidine hydrochloride; IPA, isopropanol; 2‐ME, 2‐mercaptoethanol; MeCN, acetonitrile; OG, octyl glucoside; TC, Tris⋅Cl, pH 8.5; TFA, trifluoroacetic acid.
     cRestricted digest. Note that for the 8M urea conditions, the final concentration is actually 7.3 M after addition of the buffer.
     dConcentrations higher than 2% CHAPS, octyl glucoside, and NP‐40, and 40% acetonitrile and isopropanol were not tested.

Alternate Protocol 1: Digestion of Proteins in the Presence of Urea or Guanidine·HCl

  • 8 M urea (sequenal grade, Pierce; store up to several weeks at room temperature over Bio‐Rad AG 501‐X8 mixed‐bed resin)
  • 6 M guanidine⋅HCl (sequenal grade, Pierce), prepared fresh immediately before use
  • Milli‐Q water or equivalent
  • 50°C water bath

Alternate Protocol 2: Digestion of Proteins in the Presence of SDS

  • 1× digestion buffer with 1% or 0.1% (w/v) SDS (see Table 11.1.1)
  • 10% and 1% (w/v) SDS (Bio‐Rad)
  • 10× digestion buffer (no SDS)
  • 1 M guanidine⋅HCl (sequenal grade, Pierce), prepared immediately before use
  • 60°C and 95°C water baths

Support Protocol 1: Preparing and Using Enzyme Stocks

  Materials
  • Enzymes:
  •  Trypsin (sequencing grade, Boehringer Mannheim 1047‐841 or Promega V5111)
  •  Chymotrypsin (sequencing grade, Boehringer Mannheim 1334‐131)
  •  Endoproteinase Glu‐C (endo Glu‐C; sequencing grade, Boehringer Mannheim 1047‐817)
  •  Endoproteinase Asp‐N (endo Asp‐N; sequencing grade, Boehringer Mannheim 1054‐859)
  •  Endoproteinase Lys‐C (endo Lys‐C; Wako Chemicals 129‐02541)
  •  Subtilisin (Sigma P5380)
  •  Thermolysin (Sigma P1512)
  •  Pepsin (Sigma P6887)
  •  Elastase (Sigma PE0258)
  •  Papain (Sigma P3125)
  • Milli‐Q water or equivalent
  • Trifluoroacetic acid (TFA; Pierce)
  • 5 mg/ml cytochrome c (Sigma)
  • 10× digestion buffer (see Table 11.1.1)
  • 5 mg/ml β‐lactoglobulin (Sigma)
  • 5 mg/ml triosephosphate isomerase (Sigma)
  • 8 M urea (sequenal grade, Pierce; store up to several weeks at room temperature over Bio‐Rad AG 501‐X8 mixed‐bed resin)
  • 6 M guanidine⋅HCl (sequenal grade, Pierce)
  • HPLC system and 4.6‐mm reversed‐phase column (see protocol 1)

Support Protocol 2: Reduction and S‐Alkylation of Peptides in Digest Mixtures

  Materials
  • Peptide digest mixture (see protocol 1 or protocol 2Alternate Protocols 1 or protocol 32)
  • 2‐mercaptoethanol (2‐ME; Bio‐Rad): undiluted and 10% (v/v) solution (prepare in Milli‐Q water or equivalent immediately before use)
  • 4‐vinylpyridine (Aldrich; store at −20°C), undiluted and 10% (v/v) solution (prepare in high‐purity grade ethanol immediately before use)
  • Argon (prepurified)
  • HPLC system and reversed‐phase column (see protocol 1)
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Figures

  •   FigureFigure 11.1.1 Effects of additives on proteolytic digestion of 1 nmol of cytochrome c with 1 µg trypsin (left) or 1 µg endoproteinase Lys‐C (right). Digest conditions are listed on the panels. Digest mixtures were separated by reversed‐phase HPLC on a 4.6‐mm Vydac C18 column, using gradient conditions and flow rate given in the . Abbreviations: MeCN, acetonitrile; OG, octyl glucoside.
  •   FigureFigure 11.1.2 Enzymatic digestion of protease‐resistant substrates. 2 nmol trypsin inhibitor (left panels), 1 nmol superoxide dismutase (middle panels), and 1 nmol triosephosphate isomerase (right panels) were digested by 1 µg endoproteinase Lys‐C. Digestion conditions were as follows: (A) no additives; (B) 8 M urea; (C) 1 M guanidine⋅HCl (D) 2 Mguanidine⋅HCl; (E) 1% SDS (with guanidine⋅DS precipitation). Digest mixtures were separated by reversed‐phase HPLC on a 4.6‐mm Vydac C18 column, using gradient conditions and flow rate as given in the .

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Literature Cited

Literature Cited
   Beynon, R.J. and Bond, J.S. 1989. Proteolytic Enzymes: A Practical Approach. Oxford University Press, Oxford.
   Christianson, T. and Paech, C. 1994. Peptide mapping of subtilisins as a practical tool for locating protein sequencing errors during extensive protein engineering projects. Anal. Biochem. 223:119‐129.
   Cleveland, D.W., Fischer, S.G., Kirschner, M.W., and Laemmli, U.K. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate by gel electrophoresis. J. Biol. Chem. 252:1102‐1106.
   Henzel, W.J., Billeci, T.M., Stults, J.T., Wong, S.C., Grimley, C., and Watanabe, C. 1994. Identification of 2‐D gel proteins at the femtomole level by molecular mass searching of peptide fragments in the protein sequence database. In Techniques in Protein Chemistry V (J.W. Crabb, ed.) pp. 3‐9. Academic Press, San Diego.
   Keil, B. 1981. Enzymic cleavage of proteins. In Methods in Protein Sequence Analysis (M. Elzinga, ed.) pp. 291‐304. Humana Press, Clifton,N.J.
   Keil, B. 1991. Specificity of Proteolysis. Springer‐Verlag, Heidelberg.
   Marks, A.R., Fleischer, S., and Tempst, P. 1990. Surface topography analysis of the ryanodine receptor/junctional channel complex based on proteolysis sensitivity mapping. J. Biol. Chem. 265:13143‐13149.
   Riviere, L.R., Fleming, M., Elicone, C., and Tempst, P. 1991. Study and applications of the effects of detergents and chaotropes on enzymatic proteolysis. In Techniques in Protein Chemistry II (J.J. Villafranca, ed.) pp. 171‐179. Academic Press, San Diego.
   Tempst, P. and Van Beeumen, J. 1983. The amino acid sequence of cytochrome C‐556 from Agrobacterium tumefaciens strain Apple 185. Eur. J. Biochem. 135:321‐330.
   Tempst, P., Link, A.J., Riviere, L.R., Fleming, M., and Elicone, C. 1990. Internal sequence analysis of proteins separated on polyacrylamide gels at the sub‐microgram level: Improved methods, applications and gene cloning strategies. Electrophoresis 11:537‐553.
   Vangrysperre, W., Ampe, C., Kersters‐Hilderson, H., and Tempst, P. 1989. Single active‐site histidine in D‐xylose isomerase from Streptomyces violaceoruber: Identification by chemical derivatization and peptide mapping. Biochem. J. 263:195‐199.
   Welinder, K.G. 1988. Generation of peptides suitable for sequence analysis by proteolytic cleavage in reversed‐phase high‐performance liquid chromatography solvents. Anal. Biochem. 174:54‐64.
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