Removal of N‐Terminal Blocking Groups from Proteins

Joseph W. Leone1, Brian Hampton2, Elizabeth Fowler3, Mary Moyer4, Radha G. Krishna5, Christopher C.Q. Chin5

1 Pfizer VMRD, Kalamazoo, Michigan, 2 University of Maryland School of Medicine, Baltimore, Maryland, 3 AutoImmune, Lexington, Massachusetts, 4 Glaxo Research Institute, Research Triangle Park, North Carolina, 5 University of Texas Medical School, Houston, Texas
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 11.7
DOI:  10.1002/0471140864.ps1107s63
Online Posting Date:  February, 2011
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Abstract

Two enzymatic methods commonly used in N‐terminal sequence analysis of blocked proteins are presented: one uses pyroglutamate aminopeptidase for Nα‐pyrrolidone carboxyl‐proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl‐peptide hydrolase for Nα‐acyl‐proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl‐peptide hydrolase must include fragmentation of the protein before unblocking, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N‐terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis. Curr. Protoc. Protein Sci. 63:11.7.1‐11.7.20. © 2011 by John Wiley & Sons, Inc.

Keywords: blocked proteins; hydrolase; hydrazinolysis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Removal of Pyrrolidone Carboxylic Acid with Pyroglutamate Aminopeptidase Treatment of Proteins in Solution
  • Alternate Protocol 1: Removal of Pyrrolidone Carboxylic Acid with Pyroglutamate Aminopeptidase Treatment of Electroblotted Proteins
  • Support Protocol 1: Colorimetric Assay for Pyroglutamate Aminopeptidase Activity
  • Basic Protocol 2: Removal of Acylamino Acids by Hydrolase Treatment Using Succinic Anhydride as Blocking Reagent
  • Alternate Protocol 2: Removal of Acylamino Acids by Hydrolase Treatment Using Phenylisothiocyanate/Performic Acid as Blocking Reagents
  • Basic Protocol 3: Removal of Acetyl Groups by Acid Hydrolysis
  • Alternate Protocol 3: Removal of Acetyl Groups from N‐Terminal Ser and Thr with Anhydrous Trifluoroacetic Acid
  • Alternate Protocol 4: Automated Removal of Acetyl Groups From N‐Terminal Serine and Threonine with Anhydrous Trifluoroacetic Acid
  • Alternate Protocol 5: Removal of N‐Terminal Formyl Groups and Unblocking of Pyrrolidone Carboxyl Groups with Anhydrous Hydrazine
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Removal of Pyrrolidone Carboxylic Acid with Pyroglutamate Aminopeptidase Treatment of Proteins in Solution

  Materials
  • Protein or peptide, reduced and alkylated (unit 11.1)
  • PGA digestion buffer (see recipe)
  • pfu pyroglutamate aminopeptidase (PGAP; EC 3.4.19.3; Takara Bio Inc., cat. no. 7334)
  • 0.05 M acetic acid
  • Nitrogen (research grade)
  • Screw‐cap vial
  • Magnetic stirring plate
  • Additional reagents and equipment for dialysis (unit 4.4& APPENDIX )

Alternate Protocol 1: Removal of Pyrrolidone Carboxylic Acid with Pyroglutamate Aminopeptidase Treatment of Electroblotted Proteins

  • Single protein band on PVDF membrane (unit 10.7) containing ≥50 pmol protein
  • 0.1% (v/v) N‐methylpiperidine (NMP; Applied Biosystems) in methanol
  • Methanol
  • Polyvinylpyrrolidone (mol. wt. 40,000, PVP‐40; Sigma)
  • 0.1 M acetic acid
  • pfu pyroglutamate aminopeptidase (PGAP; EC 3.4.19.3; Takara Bio Inc., cat. no. 7334)
  • 5× and 1× PGAP buffer (supplied with enzyme)
  • 0.5‐ml and 1.5‐ml microcentrifuge tubes
  • 45°C shaking water bath

Support Protocol 1: Colorimetric Assay for Pyroglutamate Aminopeptidase Activity

  Materials
  • PGA digestion buffer (see recipe)
  • PGA‐N substrate solution (see recipe)
  • 25% (v/v) trichloroacetic acid
  • 0.1% (w/v) sodium nitrite
  • 0.5% (w/v) ammonium sulfamate
  • NED solution (see recipe)
  • 13 × 100–mm test tubes
  • 37°C incubator or water bath

Basic Protocol 2: Removal of Acylamino Acids by Hydrolase Treatment Using Succinic Anhydride as Blocking Reagent

  Materials
  • 1 nmol/10 µl protein/peptide dissolved in a small volume of suitable volatile solvent (e.g., 50 to 100 mM ammonium bicarbonate or ammonium carbonate)
  • 20% (v/v) acetic acid
  • Succinic anhydride
  • 12% (v/v) triethylamine (TEA)
  • 20% (v/v) trifluoroacetic acid (TFA)
  • Ethyl ether
  • Hydrolase buffer I: 50 µM sodium phosphate buffer (pH 7.2; appendix 2E)/1 mM EDTA/2 mM MgCl 2
  • 0.5 M NaOH
  • Acylaminoacyl‐peptide hydrolase (EC 3.4.19.1; Pierce, Takara Biochemical, Boehringer Mannheim, or Sigma)
  • 70% (v/v) formic acid
  • Acylase buffer: 100 mM sodium phosphate buffer, pH 7.0 ( appendix 2E)
  • Acylase I (3.5.1.14; e.g., Sigma)
  • 0.2 M sodium citrate with pH adjusted to pH 2.2 using concentrated HCl
  • 0.9 × 7.5–cm glass test tubes
  • Additional reagents and equipment for endoprotease digestion (unit 11.1) or cyanogen bromide cleavage (unit 11.4), and reversed‐phase HPLC (unit 11.6)

Alternate Protocol 2: Removal of Acylamino Acids by Hydrolase Treatment Using Phenylisothiocyanate/Performic Acid as Blocking Reagents

  • 12.5% (v/v) trimethylamine
  • Phenylisothiocyanate
  • Benzene
  • Ethyl acetate
  • Performic acid, freshly prepared by mixing 95 µl of 99% formic acid and 5 µl of 30% hydrogen peroxide
  • Hydrolase buffer II: 10 mM sodium phosphate buffer (pH 7.2; appendix 2E)/0.1 mM dithiothreitol (DTT)
  • 0.5% (v/v) trifluoroacetic acid (TFA)
  • 0.9 × 7.5–cm glass test tubes
  • IEC CL clinical centrifuge or equivalent
  • Additional reagents and equipment for endoprotease digestion (unit 11.1) or cyanogen bromide cleavage (unit 11.4), and reversed‐phase HPLC (unit 11.6)

Basic Protocol 3: Removal of Acetyl Groups by Acid Hydrolysis

  Materials
  • 2 to 5 nmol blocked peptide
  • 1 N HCl
  • 70% (v/v) formic acid or other suitable solvent
  • 1‐ml glass vial
  • 14‐oz. propane torch
  • 110°C oven

Alternate Protocol 3: Removal of Acetyl Groups from N‐Terminal Ser and Thr with Anhydrous Trifluoroacetic Acid

  Materials
  • Polybrene solution: 100 mg/ml Polybrene in 6.7 mg/ml NaCl (0.115 M final)
  • 0.1 nmol/µl protein or peptide in a suitable volatile solvent
  • Anhydrous trifluoroacetic acid (TFA)
  • 12‐mm‐diameter glass‐fiber filter disc, treated with TFA (standard sequencer disc, Applied Biosystems)
  • 1.5‐ml polypropylene microcentrifuge tube with cap
  • 45° and 65°C oven

Alternate Protocol 4: Automated Removal of Acetyl Groups From N‐Terminal Serine and Threonine with Anhydrous Trifluoroacetic Acid

  Materials
  • At least 100 pmol of a highly pure N‐terminally blocked protein sample immobilized either on PVDF or a polybrene‐coated, TFA‐treated glass fiber filter
  • Optional:
    • Methanol
    • Hexafluoroisopropanol (HFIP; Sigma, cat. no. H8508)
    • Anhydrous TFA
  • Automated Edman sequencer such as the Applied Biosystems Procise sequencer
  • Additional reagents and equipment for electroblotting (unit 10.7; optional) and sequence analysis (unit 11.10)

Alternate Protocol 5: Removal of N‐Terminal Formyl Groups and Unblocking of Pyrrolidone Carboxyl Groups with Anhydrous Hydrazine

  Materials
  • ∼100 pmol protein, free or bound to polyvinylidine difluoride (PVDF) membrane
  • 3‐phenyl‐2‐thiohydantoin (PTH) derivatives of β‐hydrazidyl‐aspartate, γ‐hydrazidyl‐glutamate, and ornithine (standards)
  • Anhydrous hydrazine
  • 4 × 44–mm (small) test tubes
  • 13 × 100–mm (large) test tubes
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Figures

Videos

Literature Cited

   Abraham, G.N. and Podell, D.N. 1981. Pyroglutamic acid. Mol. Cell. Biochem. 38:181‐190.
   Arfin, S.M. and Bradshaw, R.A. 1988. Cotranslational processing and protein turnover in eukaryotic cells. Biochemistry 27:7979‐7984.
   Bergman, T., Gheorghe, M.T., Hjelmqvist, L., and Jörnvall, H. 1996. Alcoholytic deblocking of N‐terminally acetylated peptides and proteins for sequence analysis. FEBS Lett. 390:199‐202.
   Capecchi, M.R. 1966. Initiation of E. coli proteins. Proc. Natl. Acad. Sci. U.S.A. 55:1517‐1524.
   Carr, S.A., Biemann, K., Shoji, S., Parmelee, D.C., and Titani, K. 1982. N‐tetradecanoyl is the NH2 terminal blocking group of the catalytic subunit of cyclic AMP‐dependent protein kinase from bovine cardiac muscle. Proc. Natl. Acad. Sci. U.S.A. 79:6128‐6131.
   Chin, C.C.Q. and Wold, F. 1985. Studies on Nα‐acetylated proteins: The N‐terminal sequences of two muscle enolases. Biosci. Rep. 5:847‐854.
   Chin, C.C.Q. and Wold, F. 1986. Reinventing the wheel: General approaches to the elucidation of blocked N‐terminal sequences. In Methods in Protein Sequence Analysis (K.A. Walsh, ed.) pp. 505‐512. Humana Press, Clifton, N.J.
   Crimmins, D.L., McCourt, D.W., and Schwartz, B.D. 1988. Facile analysis and purification of deblocked N‐terminal pyroglutamyl peptides with a strong cation‐exchange sulfoethyl aspartamide column. Biochem. Biophys. Res. Comm. 156:910‐916.
   Dimaline, R. and Reeve, J.R. Jr. 1983. Reversed‐phase high‐performance liquid chromatography used to monitor enzymatic cleavage of pyrrolidone carboxylic acid from regulatory peptides. J. Chromatogr. 257:355‐360.
   Doolittle, R.F. 1972. Terminal pyrrolidonecarboxylic acid: Cleavage with enzymes. Methods Enzymol. 25:231‐244.
   Doolittle, R.F. and Armentrout, R.W. 1968. Pyrrolidonyl peptidase. An enzyme for selective removal of pyrrolidonecarboxylic acid residues from polypeptides. Biochemistry 7:516‐521.
   Farries, T.C., Harris, A., Auffret, A.D., and Aitken, A. 1991. Removal of N‐acetyl groups from blocked peptides with acylpeptide hydrolase: Stabilization of the enzyme and its application to protein sequencing. Eur. J. Biochem. 196:679‐685.
   Gade, W. and Brown, J.L. 1987. Purification and partial characterization of N‐acyl peptide hydrolase from liver. J. Biol. Chem. 253:5012‐5018.
   Hirano, H., Komatsu, S., Kajiwara, H., and Tsunasawa, S. 1993. Microsequence analysis of the N‐terminally blocked proteins immobilized on polyvinylidene difluoride membrane by Western blotting. Electrophoresis 14:839‐846.
   Huynh, Q.K., Vaaler, G.L., Recsei, P.A., and Snell, E.E. 1984. Histidine carboxylase of Lactobacillus 30a. Sequences of the cyanogen bromide peptides from the α chain. J. Biol. Chem. 259:2826‐2832.
   Kaji, H. 1976. Amino‐terminal arginylation of chromosomal proteins of arginyl‐tRNA. Biochemistry 15:5121‐5125.
   Kennedy, L. and Baynes, J.W. 1984. Nonenzymatic glycosylation and the chronic complications of diabetes: An overview. Diabetologia 26:93‐98.
   Kobayashi, K. and Smith, J. 1987. Ac‐peptide hydrolase from rat liver: Characterization of enzyme reaction. J. Biol. Chem. 262:11435‐11445.
   Kolattukudy, P.E. 1984. Detection of an N‐terminal glucuronamide linkage in proteins. Methods Enzymol. 106:210‐217.
   Krishna, R.G. and Wold, F. 1992. Specificity determinants of acetylaminoacyl‐peptide hydrolase. Protein Sci. 1:582‐589.
   Krishna, R.G. and Wold, F. 1993. Post‐translational modification of proteins. Adv. Enzymol. Relat. Areas Mol. Biol. 67:265‐298.
   Landon, M. 1977. Cleavage at aspartyl‐prolyl bonds. Methods Enzymol. 47:145‐149.
   McDonald, J.K. and Barrett, A.J. 1986. Pyroglutamyl peptidase I. In Mammalian Proteases: A Glossary and Bibliography, Vol. 2. Exopeptidases (J.K. McDonald and A.J. Barrett, eds.) pp. 305‐307. Academic Press, New York.
   Miyatake, N., Kamo, M., Satake, K., Uchiyama, Y., and Tsugita, A. 1993. Removal of N‐terminal formyl groups and deblocking of pyrrolidone carboxylic acid of proteins with anhydrous hydrazine vapor. Eur. J. Biochem. 212:785‐789.
   Moscarello, M.A., Pang, H., Pace‐Asciak, C.R., and Wood, D.D. 1992. The N‐terminus of human myelin basic protein consists of C2, C4, C6, and C8 alkyl carboxylic acids. J. Biol. Chem. 267:9779‐9782.
   Nagasawa, H., Maruyama, K., Sato, B., Hietter, H., Isogai, A., Tamura, S., Ishizaki, H., Semba, R., and Suzuki, A. 1988. Structure and synthesis of bombesin from the silkworm Bombyx mori. In Peptide Chemistry (T. Shiba and S. Sakakibara, eds.) pp. 123‐126. Protein Research Foundation, Osaka, Japan.
   Neubert, T.A., Johnson, R.S., Hurley, J.B., and Walsh, K.A. 1992. The rod transducin subunit amino terminus is heterogeneously fatty acylated. J. Biol. Chem. 267:18274‐18277.
   Orlowski, M. and Meister, A. 1971. Enzymology of pyrrolidone carboxylic acid. Enzymes 4:123‐151.
   Recsei, P.A. and Snell, E.E. 1984. Pyruvoyl enzymes. Annu. Rev. Biochem. 53:357‐387.
   Sakiyama, F. 1990. New tools for protein sequence analysis. Trends Biotech. 8:282‐288.
   Schultz, J. 1967. Cleavage at aspartic acid. Methods Enzymol. 11:255‐263.
   Siegel, F.L. 1988. Enzymatic N‐methylation of calmodulin. In Advances in Post‐translational Modification of Proteins and Aging (V. Zappia, P. Galletti, R. Porta, and F. Wold, eds.) pp. 341‐352. Plenum Press, New York.
   Sokolik, C.W., Liang, T.C., and Wold, F. 1994. Studies on the specificity of acetylaminoacyl‐peptide hydrolase. Protein Sci. 2:126‐131.
   Szewczuk, A. and Kwiatkowska, J. 1970. Pyrrolidonyl peptidase in animal, plant and human tissues: Occurrence and some properties of the enzyme. Eur. J. Biochem. 15:92‐96.
   Tsunasawa, S., Takakura, H., and Sakiyama, F. 1990. Microsequence analysis of N‐acetylated proteins. J. Prot. Chem. 9:265‐266.
   Wellner, D., Panneerselvam, C., and Horecker, B.L. 1990. Sequencing of peptides and proteins with blocked N‐terminal amino acids: N‐acetylserine or N‐acetylthreonine. Proc. Natl. Acad. Sci. U.S.A. 87:1947‐1949.
Key References
   Chin and Wold, 1986. See above.
  Describes chemical unblocking using aqueous acid.
   Crimmins et al., 1988. See above.
  Describes digestion of peptides and a cation‐exchange method for isolating digestion products.
   Dimaline and Reeve, 1983. See above.
  Describes digestion of small amounts of peptides and a reversed‐phase HPLC method for monitoring digestion and isolating digestion products.
   Hirano et al., 1993. See above.
  A general overview of unblocking.
   Krishna, R.G. 1992. N‐Acylaminoacyl‐peptide hydrolase: Specificity and use to unblock N‐acetylated proteins. In Techniques in Protein Chemistry III (R.H. Angeletti, ed.) pp. 77‐84. Academic Press, San Diego.
  Describes the use of hydrolase with succinic acid blocking.
   Krishna, R.G., Chin, C.C.Q., and Wold, F. 1991. N‐terminal sequencing of N‐acetylated proteins after unblocking with N‐acetylaminoacyl‐peptide hydrolase. Anal. Biochem. 199:45‐50.
  Describes the use of hydrolase with succinic acid blocking.
   Miyatake et al., 1993. See above.
  Describes chemical unblocking using hydrazine.
   Moyer, M., Harper, A., Payne, G., Ryals, J., and Fowler, E. 1990. In situ digestion with pyroglutamate aminopeptidase for N‐terminal sequencing of electroblotted proteins. J. Prot. Chem. 9:282‐283.
  Describes enzymatic unblocking of electroblotted proteins.
   Podell, D.N. and Abraham, G.N. 1978. A technique for the removal of pyroglutamic acid from the amino terminus of proteins using calf liver pyroglutamate aminopeptidase. Chem. Biophys. Res. Comm. 81:176‐185.
  Describes the basic method for digestion of milligram quantities of protein.
   Tsunasawa et al., 1990. See above.
  Describes hydrolysis with phenylcarbamate blocking.
   Van Beeumen, J., Van Driessche, G., Huitema, F., Duine, J.A., and Canters, G.W. 1993. N‐terminal heterogeneity of methylamine dehydrogenase from Thiobacillus versutus. FEBS Lett. 333:188‐192.
  Describes digestion and subsequent sequencing of microgram quantities of protein.
   Wellner et al., 1990. See above.
  Describes chemical unblocking with anhydrous acid.
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