C‐Terminal Sequence Analysis

Tomas Bergman1, Ella Cederlund1, Hans Jörnvall1, Elizabeth Fowler2

1 Karolinska Institutet, Stockholm, 2 Millennium Pharmaceuticals, Cambridge, Massachusetts
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 11.8
DOI:  10.1002/0471140864.ps1108s31
Online Posting Date:  May, 2003
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Abstract

Carboxy‐terminal (C‐terminal) sequence analysis is used for direct confirmation of the C‐terminal sequence of native and expressed proteins, for detection and characterization of protein processing at the C‐terminus, for identification of post‐translational proteolytic cleavages, and for obtaining partial sequence information on N‐terminally blocked protein samples in order to facilitate design of oligonucleotide probes for gene cloning. This unit describes an automated chemical method and a manual enzymatic (carboxypeptidase digestion) method for determining C‐terminal sequence information. Carboxypeptidase digestion requires only a standard amino acid analysis method.

     
 
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Table of Contents

  • Basic Protocol 1: Automated C‐Terminal Sequence Analysis
  • Basic Protocol 2: Manual C‐Terminal Sequencing Using Carboxypeptidases Followed by Mass Spectrometry
  • Basic Protocol 3: C‐Terminal Sequencing Using Carboxypeptidases Followed by Amino Acid Analysis
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Automated C‐Terminal Sequence Analysis

  Materials
  • Gel‐separated protein sample (one‐ or two‐dimensional separations; units 10.1 10.4)
  • Coomassie blue (unit 10.5)
  • Analytical‐grade chemicals for N‐ and C‐terminal sequence analysis (Table 11.8.1 provides a list of reagents and solutions employed in the C‐terminal sequencer; Applied Biosystems)
  • Methanol
  • 0.5% and 0.1% (v/v) trifluoroacetic acid (TFA)
  • 1.25% (v/v) diisopropylethylamine (DIEA) in heptane
  • 1% (v/v) phenylisocyanate (PIC; Sigma) in acetonitrile
  • PVDF membranes (e.g., Immobilon‐P, Millipore; ProBlott, Applied Biosystems)
  • 1.5‐ml microcentrifuge tubes
  • ProSorb cartridges for sample immobilization and desalting (Applied Biosystems)
  • Petri dishes
  • 60°C heating block
  • For C‐terminal sequencer degradation: Procise C instrument (Applied Biosystems) equipped with a blot cartridge (Applied Biosystems) and a 2 × 220–mm reversed‐phase (C 18) column (Brownlee Spheri‐5 PTC, 5 µm) operated at 0.3 ml/min and with detection at 257 nm
  • For sequential N‐terminal and then C‐terminal degradation: Procise HT instrument (Applied Biosystems) equipped with the same C 18 column described for the Procise C instrument, but with detection at 269 nm
  • Additional reagents and equipment for electroblotting (unit 10.7), N‐terminal degradation (unit 11.10)
NOTE: Only the modifications of the standard program are given here. The standard program comes with the instrument and the operator has only to include the extra points into the existing program.
Table 1.8.1   Materials   Reagents, Solutions, and Conditions for C‐Terminal Sequencer Analysis a   Reagents, Solutions, and Conditions for C‐Terminal Sequencer Analysis

Parameter changed Manufacturer's protocol Modified protocol
Dilution
(C1) b ATH aa standard 100 pmol/injection 10 pmol/injection
(C3) bN‐methylimidazole/acetonitrile Use as delivered Dilute 1:1 with acetonitrile
(C4) b piperidine thiocyanate/acetonitrile Use as delivered Dilute 1:1 with acetonitrile
(C6) b acetic anhydride/lutidine/acetonitrile Use as delivered Dilute 1:1 with acetonitrile
(C8) b bromomethyl‐naphthalene/acetonitrile Use as delivered Dilute 1:1 with acetonitrile
(C10) b tetrabutylammonium thiocyanate/acetonitrile Use as delivered Dilute 1:1 with acetonitrile
(C11) b DIEA/heptane Use as delivered Diluted 1:1 with heptane
2% phenylisocyanate/acetonitrile According to Applied Biosystems recommendation Dilute 1:1 with acetonitrile
Chromatography
Solvent A 3.5% THF in 82.5 mM sodium acetate pH 3.8, DIEA/acetone 3.8% ethanol in 30 mM sodium acetate, pH 3.8, acetone
Solvent B 18% THF in acetonitrile 7.1% ethanol in acetonitrile
Temperature
Column 45°C 40°C
Transfer flask 45°C 40°C

 aParameters were modified in relation to the manufacturer's protocol to improve sensitivity and length of degradation.
 bLabel (bottle position) according to Applied Biosystems nomenclature.

Basic Protocol 2: Manual C‐Terminal Sequencing Using Carboxypeptidases Followed by Mass Spectrometry

  Materials
  • Peptide or protein of interest (1 to 3 pmol, ≥90% purity)
  • ∼2 × 10−3 U/µl carboxypeptidase Y (CPY) in 30 mM ammonium citrate, pH 6.1
  • ∼2 × 10−3 U/µl carboxypeptidase P (CPP) in 30 mM ammonium citrate, pH 4.5
  • Sequazyme kit (Applied Biosystems) containing (or individual reagents):
  •  MALDI matrix: α‐cyano‐4‐hydroxy cinnamic acid
  •  MALDI matrix diluent: 50% acetonitrile in 0.3% trifluoroacetic acid (TFA)
  • 1 pmol/µl ACTH 18‐39 peptide sequencing standard prepared in HPLC‐grade water
  • 1 pmol/µl ACTH 18‐39 calibration standard prepared in HPLC‐grade water
  • 1 pmol/µl angiotensin I calibration standard prepared in HPLC‐grade water
  • C‐terminal sequencer
  • Sequence analysis software
NOTE: If the peptide of interest is considerably larger than the recommended standards, additional calibration standards in the appropriate mass range should be used.

Basic Protocol 3: C‐Terminal Sequencing Using Carboxypeptidases Followed by Amino Acid Analysis

  Materials
  • Purified protein
  • Digestion buffer: 100 mM N‐ethylmorpholine (NEM, Aldrich), adjusted to pH 6.0 with acetic acid; store at 4°C
  • Aminobutyric acid (Sigma)
  • Carboxypeptidases A, B, P, and/or Y (20 µg/vial sequencing grade, Boehringer Mannheim)
  • Trifluoroacetic acid (TFA, Fluka)
  • Titertubes with plugs (Bio‐Rad)
  • 5‐kDa‐MWCO and (optionally) 30‐ and 10‐kDa‐MWCO ultrafree filtration devices (Millipore)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and amino acid analysis (unit 3.2)
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Figures

Videos

Literature Cited

Literature Cited
   Ambler, R.P. 1972. Enzymatic hydrolysis with carboxypeptidases. Methods Enzymol. 25:143‐154.
   Bergman, T., Cederlund, E., and Jörnvall, H. 2001. Chemical C‐terminal protein sequence analysis: Improved sensitivity, length of degradation, proline passage, and combination with Edman degradation. Anal. Biochem. 290:74‐82.
   Bergman, T., Cederlund, E., and Jörnvall, H. 2002. Carboxy‐terminal sequence analysis. In Proteins and Proteomics: A Laboratory Manual. (R. Simpson, ed.) Cold Spring Harbor Laboratory Press. Cold Spring Harbor. N.Y. pp. 310‐317 and 332‐335.
   Boyd, V.L., Bozzini, M., Guga, P.J., DeFranco, R.J., and Yuan, P.‐M. 1995. Activation of the carboxy terminus of a peptide for carboxy‐terminal sequencing. J. Organic Chem. 60:2581‐2587.
   Boyd, V.L., Bozzini, M., Zon, G., Noble, R.L., and Mattaliano, R.J. 1992. Sequencing of peptides and proteins from the carboxy terminus. Anal. Biochem. 206:344‐352.
   Geng, M., Zhang, X., Bina, M., and Regnier, F. 2001. Proteomics of glycoproteins based on affinity selection of glycopeptides from tryptic digests. J. Chromatography B 752:293‐306.
   Gheorghe, M.T., Jörnvall, H., and Bergman, T. 1997. Optimized alcoholytic deacetylation of N‐acetyl‐blocked polypeptides for subsequent Edman degradation. Anal. Biochem. 254:119‐125.
   Hayashi, R. 1977. Carboxypeptidase Y in sequence determination of peptides. Methods Enzymol. 47:84‐93.
   Hofmann, T. 1976. Penicillocarboxy peptidases S‐1 and S‐2. Methods Enzymol. 45:587‐599.
   Horn, M.J., Mayhew, J.W., and O'Dea, K.C. 1995. A method for the characterization of the C‐terminus of peptides and proteins. Protein Sci. 2:155.
   Isobe, T., Ichimura, T., and Okuyama, T. 1986. Identification of the C‐terminal portion of a protein by comparative peptide mapping. Anal. Biochem. 155:135‐140.
   Jones, B.N. 1986. Microsequence analysis by enzymatic methods. In Methods of Protein Microcharacterization. (J.E. Shively, ed.) pp. 337‐361. Humana Press, Totowa, N.J.
   Jonsson, A.P., Griffiths, W.J., Bratt, P., Johansson, I., Strömberg, N., Jörnvall, H., and Bergman, T. 2000. A novel Ser O‐glucuronidation in acidic proline‐rich proteins identified by tandem mass spectrometry. FEBS Lett. 475:131‐134.
   Lu, H.S., Klein, M.L., and Lai, P.H. 1988. Narrowbore high‐performance liquid chromatography of phenylthiocarbamyl amino acids and carboxypeptidase P digestion for protein C‐terminal sequence analysis. J. Chromatogr. 447:335‐364.
   Michel, G., Chauvet, M.T., Clarke, C., Bern, H., and Acher, R. 1993. Chemical identification of the mammalian oxytocin in a holocephalian fish, the ratfish (Hydrolagus colliei). Gen. Comp. Endocrinol. 92:260‐268.
   Mondino, A., Bongiovanni, G., Fumero, S., and Rossi, L. 1972. An improved method of plasma deproteination with sulphosalicylic acid for determining amino acids and related compounds. J. Chromatogr. 74:255‐263.
   Patterson, D.H., Tarr, G., Regnier, F.E., and Martin, S.A. 1995. C‐terminal ladder sequencing via matrix‐assisted laser‐desorption mass spectrometry coupled with carboxypeptidase Y time‐dependent and concentration‐dependent digestions. Anal. Chem. 67:3971‐3978.
   Patterson, D.H., Tarr, G.E., Hine, W.M., Vestal, M.L. and Martin, S.A. 1996. Proceedings from the 1996 ASMS conference.
   Rosnack, K.J. and Stroh, J.G. 1992. C‐terminal sequencing of peptides using electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 6:637‐640.
   Sechi, S. and Chait, B.T. 2000. A method to define the carboxyl terminal of proteins. Anal. Chem. 72:3374‐3378.
   Stark, G.R. 1968. Sequential degradation of peptides from their carboxyl termini with ammonium thiocyanate and acetic anhydride. Biochemistry. 7:1796‐1807.
Key References
   Boyd et al., 1992. See above.
  Describes the application of the thiohydantoin chemistry to a sequencer instrument and presents the one‐time‐activation and S‐alkylation for improved yield and recovery.
   Bergman et al., 2001. See above.
  Describes the current modifications of the original sequencer system (Boyd et al. , ) and provides details about performance and applications.
   Jones, B.N. 1986. See above.
  A detailed discussion of enzymatic methods for C‐ and N‐terminal sequence determination; includes examples and information about enzyme properties, kinetic studies, and analytical methods.
   Patterson, et al. 1995. See above.
  Comparison of results generated by liquid phase and on‐plate digestions; includes discussion of a statistical analysis strategy for ladder sequencing data.
   Ward, C.W. 1986. Carboxy terminal sequence analysis. In Practical Protein Chemistry: A Handbook (A. Darbee, ed.) pp. 517‐522. John Wiley & Sons, New York.
  General description of the techniques and carboxypeptidases.
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