N‐Terminal Sequence Analysis of Proteins and Peptides

Kaye D. Speicher1, Nicole Gorman1, David W. Speicher1

1 The Wistar Institute, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 11.10
DOI:  10.1002/0471140864.ps1110s57
Online Posting Date:  August, 2009
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Automated N‐terminal sequence analysis involves a series of chemical reactions that derivatize and remove one amino acid at a time from the N‐terminus of purified peptides or intact proteins. At least several picomoles of a purified protein or 10 to 20 pmol of a purified peptide with an unmodified N‐terminus is required to obtain useful sequence information. In recent years, the demand for N‐terminal sequencing has decreased substantially as some applications for protein identification and characterization can now be more effectively performed using mass spectrometry. However, N‐terminal sequencing remains the method of choice for verifying the N‐terminal boundary of recombinant proteins, determining the N‐terminus of protease‐resistant domains, identifying proteins isolated from species where most of the genome has not yet been sequenced, and mapping modified or crosslinked sites in proteins that prove to be refractory to analysis by mass spectrometry. Curr. Protoc. Protein Sci. 57:11.10.1‐11.10.31. © 2009 by John Wiley & Sons, Inc.

Keywords: N‐terminal sequencing; Edman sequencing; protein structure; blocked N‐terminal; protein modifications

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Sequencing Liquid Samples on Glass Fiber Filters
  • Basic Protocol 2: Sequencing PVDF‐Bound Samples Using a Blott Cartridge
  • Support Protocol 1: Optimizing Separation of PTH Amino Acids
  • Support Protocol 2: Sequence Data Interpretation
  • Support Protocol 3: Optimizing Sequencer Performance
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Sequencing Liquid Samples on Glass Fiber Filters

  Materials
  • Liquid peptide or protein sample(s) to be sequenced
  • Volatile solvents for reversed‐phase chromatography: e.g., 0.1% trifluoroacetic acid (TFA) and acetonitrile (unit 11.6)
  • 1% SDS
  • Argon or nitrogen gas source
  • 50% acetonitrile/0.1% TFA
  • Methanol
  • Polybrene solution (Biobrene Plus from Applied Biosystems; store in original container up to 3 months at 4°C; discard if contamination is suspected; alternatively store 30‐ to 100‐µl aliquots in clean microcentrifuge tubes up to 1 year at −20°C)
  • Sequencer solvent/reagent kit (Applied Biosystems) including:
    • R1 (phenylisothiocyanate)
    • R2C (n‐methylpiperidine)
    • R3 (trifluoroacetic acid)
    • R4A (25% trifluoroacetic acid)
    • R5 (PTH sequencing standard)
    • S2B (ethyl acetate)
    • S3 (n‐butyl chloride)
    • S4B (20% acetonitrile)
  • Premix PTH analyzer kit (Applied Biosystems) including:
    • HPLC solvent A3 (3.5% tetrahydrofuran)
    • HPLC solvent B2 (isopropanol/acetonitrile)
    • Premix buffer concentrate
  • 1.0‐ or 2.1‐mm i.d. reversed‐phase column (unit 11.6)
  • TFA‐treated glass fiber filters (GFF; Applied Biosystems)
  • Cartridge seals (Applied Biosystems)
  • Powder‐free gloves
  • Applied Biosystems Procise sequencing system, e.g., model 494 including:
    • Sequencer module with glass sample cartridge blocks
    • On‐line PTH analyzer (dedicated HPLC and detector)
    • Computer controller
  • Stainless steel or Teflon‐coated forceps
  • Additional reagents and equipment for reversed‐phase purification of peptides (unit 11.6), concentration of proteins and microdialysis (unit 4.4), quantitation of proteins/peptides by amino acid analysis (units 3.2& 11.9), and spectrophotometric quantitation of protein (unit 3.4)
NOTE: Prepare solutions with Milli‐Q water or equivalent taken directly from the purification unit. Water stored for any length of time in glass or plastic containers supports microbial and algal growth, which results in high amino acid background in early sequencer cycles.

Basic Protocol 2: Sequencing PVDF‐Bound Samples Using a Blott Cartridge

  Materials
  • PVDF‐bound sample(s) to be sequenced
  • Methanol in wash bottle
  • Stainless steel scalpel and stainless steel or Teflon‐coated forceps
  • Glass gel plate
  • Bath sonicator
  • Blott cartridge for Applied Biosystems Procise sequencer (Applied Biosystems)
  • Additional reagents and equipment for sequencing (see protocol 1)
NOTE: Prepare solutions with Milli‐Q water or equivalent taken directly from the purification unit. Water stored for any length of time in glass or plastic containers supports microbial and algal growth, which results in high amino acid background in early sequencer cycles.

Support Protocol 1: Optimizing Separation of PTH Amino Acids

  • 1% (v/v) acetone in Milli‐Q water
  • 1.0 M KH 2PO 4 in Milli‐Q water
  • PTH analyzer standard mixture (standard mixture of PTH amino acids; Applied Biosystems)
  • 1‐liter bottles and three‐valve caps (Rainin)
NOTE: Prepare solutions with Milli‐Q water or equivalent taken directly from the purification unit. Water stored for any length of time in glass or plastic containers supports microbial and algal growth, which results in high amino acid background in early sequencer cycles.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Atherton, D., Fernandez, J., DeMott, M., Andrews, L., and Mische, S.M. 1993. Routine protein sequence analysis below ten picomoles: One sequencing facility's approach. In Techniques in Protein Chemistry IV (R. Angelletti, ed.) pp. 409‐418. Academic Press, San Diego.
   Beyer, K., Bardina, L., Grishina, G., and Sampson, H.A., 2002. Identification of sesame seed allergens by 2‐dimensional proteomics and Edman sequencing: Seed storage proteins as common food antigens. J. Allergy Clin. Immunol. 110:154‐159.
   Brown, J.L. and Roberts, W.K. 1976. Evidence that ∼80% of the soluble proteins from Ehrlich ascites cells are N‐alpha acetylated. J. Biol. Chem. 251:1009‐1014.
   Edman, P. 1949. A method for the determination of the amino acid sequence in peptides. Arch. Biochem. Biophys. 22:475‐480.
   Edman, P. and Begg, G. 1967. A protein sequenator. Eur. J. Biochem. 1:80‐91.
   Erdjument‐Bromage, H., Geromanos, S., Chodera, A., and Tempst, P. 1993. Successful peptide sequencing with femtomole level PTH‐analysis: A commentary. In Techniques in Protein Chemistry IV. (R. Angelletti, ed.) pp. 419‐426. Academic Press, San Diego.
   Hewick, R.M., Hunkapiller, M.W., Hood, L.E., and Dryer, W.J. 1981. A gas‐liquid solid phase peptide and protein sequencer. J. Biol. Chem. 256:7990‐7997.
   Kaju, K., Tomino, S., and Asano, T. 2009. A serine protease in the midgut of the silkworm, Bombyx mori: Protein sequencing, identification of cDNA, demonstration of its synthesis as zymogen form and activation during midgut remodeling. Insect Biochem. Mol. Biol. 39:207‐217.
   Mozdzanowski, J. and Speicher, D.W. 1992. Microsequence analysis of electroblotted proteins I. Comparison of electroblotting recoveries using different types of PVDF membranes. Anal. Biochem. 207:11‐18.
   Reim, D.F. and Speicher, D.W. 1994. A method for high‐performance sequence analysis using polyvinylidene difluoride membranes with a biphasic reaction column sequencer. Anal. Biochem. 216:213‐222.
   Sheer, D.G., Yuen, S., Wong, J., Wasson, J., and Yuan, P.M. 1991. A modified reaction cartridge for direct sequencing on polymeric membranes. Biotechniques 11:526‐534.
   Speicher, D.W. 1994. Methods and strategies for the sequence analysis of proteins on PVDF membranes. Methods 6:262‐273.
   Tempst, P. and Riviere, L. 1989. Examination of automated polypeptide sequencing using standard phenylisothiocyanate reagent and subpicomole high‐performance liquid chromatographic analysis. Anal. Biochem. 183:290‐300.
   Tempst, P., Geromanos, S., Elicone, C., and Erdjument‐Bromage, H. 1994. Improvements in microsequencer performance for low picomole sequence analysis. Methods 6:248‐261.
   Yuksel, K.U., Grant, G.A., Mende‐Muller, L., Niece, R.L., Williams, K.R., and Speicher, D.W. 1991. Protein sequencing from polyvinylidenefluoride membranes: Design and characteristics of a test sample (ABRF‐90SEQ) and evaluation of results. In Techniques in Protein Chemistry II. (J.J. Villafranca, ed.) pp. 151‐162. Academic Press, San Diego.
Key References
   Edman and Begg, 1967. See above.
  First description of the basic Edman chemistry.
   Hewick et al., 1981. See above.
  Describes the first sequencer capable of picomole‐level sequencing.
   Tempst et al., 1994. See above.
  Review containing additional suggestions for improving sequencing sensitivity.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library