Enzymatic Approaches for Obtaining Amino Acid Sequence: On‐Target Ladder Sequencing

C.R. Jiménez1, L. Huang1, Y. Qiu1, A.L. Burlingame1

1 University of California, San Francisco, San Francisco
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 16.7
DOI:  10.1002/0471140864.ps1607s15
Online Posting Date:  May, 2001
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Abstract

Peptide sequencing by mass spectrometry (MS) is usually based on detecting mass differences associated with various amino acids in the polymer chain. Post‐source decay (PSD) and MS/MS spectra may yield internal peptide sequences. However, determination of the order of the first two, and sometimes the last few, amino acids in the peptide is often problematic without additional experiments. Several enzymatic approaches have proven useful for identifying the N‐ and C‐terminal residues. They involve the use of carboxypeptidases and aminopeptidases to produce peptide ladders for rapid analysis by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). This unit describes the ladder sequence method to generate amino acid sequence information from low‐ to subpicomole quantities of peptides. It can be performed directly on the sample stage, thus minimizing potential sample losses to vials and through sample transfer.

     
 
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Table of Contents

  • Commentary
     
 
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Materials

Basic Protocol 1:

  Materials
  • Carboxypeptidase P, Y, and A
  • 50 mM sodium citrate buffer, pH 6.0
  • Aminopeptidase M
  • 50 mM ammonium carbonate buffer, pH 7.0
  • ∼1 pmol/µl peptide substrate in 0.1% (v/v) trifluoroacetic acid (TFA)
  • Matrix solution (unit 16.3, protocol 1Basic Protocol)
  • 10% (v/v) formic acid, cold
  • Matrix‐assisted laser desorption/ionization (MALDI) mass spectrometer
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Figures

Videos

Literature Cited

Literature Cited
   Patterson, D.H., Tarr, G.E., Regnier, F.E., and Martin, S.A. 1995. C‐terminal ladder sequencing via matrix‐assisted laser desorption ionization mass spectrometry coupled with carboxypeptidase Y time‐dependent and concentration‐dependent digestions. Anal. Chem. 67: 3971‐3978.
   Thiede, B., Wittman‐Liebold, B., Bienert, M., and Krause, E. 1995. MALDI‐MS for C‐terminal sequence determination of peptides and proteins degraded by carboxypeptidase Y and P. FEBS Lett. 357: 65‐69.
   Woods, A.S., Huang, A.Y.C., Cotter, R.J., Pasternack, G.R., Pardoll, D.M., and Jaffee, E.M. 1995. Simplified high‐sensitivity sequencing of a major histocompatibility complex class I‐associated immunoreactive peptide using matrix‐assisted laser desorption ionization mass spectrometry. Anal. Biochem. 226: 15‐25.
Key Reference
   Patterson et al., 1995. See above.
  This article provides an elaborate description of both time‐dependent and concentration‐dependent digestion strategies. In addition, it provides a protocol for statistical analyses of the resulting peptide mass ladder.
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