Sample Preparation for MALDI Mass Analysis of Peptides and Proteins

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 16.3
DOI:  10.1002/0471140864.ps1603s14
Online Posting Date:  May, 2001
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Abstract

Sample preparation may be the most crucial step in mass spectrometric analysis of peptides and proteins by MALDI. The free dipolar ionic peptides or proteins must be incorporated into a crystalline solid solution that is comprised of excess solvent (the matrix), which modulates desorption/ionization by preferentially absorbing the laser energy at the irradiation wavelength and laser pulse intensity being employed. Usually the cocrystalline sample and matrix are prepared by mixing solutions of each component and permitting the mixture to crystallize through evaporation of the solvents. Two methods employing this approach are presented in this unit: the dried drop method and the rapid evaporation method.

     
 
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Table of Contents

  • Basic Protocol 1: Dried Droplet Method
  • Basic Protocol 2: Rapid Evaporation Method
  • Support Protocol 1: Purification/Recrystallization of HCCA
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Dried Droplet Method

  Materials
  • 100% methanol
  • Peptide or protein sample of interest: ∼1 pmol/µl in 0.1% (v/v) trifluoroacetic acid (TFA)
  • recipePeptide or recipeprotein matrix solution (see reciperecipes)
  • recipePeptide or recipeprotein standard mixture (see reciperecipes)
  • Matrix‐assisted laser desorption/ionization (MALDI) mass spectrometer

Basic Protocol 2: Rapid Evaporation Method

  Materials
  • 100% methanol
  • recipePeptide matrix/nitrocellulose solution (see recipe)
  • Peptide sample of interest: ∼1 pmol/µl in 0.1% (v/v) trifluoroacetic acid (TFA)
  • 10% (v/v) formic acid, 0° to 4°C
  • recipePeptide standard mixture (see recipe)
  • Matrix‐assisted laser desorption/ionization (MALDI) mass spectrometer

Support Protocol 1: Purification/Recrystallization of HCCA

  Materials
  • α‐Cyano‐4‐hydroxy‐trans‐cinnamic acid (HCCA; Sigma or Aldrich)
  • 95% (v/v) ethanol: 50°C, room temperature, and 4°C
  • Class C and E Whatman glass filters
  • 50°C water bath
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Literature Cited

Literature Cited
   Clauser, K.R., Baker, P. and, Burlingame, A.L. 1996. Peptide fragment‐ion tags from MALDI/PSD for error‐tolerant searching of genomic databases. In Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, p. 365. American Society for Mass Spectrometry, Portland, Oreg.
   Cohen, S.L. and Chait, B.T. 1996. Influence of matrix solution conditions on the MALDI‐MS analysis of peptides and proteins. Anal. Chem. 68:31‐37.
   Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. 1996. Mass spectrometric sequencing of proteins from silver‐stained polyacrylamide gels. Anal. Chem. 68:850‐858.
   Vestal, M.L., Juhasz, P., and Martin, S.A. 1995. Delayed extraction matrix‐assisted laser desorption time of flight mass spectrometry. Rapid Commun. Mass Spectrom. 9:1044‐1050.
   Vorm, O., Roepstorff, P., and Mann, M. 1994. Improved resolution and very high sensitivity in MALDI TOF of matrix surfaces made by fast evaporation. Anal. Chem. 66:3281‐3287.
Key References
   Burlingame, A.L. and Carr, S. (eds.) 1996. Mass Spectrometry in the Biological Sciences. Humana Press, Totawa, N.J.
  This book may serve as an introduction to the field of biological mass spectrometry.
   Burlingame, A.L., Boyd, R.K. and Gaskell, S.G. 1998. Mass spectrometry. Anal. Chem. 70:647‐716.
  An extensive overview of the literature on the use of mass spectrometry for protein studies between 1997 and mid‐1998.
   Mann, M. and Talbo, G. 1996. Developments in matrix‐assisted laser desorption/ionization mass spectrometry. Curr. Opin. Biotechnol. 7:11‐19.
  A review of the progress of MALDI‐MS up to 1996.
   Roepstorff, P. 1997. Mass spectrometry in protein studies from genome to function. Curr. Opin. Biotechnol. 8:6‐13.
  A comprehensive review on applications and recent developments in MS instrumentation.
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