Rapid Detergent Removal from Peptide Samples with Ethyl Acetate for Mass Spectrometry Analysis

E. Richard Stanley1

1 Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 16.12
DOI:  10.1002/0471140864.ps1612s59
Online Posting Date:  February, 2010
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Detergents are required for the extraction of hydrophobic proteins and for the maintenance of their solubility in solution. However, the presence of detergents in the peptide samples severely suppresses ionization in mass spectrometry (MS) analysis and decreases chromatographic resolution in LC‐MS. Thus, detergents must be removed for sensitive detection of peptides by MS. This unit describes a rapid protocol in which ethyl acetate extraction is used to remove octylglucoside from protease digests without loss of peptides. This procedure can also be used to reduce interference by sodium dodecyl sulfate, Nonidet P‐40, or Triton X‐100 in peptide samples for MS analysis. Curr. Protoc. Protein Sci. 59:16.12.1‐16.12.5. © 2010 by John Wiley & Sons, Inc.

Keywords: detergent removal; mass spectrometry; ethyl acetate extraction; octylglucoside

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Table of Contents

  • Introduction
  • Basic Protocol 1: Extraction of Detergent from Peptide Samples
  • Support Protocol 1: Preparation of Water‐Saturated Ethyl Acetate
  • Commentary
  • Literature Cited
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Basic Protocol 1: Extraction of Detergent from Peptide Samples

  • Water‐saturated ethyl acetate (see protocol 2)
  • Peptide‐containing solution
  • Acetic acid, formic acid, or trifluoroacetic acid (HPLC grade or better)
  • Vortex mixer
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge (13,000 to 16,000 × g maximum speed)
  • 200 µl round gel‐loading micropipet tips
  • Speed vacuum system with a Savant SpeedVac, refrigerated vapor trap, vacuum gauge and vacuum pump, or equivalent system
  • pH 1 to 12 pH paper
NOTE: Extraction can be performed immediately after digestion or on stored samples.

Support Protocol 1: Preparation of Water‐Saturated Ethyl Acetate

  • Milli‐Q (Millipore) or glass double‐distilled water
  • Ethyl acetate (HPLC grade or better, in a glass bottle)
  • 125‐ml glass bottle with glass stopper
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Literature Cited

Literature Cited
   Chen, E.I., Cociorva, D., Norris, J.L., and Yates, J.R. 2007. Optimization of mass spectrometry‐compatible surfactants for shotgun proteomics. J. Proteome Res. 6:2529‐2538.
   Cravatt, B.F., Simon, G.M., and Yates, J.R. III. 2007. The biological impact of mass‐spectrometry‐based proteomics. Nature 450:991‐1000.
   Katayama, H., Nagasu, T., and Oda, Y. 2001. Improvement of in‐gel digestion protocol for peptide mass fingerprinting by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Rapid Commun. Mass Spectrom. 15:1416‐1421.
   Katayama, H., Tabata, T., Ishihama, Y., Sato, T., Oda, Y., and Nagasu, T. 2004. Efficient in‐gel digestion procedure using 5‐cyclohexyl‐1‐pentyl‐β‐D‐Maltoside as an additive for gel‐based membrane proteomics. Rapid Commun. Mass Spectrom. 18:2388‐2394.
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   Nomura, E., Katsuta, K., Ueda, T., Toriyama, M., Mori, T., and Inagaki, N. 2004. Acid‐labile surfactant improves in‐sodium dodecyl sulfate polyacrylamide gel protein digestion for marrix‐assisted laser desorption/ionization mass spectrometric peptide mapping. J. Mass Spectrom. 39:202‐207.
   Yeung, Y.G., Nieves, E., Angeletti, R.H. and Stanley, E.R. 2008. Removal of detergents from protein digests for mass spectrometry analysis. Anal. Biochem. 382:135‐137.
   Zhang, N. and Li, L. 2004. Effects of common surfactants on protein digestion and matrix‐assisted laser desorption/ionization mass spectrometric analysis of the digested peptides using two‐layer sample preparation. Rapid Commun. Mass Spectrom. 18:889‐896.
   Zhang, G. and Neubert, T.A. 2006. Use of detergents to increase selectivity of Immunoprecipitation of tyrosine phosphorylated peptides prior to identification by MALDI quadruple‐TOF MS. Proteomics 6:571‐578.
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