Extraction of Proteins from Formalin‐Fixed, Paraffin‐Embedded Tissue Using the Qproteome Extraction Technique and Preparation of Tryptic Peptides for Liquid Chromatography/Mass Spectrometry Analysis

Thibault Geoui1, Henning Urlaub2, Uwe Plessmann2, Peter Porschewski1

1 QIAGEN GmbH, Hilden, Germany, 2 Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.27
DOI:  10.1002/0471142727.mb1027s90
Online Posting Date:  April, 2010
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Abstract

This unit provides a robust, reliable, and easy‐to‐use kit‐based method for extraction of intact, non‐degraded proteins from formalin‐fixed, paraffin‐embedded (FFPE) tissue, and their subsequent use for analysis by liquid chromatography/mass spectrometry (LC/MS). After deparaffinization, proteins are extracted from unstained sections of FFPE rat liver tissue. After a simple cleanup step using organic extraction, the sample is transferred into a buffer optimized for trypsin digestion of the extracted proteins. Subsequently, LC/MS is used to identify the proteins that gave rise to the tryptic peptides. Comparing formalin‐fixed and frozen tissues, good correlation is observed in the mass spectrometric pattern attributable to the tryptic peptides and number of identified proteins. Since FFPE tissues are generally available in clinical practice, this method can be used to analyze biomarkers in different pathological situations (e.g., healthy vs. diseased). The method can also be used for protein extraction from fresh‐frozen tissue. Curr. Protoc. Mol. Biol. 90:10.27.1‐10.27.12. © 2010 by John Wiley & Sons, Inc.

Keywords: formalin‐fixed tissue; mass spectrometry; proteomics; protein extraction

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Deparaffinization of Sections from an FFPE Tissue Block and Extraction of Total Protein
  • Basic Protocol 2: Tryptic Digestion of Extracted Proteins for Mass Spectrometry
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Deparaffinization of Sections from an FFPE Tissue Block and Extraction of Total Protein

  Materials
  • FFPE tissue block: e.g., rat liver fixed for 24 hr in 10% neutral buffered formalin solution (PBS‐buffered) prior to paraffin embedding
  • Fresh‐frozen tissue (optional; stored at –80°C)
  • Qproteome FFPE Tissue 2D‐Page Kit (#37633; QIAGEN) including:
    • Extraction Buffer EXB
    • FFPE solvents (including heptane, methanol, and chloroform)
    • 1.5‐ml collection tubes with sealing clips
  • Protein quantification kit (e.g., Bio‐Rad DC Protein Assay Kit 1 [#500‐0001] for Lowry method or Pierce Micro BCA Protein Assay Kit [#23235])
  • Monoclonal antibody for protein assessment by immunoblotting (e.g., anti‐α‐tubulin, #MAB1501, Millipore, or anti‐β‐actin, #T5168, Sigma‐Aldrich)
  • Acetone, HPLC grade
  • Microtome
  • 100°C water bath or heating block
  • Thermomixer (e.g., Eppendorf)
  • 0.5‐ml microcentrifuge tubes (e.g., Eppendorf Safe‐Lock tubes, colorless)
  • Additional reagents and equipment for 1‐D SDS‐PAGE (unit 10.2) and immunoblotting (unit 10.8)
NOTE: When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheet (MSDS), available from the product supplier. Procedures using heptane, methanol, and chloroform should be performed in a well‐ventilated fume hood. All organic solvents should be disposed of according to applicable environmental regulations.NOTE: Unless otherwise noted, use analytical‐grade reagents for preparation of all buffers and solutions.NOTE: For each pipetting step, use a fresh tip or a multi‐step dispenser, depending on the number of samples.

Basic Protocol 2: Tryptic Digestion of Extracted Proteins for Mass Spectrometry

  Materials
  • Extracted protein sample (25 µg protein pellet; see protocol 1)
  • 25 mM NH 4HCO 3, analytical grade
  • 0.1% (w/v) RapiGest SF (Waters) in 25 mM NH 4HCO 3
  • 50 mM dithiothreitol (DTT) in 25 mM NH 4HCO 3 (Calbiochem), analytical grade
  • 100 mM iodoacetamide in 25 mM NH 4HCO 3 (Sigma‐Aldrich), analytical grade
  • 0.25 µg/µl trypsin in 25 mM NH 4HCO 3 (sequencing‐grade trypsin, Promega)
  • 5% (v/v) trifluoroacetic acid (TFA; Sigma‐Aldrich), analytical grade
  • HPLC‐grade acetonitrile (ACN, Acetonitrile LiChrosolv, gradient grade #1.0030.2500, Merck KGgA)
  • HPLC‐grade formic acid (FA, for LC/MS, Sigma‐Aldrich)
  • HPLC‐grade H 2O (Water LiChrosolv #1.15333.2500, Merck KGgA)
  • Chromatography solvent A: 0.1 % (v/v) FA in H 2O
  • Chromatography solvent B: 95% (v/v) ACN/0.1% (v/v) FA
  • Thermomixer (e.g., Eppendorf)
  • 0.5‐ml microcentrifuge tubes (e.g., Eppendorf Safe‐Lock tubes, colorless)
  • Speedvac evaporator (with rotor/adapters for 0.5‐ml tubes)
  • Agilent 1100 chromatography system (Agilent Technologies) or equivalent, with:
    • In‐house‐packed pre‐columns (10–20 × 0.15 mm, C 18, 5 µm, 120 Å) or purchased pre‐columns (e.g., #r15aq.v000.3T, 10 × 0.3 mm; Dr Maisch HPLC GmbH, http://www.dr‐maisch.com)
    • In‐house‐packed analytical columns (100 × 0.075 mm, C 18, 5 µm, 120 Å) or purchased analytical columns (e.g., #r15aq.s.150075, 150 × 0.075 mm; Dr Maisch HPLC GmbH)
  • Linear Trap Quadrupole (LTQ) Orbitrap XL mass spectrometer (Thermo Fisher Scientific)
  • Mascot Daemon version 2.2.2 (http://www.matrixscience.com)
  • Mascot version 2.2.06 (http://www.matrixscience.com)
  • NCBInr FASTA database (http://blast.ncbi.nlm.nih.gov)
NOTE: Unless otherwise noted, use HPLC‐grade water (e.g., Water LiChrosolv) and analytical‐grade reagents for preparation of all buffers and solutions.
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Figures

Videos

Literature Cited

Literature Cited
   Ahram, M., Flaig, M.J., Gillespie, J.W., Duray, P.H., Linehan, W.M., Ornstein, D.K., Niu, S., Zhao, Y., Petricoin, E.F. III, and Emmert‐Buck, M.R. 2003. Evaluation of ethanol‐fixed, paraffin‐embedded tissues for proteomic applications. Proteomics 3:413‐421.
   Becker, K.F., Schott, C., Hipp, S., Metzger, V., Porschewski, P., Beck, R., Nährig, J., Becker, I., and Höfler, H. 2007. Quantitative protein analysis from formalin‐fixed tissues: Implications for translational clinical research and nanoscale molecular diagnosis. J. Pathol. 211:370‐378.
   Becker, K.F., Schott, C., Becker, I., and Höfler, H. 2008. Guided protein extraction from formalin‐fixed tissues for quantitative multiplex analysis avoids detrimental effects of histological stains. Proteomics Clin. Appl. 2:737‐743.
   Carson, F.L. and Hladik, C. 2009. Histotechnology: A Self‐Instructional Text, 3rd ed. ASCP Press, Chicago.
   de Godoy, L.M., Olsen, J.V., Cox, J., Nielsen, M.L., Hubner, N.C., Fröhlich, F., Walther, T.C., and Mann, M. 2008. Comprehensive mass‐spectrometry‐based proteome quantification of haploid versus diploid yeast. Nature 455:1251‐1254.
   Guo, T., Wang, W., Rudnick, P.A., Song, T., Li, J., Zhuang, Z., Weil, R.J., DeVoe, D.L., Lee, C.S., and Balgley, B.M. 2007. Proteome analysis of microdissected formalin‐fixed and paraffin‐embedded tissue specimens. J. Histochem. Cytochem. 55:763‐772.
   Hewitt, S.M., Lewis, F.A., Cao, Y., Conrad, R.C., Cronin, M., Danenberg, K.D., Goralski, T.J., Langmore, J.P., Raja, R.G., Williams, P.M., Palma, J.F., and Warrington, J.A. 2008 Tissue handling and specimen preparation in surgical pathology: Issues concerning the recovery of nucleic acids from formalin‐fixed, paraffin‐embedded tissue. Arch. Pathol. Lab. Med. 132:1929‐1935.
   Masuda, J., Maynard, D.M., Nishimura, M., Ueda, T., Kowalak, J.A., and Markey, S.P. 2005. Fully automated micro‐ and nanoscale one‐ or two‐dimensional high‐performance liquid chromatography system for liquid chromatography‐mass spectrometry compatible with non‐volatile salts for ion exchange chromatography. J. Chromatogr. 21:57‐69.
   Prophet, E.B. 1992. Fixation. In Laboratory Methods in Histotechnology, 1st ed. (E.B. Prophet, B. Mills, J.B. Arrington, and L.H. Sobin, eds.) pp. 25‐29. American Registry of Pathology, Washington, D.C.
   Puchtler, H. and Meloan, S.N. 1985. On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochemistry 82:201‐204.
   Werner, M., Chott, A., Fabiano, A., and Battifora, H. 2000. Effect of formalin tissue fixation and processing on immunohistochemistry. Am. J. Surg. Pathol. 24:1016‐1019.
   Wright, M.E., Han, D.K., and Aebersold, R. 2005. Mass spectrometry‐based expression profiling of clinical prostate cancer. Mol. Cell. Proteomics 4:545‐554.
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