Isolation of Proteins by Heat‐Induced Extraction from Formalin‐Fixed, Paraffin‐Embedded Tissue and Preparation of Tryptic Peptides for Mass Spectrometric Analysis

Yuan Tian1, Hui Zhang1

1 Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.26
DOI:  10.1002/0471142727.mb1026s90
Online Posting Date:  April, 2010
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This unit describes a method of isolating of proteins from formalin‐fixed and paraffin‐embedded (FFPE) tissue for mass spectrometry analysis. Heat‐induced antigen retrieval is the basis of the protein extraction strategy presented in this protocol. This protocol may be used to identify nuclear, cytosolic, and membrane proteins from FFPE tissues extracted from tissue blocks or slides. Curr. Protoc. Mol. Biol. 90:10.26.1‐10.26.7. © 2010 by John Wiley & Sons, Inc.

Keywords: peptide extraction; formalin‐fixed and paraffin‐embedded tissue; mass spectrometry; protein identification

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • FFPE tissue block(s) or slide(s)
  • Xylene
  • 80%, 90%, and absolute ethanol
  • 10 mM sodium citrate buffer containing 0.05% SDS (pH 6.0), 95°C
  • 5 mM phosphate buffer
  • Trifluoroethanol (TFE; Sigma‐Aldrich)
  • Tributylphosphine (TBP; Sigma‐Aldrich)
  • Iodoacetamide (Sigma‐Aldrich)
  • 50 mM NH 4HCO 3, pH 7.8
  • Trypsin (sequencing‐grade, lyophilized 20 µg/tube trypsin; Promega)
  • Silver staining kit (Pierce)
  • BCA protein assay kit (Pierce)
  • 6 N hydrochloric acid (HCl)
  • C18 Sep‐Pak cartridges (1‐ml, Sep‐Pak Vac; Waters)
  • 0.1% (v/v) trifluoroacetic acid (TFA) in 80% acetonitrile
  • 0.1% (v/v) trifluoroacetic acid (TFA) in water
  • iTRAQ Reagent‐8Plex (Applied Biosystems)
  • 0.4% (v/v) acetic acid in water
  • HPLC solution A: 0.1% (v/v) formic acid in water
  • HPLC solution B: 0.1% (v/v) formic acid, 5% isopropanol in acetonitrile
  • α‐Cyano‐4‐cinnamic acid (CHCA; Aligent)
  • Petri dishes
  • 60°C oven
  • Glass boxes with glass lids
  • Kimwipes
  • Razor blades
  • 1.5‐ml microcentrifuge tubes
  • Vortex apparatus
  • Sonicator
  • Ice‐water, 37°, and 60°C water baths
  • Centrifuge
  • pH paper
  • 0.7‐ml glass vial inserts without caps (Waters)
  • SpeedVac with rotors suitable for microcentrifuge tubes and glass vial insert
  • Nano‐scale C‐18 reversed‐phase column (75‐µm i.d. × 15‐cm length)
  • NanoLC‐2D pump (Eksigent)
  • Nano‐scale spotter (Leap Technologies)
  • ABI 4800 MALDI‐TOF/TOF analyzer (Applied Biosystems)
  • Peptide cartridge packed with Magic C18 (75‐µm i.d. × 15‐cm length, directly connected to ESI‐QSTAR; Michrom Bioresources)
  • Electrospray ionization (ESI)‐QSTAR mass spectrometer (Applied Biosystems)
  • Software for sequence assignment from MS/MS data: GPS Explore (Applied Biosystems) and SEQUEST (Eng et al., )
  • Software for statistical evaluation of peptide sequence assignment: PeptideProphet (Keller et al., )
  • Additional reagents and equipment for 1‐D SDS‐PAGE gel electrophoresis (unit 10.2)
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Literature Cited

Literature Cited
   Becker, K.F., Schott, C., Hipp, S., Metzger, V., Porschewski, P., Beck, R., Nahrig, J., Becker, I., and Hofler, H. 2007. Quantitative protein analysis from formalin‐fixed tissues: Implications for translational clinical research and nanoscale molecular diagnosis. J. Pathol. 211:370‐378.
   Bodovitz, S. and Joos, T. 2004. The proteomics bottleneck: Strategies for preliminary validation of potential biomarkers and drug targets. Trends Biotechnol. 22:4‐7.
   Crockett, D.K., Lin, Z., Vaughn, C.P., Lim, M.S., and Elenitoba‐Johnson, K.S. 2005. Identification of proteins from formalin‐fixed paraffin‐embedded cells by LC‐MS/MS. Lab. Invest. 85:1405‐1415.
   Eng, J., McCormack, A.L., and Yates, J.R. 3rd. 1994. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5:976‐989.
   Fowler, C.B., Cunningham, R.E., O'Leary, T.J., and Mason, J.T. 2007. ‘Tissue surrogates’ as a model for archival formalin‐fixed paraffin‐embedded tissues. Lab. Invest. 87:836‐846.
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