Peptide Extraction from Formalin‐Fixed Paraffin‐Embedded Tissue

Katherine J. Heaton1, Stephen R. Master2

1 Phoenix S&T, Chester, Pennsylvania, 2 University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 23.5
DOI:  10.1002/0471140864.ps2305s65
Online Posting Date:  August, 2011
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This unit describes how to extract tryptic peptides from formalin‐fixed, paraffin‐embedded (FFPE) tissues for analysis using nano‐reverse‐phase liquid chromatography/tandem mass spectrometry (nRPLC‐MS/MS). The tissues are deparaffinized in the first protocol. Following deparaffinization, the cells are harvested via one of two methods: needle dissection or laser capture microdissection (LCM). Needle dissection is performed using hydrated, unstained tissue, whereas LCM is performed with dehydrated, hematoxylin‐stained tissue. Heat is applied to the collected cells to reverse the cross‐links that have formed during the formalin fixation process. Finally, the cells are digested using filter‐aided sample preparation, in which buffers are exchanged throughout the process. An alternate protocol using commercially available Liquid Tissue is also described. These samples are then ready for mass spectrometric analysis. Curr. Protoc. Protein Sci. 65:23.5.1‐23.5.19. © 2011 by John Wiley & Sons, Inc.

Keywords: FFPE; proteomics; FASP; antigen retrieval; protein extraction

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Table of Contents

  • Introduction
  • Basic Protocol 1: Deparaffinization and Rehydration of the FFPE Tissues
  • Basic Protocol 2: Harvesting Cells Using Needle Dissection
  • Alternate Protocol 1: Harvesting Cells Using Laser Capture Microdissection (LCM)
  • Basic Protocol 3: Antigen Retrieval and Tryptic Digestion Using Filter‐Aided Sample Preparation (FASP)
  • Alternate Protocol 2: Liquid Tissue Protocol
  • Support Protocol 1: Preparation of StageTips
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Deparaffinization and Rehydration of the FFPE Tissues

  • Glass slide with unstained FFPE tissue section
  • Xylene (Histo grade)
  • 100% (v/v) ethanol
  • 95% (v/v) ethanol (see recipe)
  • 70% (v/v) ethanol (see recipe)
  • Milli‐Q Millipore water or equivalent
  • Oven set to 58°C
  • Six containers with lids that can hold the above solvents
  • Holder for the slide(s)
NOTE: Unless otherwise noted, all reagents should be of analytical grade.

Basic Protocol 2: Harvesting Cells Using Needle Dissection

  • Lysis buffer: 4% SDS, 100 mM Tris⋅Cl, pH 7.6
  • Deparaffinized and rehydrated slide‐mounted tissue section (from protocol 1)
  • Stained slide of a serial section of tissue to use as a guide
  • 1.5‐ml low‐protein‐binding centrifuge tubes
  • Centrifuge
  • Kimwipe
  • Beveled syringe needle (18‐ to 25‐G), ideally >4‐cm long

Alternate Protocol 1: Harvesting Cells Using Laser Capture Microdissection (LCM)

  • Milli‐Q Millipore water
  • 70% (v/v) ethanol (see recipe)
  • 95% (v/v) ethanol (see recipe)
  • 100% ethanol
  • Xylene (histological grade)
  • Hydrated, unstained tissue attached to 1.0‐mm nuclease‐free PEN‐coated Membrane Slides (Carl Zeiss, cat. no. 415190‐9081‐000; see protocol 1)
  • 10% (v/v) Mayer's hematoxylin stain (see recipe)
  • Seven or eight containers with lids to hold the solutions
  • Kimwipes
  • Holder for the slide(s)
  • PALM LCM Instrument (Zeiss)
  • 500‐µl centrifuge tubes with opaque adhesive caps (Carl Zeiss, cat. no. 415190‐9201‐000)
  • 500‐µl low‐protein‐binding centrifuge tube
  • Vortex

Basic Protocol 3: Antigen Retrieval and Tryptic Digestion Using Filter‐Aided Sample Preparation (FASP)

  • Tube(s) containing harvested cells (from protocol 2 or protocol 3)
  • 1 M Dithiothreitol (DTT)
  • Lysis buffer: 4% SDS, 100 mM Tris⋅Cl, pH 7.6
  • Urea (UA) buffer: 8 M urea in 0.1 M Tris⋅Cl, pH 8.5
  • IAA solution: 0.05 M iodoacetamide (see recipe) in UA buffer (see recipe)
  • ABC buffer: 0.05 M ammonium bicarbonate (see recipe)
  • Trypsin solution (see recipe)
  • 0.5 M sodium chloride (NaCl; see recipe)
  • 2% Trifluoroacetic acid (TFA; see recipe)
  • Methanol
  • Buffer B: 80% ACN with 0.5% (v/v) acetic acid (see recipe)
  • Buffer A: 0.5% (v/v) acetic acid (see recipe)
  • 2% ACN with 0.1% formic acid in water (see recipe)
  • Heating block set to 99°C
  • 30‐kDa Microcon Ultracel YM‐30 Filters (Millipore, cat. no. 42410)
  • 1.5‐ml microcentrifuge tubes (Millipore)
  • Fixed‐angle microcentrifuge capable of 14,000 × g (Thermo Fisher, cat. no. 5417C)
  • Mixer capable of 600 rpm (Eppendorf Thermomixer R, cat. no. 022670107, with 1.5‐ml interchangeable block, cat. no. 022670522)
  • Aluminum foil
  • 37°C water bath and floating tube rack, optional
  • C‐18 StageTips (see protocol 6)
  • 1‐ml syringe
  • SpeedVac
  • Additional reagents and equipment for nano‐reverse‐phase liquid chromatography/tandem mass spectrometry to analyze the samples (units 16.9& 23.1)
NOTE: If the V‐shaped Microcon filters are used, the protocol should be altered to increase the volume at each step (e.g., 200 µl of UA buffer instead of 100 µl; see steps 4, 7, 13, 16, and 18). The final elution in step 23 can also be performed in a large volume, since the sample will be concentrated during the desalting on StageTips. If V‐shaped Microcon filters are used, it is important to check yields during pilot experiments. No change to the protocol is required for PALL filter units.

Alternate Protocol 2: Liquid Tissue Protocol

  • Centrifuge tubes containing the tissue (see protocol 3 or protocol 2)
  • Ice
  • Liquid Tissue MS Protein Prep Kit (Expression Pathology; contains all reagents needed with instructions) containing:
    • Liquid Tissue buffer
    • Trypsin diluent
    • Trypsin, lyophilized
    • Reduction agent (100 mM DTT)
  • Heating block set to 95°C
  • Centrifuge capable of 10,000 × g
  • 37°C water bath
  • Vortex
  • Additional reagents and equipment for nano‐reverse‐phase liquid chromatography/tandem mass spectrometry to analyze the samples (units 16.9& 23.1)
NOTE: After harvesting cells with either protocol 2 or protocol 3, place the tissue in 20 µl Liquid Tissue buffer. If more than 45,000 cells are collected, the protocol suggests scaling the amount of buffer used appropriately.

Support Protocol 1: Preparation of StageTips

  • Flat tweezers, optional
  • C18 (Octadecyl) 47‐mm Empore High Performance Extraction Disks (3 M, cat. no. 2215)
  • 60‐mm petri dishes
  • Cutter tube (Hamilton, cat. no. 90516)
  • 200‐µl non‐barrier pipet tips
  • Plunger (Hamilton, cat. no. 1122‐01)
  • Pipet box
NOTE: This protocol can be stopped and started again after each tip is made as long as the C18 Empore disk is stored in a clean, dust‐free environment (petri dish).
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  •   FigureFigure 23.5.1 Pictures of harvesting cells via needle dissection. (A) The hematoxylin and eosin–stained guide slide is seen at the top of the photo, the hydrated tissue section is on the bottom, and the syringe needle (15‐G) is on the right. (B) The hydrated tissue section slide is on top of the stained guide slide. (C) and (D) show harvesting cells from tissue. (D) Depicts the tissue being moist without a drop of water on top of the section being collected. (E) Tissue collected on tip of needle. (F) Placement of the needle inside lysis buffer to remove tissue.
  •   FigureFigure 23.5.2 20× photos of focal in‐situ tumor in ovarian tissue. (A) Unprocessed hematoxylin and eosin (H&E)–stained slide. (B) and (C) show a deparaffinized, stained slide before (B) and after (C) cell harvesting by laser‐capture microdissection (LCM).
  •   FigureFigure 23.5.3 Graph depicting comparative yields of peptides from PALL Nanosep 30K Centrifugal device, flat‐bottom Microcon 30K filter, and V‐shaped Microcon 30K filter with and without mixing. The FASP protocol was performed using 50 µg BSA. NOTE: For each FASP step with the V‐shaped filter, 200 µl of buffer was added. Incubation of the digestion step was performed either in a 37°C water bath without mixing or in a thermomixer (Eppendorf Thermomixer R) at 37°C with mixing for 1 min/hr, as indicated in the figure. Absorbance measurements were taken after digestion and prior to sample clean‐up with StageTips.


Literature Cited

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   Berg, D., Hipp, S., Malinowsky, K., Bollner, C., and Becker, K. 2010. Molecular profiling of signaling pathways in formalin‐fixed and paraffin‐embedded cancer tissues. Euro. J. Cancer 46:47‐55.
   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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   Huang, S.K., Darfler, M.M., Nicholl, M.B., You, J., Bemis, K.G., Tegeler, T.J., Wang, M., Wery, J., Nguyen, L., Scolyer, R.A., and Hoon, D.S.B. 2009. LC/MS‐ based quantitative proteomic analysis of paraffin‐embedded archival melanomas reveals potential proteomic biomarkers associated with metastasis. PLoS ONE 4:e4430.
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   Ostasiewicz, P., Zielinska, D.F., Mann, M., and Wisniewski, J.R. 2010. Proteome, phosphoproteome, and N‐glycoproteome are quantitatively preserved in formalin‐fixed paraffin‐embedded tissue and analyzable by high‐resolution mass spectrometry. J. Prot. Res. 9:3688‐3700.
   Palmer‐Toy, D.E., Krastins, B., Sarracin, D.A., Nadol, J.B. Jr., and Merchant, S.N. 2005. Efficient method for the proteomic analysis of fixed and embedded tissues. J. Prot. Res. 4:2404‐2411.
   Patel, V., Hood, B.L., Molinolo, A.A., Lee, N.H., Conrads, T.P., Braisted, J.C., Krizman, D.B., Veenstra, T.D., and Gutkind, J.S. 2008. Proteomic analysis of laser‐captured paraffin‐embedded tissues: A molecular portrait of head and neck cancer progression. Clin. Cancer Res. 14:1002‐10014.
   Prieto, D.A., Hood, B.L., Darfler, M.M., Guiel, T.G., Lucas, D.A., Conrads, T.P., Veenstra, T.D., and Krizman, D.B. 2005. Liquid Tissue™: Proteomic profiling of formalin‐fixed tissues. Biotechniques 38:S32‐S35.
   Rappsilber, J., Mann, M., and Ishihama, Y. 2007. Protocol for micro‐purification, enrichment, pre‐fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2:1896‐1906.
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   Shi, S.R., Key, M.E., and Kalra, K.L. 1991. Antigen retrieval in formalin‐fixed, paraffin‐embedded tissues: An enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 39:741‐748.
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Key References
   Ostasiewicz et al., 2010. See above.
  This recent reference, from the group that developed FASP, also describes its application to FFPE material. There are minor differences between their final protocol and the description provided within this unit. This publication also describes extensive validation of this strategy for characterizing post‐translational modifications, such as phosphorylation.
  Hood et al., 2005. See above.
  This paper (by the group that developed Liquid Tissue) provides a good introduction to the utility of using FFPE samples for biomarker discovery.
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