Laser Capture Microdissection for Proteome Analysis

Rachel A. Craven1, Rosamonde E. Banks1

1 St. James's University Hospital, Leeds
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 22.3
DOI:  10.1002/0471140864.ps2203s31
Online Posting Date:  May, 2003
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Abstract

Laser capture microdissection is being employed increasingly to isolate specific cell types from tissues, thus overcoming problems of experimental interpretation due to tissue heterogeneity of samples. This unit describes protocols which have been optimized to allow laser capture microdissection of tissues with minimal effect on protein integrity, and their subsequent analysis by techniques including 2D‐PAGE, immunoblotting and SELDI, and discusses the relative merits of this approach.

     
 
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Table of Contents

  • Basic Protocol 1: Laser Capture Microdissection (LCM)
  • Alternate Protocol 1: Immunolabeling of Tissue Sections for LCM
  • Support Protocol 1: Collection and Storage of Tissue for Analysis by LCM
  • Support Protocol 2: Quantification of Protein in Material Captured by LCM Using Sypro Ruby
  • Basic Protocol 2: Two‐Dimensional Gel Electrophoresis of Material Captured by LCM
  • Basic Protocol 3: Analysis of Material Captured by LCM Using Immunoblotting
  • Basic Protocol 4: Seldi Mass Spectrometry
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Laser Capture Microdissection (LCM)

  Materials
  • OCT‐embedded tissue blocks (see protocol 3)
  • 70% and 100% (v/v) ethanol
  • recipeMayer's hematoxylin (see recipe)
  • recipeScott's water (see recipe)
  • 1% (w/v) eosin (BDH Chemicals)
  • Complete Mini Protease Inhibitor Tablets (Roche Molecular Biologicals)
  • Xylene
  • Solubilization buffer compatible with protein analysis to be performed
  • Cryostat (Leica CM1850 or equivalent)
  • Glass slides, cleaned by dipping in ethanol
  • Arcturus PixCell II laser capture microdissection system
  • CapSure LCM caps or CapSure HS caps (Arcturus)
  • Humidified chamber (prepared by placing wet tissues inside the base of a plastic pipet‐tip box)
  • 0.5‐ml Eppendorf Safe‐Lock tubes

Alternate Protocol 1: Immunolabeling of Tissue Sections for LCM

  • recipeTBS (see recipe)
  • Complete Mini Protease Inhibitor Tablets (Roche Molecular Biologicals)
  • Acetone
  • Primary antibody against protein of interest
  • Gold‐labeled secondary antibody (British BioCell International)
  • Silver‐enhancing kit (British BioCell International)

Support Protocol 1: Collection and Storage of Tissue for Analysis by LCM

  Materials
  • Tissue of interest freshly obtained from patient
  • recipeTransport medium (see recipe), ice‐cold
  • RPMI 1640 medium (Life Technologies)
  • Complete Mini Protease Inhibitor Tablets (Roche Molecular Biologicals)
  • recipePBS (see recipe), ice‐cold
  • Isotonic (250 mM) sucrose (8.6 g sucrose, H 2O to 100 ml; prepare immediately before use), ice‐cold
  • OCT embedding medium (BDH)
  • Liquid nitrogen
  • Sterile disposable scalpels
  • Cryostat chucks, precooled on dry ice

Support Protocol 2: Quantification of Protein in Material Captured by LCM Using Sypro Ruby

  Materials
  • 10 mM Tris⋅Cl, pH 8.0 ( appendix 2E)
  • 10 mM Tris⋅Cl, pH 8.0 ( appendix 2E)/ 0.1% (w/v) NP‐40 (prepare fresh; add NP‐40 from 10% w/v stock solution)
  • Bovine serum albumin (BSA)
  • Samples from LCM (see protocol 1) in 10 mM Tris⋅Cl, pH 8.0/1% NP‐40
  • SYPRO Ruby protein blot stain (Molecular Probes)
  • MultiScreen‐HA sterile 96‐well plate (mixed cellulose ester membrane; Millipore)
  • Fluorescence plate reader (BMG FLUOstar or equivalent reader with excitation filter at either 280 or 450 nm and emission filter at 618 nm)

Basic Protocol 2: Two‐Dimensional Gel Electrophoresis of Material Captured by LCM

  Materials
  • Cells of interest captured by LCM (see protocol 1)
  • recipe2‐D PAGE lysis buffer (see recipe)
  • recipeRe‐swell buffer (see recipe)
  • DryStrip cover fluid (Amersham Pharmacia Biotech)
  • Molecular‐weight markers (Novex Mark12 unstained standards, or equivalent)
  • recipe1% LMP agarose (see recipe)
  • recipeEquilibration buffer (see recipe) containing 10 mg/ml dithiothreitol (DTT) and a trace of bromphenol blue
  • recipeEquilibration buffer (see recipe) containing 40 mg/ml iodoacetamide and a trace of bromphenol blue
  • recipeSDS‐PAGE running buffer (see recipe)
  • 50% (v/v) methanol/10% (v/v) acetic acid
  • OWL silver stain (Autogen Bioclear)
  • 0.5‐ml Eppendorf Safe‐Lock tubes
  • 18‐cm pH 3‐10NL IPG strips (Amersham Pharmacia Biotech)
  • IPGphor and 18‐cm strip holders (Amersham Pharmacia Biotech)
  • Equilibration tray
  • ISO‐DALT tank, casting unit, and glass plates (Amersham Pharmacia Biotech)
  • Gel scanner (Molecular Dynamics Personal Densitometer or equivalent)
  • Additional reagents and equipment for 2D‐PAGE (unit 10.4)

Basic Protocol 3: Analysis of Material Captured by LCM Using Immunoblotting

  Materials
  • Cells of interest captured by LCM (see protocol 1)
  • recipeSDS‐PAGE lysis buffer (see recipe)
  • recipeSDS‐PAGE running buffer (see recipe)
  • Molecular weight markers (Invitrogen MultiMark multicolored standards, or equivalent)
  • recipeTowbin's transfer buffer (see recipe)
  • recipeTBS‐T (see recipe) containing 1%, 5%, and 10% (w/v) nonfat dry milk
  • Primary antibody against protein of interest
  • recipeTBS‐T (see recipe)
  • Envision kit (Dako; available for use with rabbit or mouse primary antibodies)
  • Super Signal (PerBio)
  • 0.5‐ml Eppendorf Safe‐Lock tubes
  • Minigel system (BioRad mini‐PROTEAN II and Mini Trans‐Blot cell or equivalent)
  • Whatman filter paper
  • Hybond C Super nitrocellulose membrane (Amersham Pharmacia Biotech)
  • Additional reagents and equipment for one‐dimensional SDS‐PAGE (unit 10.1) and immunoblotting (unit 10.10)

Basic Protocol 4: Seldi Mass Spectrometry

  Materials
  • Cells of interest captured by LCM (see protocol 1)
  • 10 mM Tris⋅Cl, pH 8.0 ( appendix 2E)/ 0.1% (w/v) NP‐40 (prepare fresh; add NP‐40 from 10% w/v stock solution)
  • recipe20% (v/v) acetonitrile/0.2% (v/v) trifluoroacetic acid (see recipe)
  • recipeSinapinic acid matrix solution, saturated (see recipe)
  • recipe50% acetonitrile (see recipe)
  • 10% (v/v) acetonitrile/0.1% v/v trifluoroacetic acid (see recipe for recipe20% acetonitrile/0.2% TFA; dilute 1:1 with H 2O)
  • 10 mM HCl
  • recipe100 mM ammonium acetate pH 6.5 (see recipe)
  • recipe50 mM nickel sulfate (see recipe)
  • recipe2× PBS/NaCl (see recipe)
  • SlickSeal low‐protein‐binding tubes (National Scientific Supply)
  • NP2, H4, SAX2, WCX2, and/or IMAC3 ProteinChips (Ciphergen)
  • SELDI mass spectrometer (Ciphergen)
  • Humidified chamber (prepared by placing wet tissues inside the base of a plastic pipet‐tip box)
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Figures

Videos

Literature Cited

Literature Cited
   Banks, R.E., Dunn, M.J., Forbes, M.A., Stanley, A., Pappin, D., Naven, T., Gough, M., Harnden, P., and Selby, P.J. 1999. The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis: Preliminary findings. Electrophoresis 20:689‐700.
   Cornea, A. and Mungensat, A. 2002. Comparison of current equipment. Methods Enzymol. 356:3‐12.
   Craven, R.A. and Banks, R.E. 2002. Use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis. Methods Enzymol. 356:33‐49.
   Craven, R.A., Totty, N., Harnden, P., Selby, P.J., and Banks, R.E. 2002. Laser capture microdissection and two‐dimensional polyacrylamide gel electrophoresis: Evaluation of tissue preparation and sample limitations. Am. J. Pathol. 160:815‐822.
   Emmert‐Buck, M.R., Bonner, R.F., Smith, P.D., Chuaqui, R.F., Zhuang, Z., Goldstein, S.R., Weiss, R.A., and Liotta, L.A. 1996. Laser capture microdissection. Science 274:998‐1001.
   Emmert‐Buck, M.R., Gillespie, J.W., Paweletz, C.P., Ornstein, D.K., Basrur, V., Appella, E., Wang, Q.H., Huang, J., Hu, N., Taylor, P., and Petricoin, E.F. 2000. An approach to proteomic analysis of human tumors. Mol. Carcino. 27:158‐165.
   Hofman, F. 2002. Immunohistochemistry. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober) 21.4.1‐21.4.23. John Wiley & Sons, New York.
   Knezevic, V., Leethanakul, C., Bichsel, V.E., Worth, J.M., Prabhu, V.V., Gutkind, J.S., Liotta, L.A., Munson, P.J., Petricoin, E.F., and Krizman, D.B. 2001. Proteomic profiling of the cancer microenvironment by antibody arrays. Proteomics 1:1271‐1278.
   Kolble, K. 2000. The LEICA microdissection system: Design and applications. J. Mol. Med. 78:B24‐B25.
   Palmer‐Toy, D.E., Sarracino, D.A., Sgroi, D., LeVangie, R., and Leopold, P.E. 2000. Direct acquisition of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectra from laser capture microdissected tissues. Clin. Chem. 46:1513‐1516.
   Paweletz, C.P., Ornstein, D.K., Roth, M.J., Bichsel, V.E., Gillespie, J.W., Calvert, V.S., Vocke, C.D., Hewitt, S.M., Duray, P.H., Herring, J., Wang, Q.H., Hu, N., Linehan, W.M., Taylor, P.R., Liotta, L.A., Emmert‐Buck, M.R., and Petricoin, E.F. III. 2000a. Loss of annexin 1 correlates with early onset of tumorigenesis in esophageal and prostate carcinoma. Cancer Res. 60:6293‐6297.
   Paweletz, C.P., Gillespie, J.W., Ornstein, D.K., Simone, N.L., Brown, M.R., Cole, K.A., Wang, Q.‐H., Huang, J., Hu, N., Yip, T.‐T., Rich, W.E., Kohn, E.C., Linehan, W.M., Weber, T., Taylor, P., Emmert‐Buck, M.R., Liotta, L.A., and Petricoin, E.F. 2000b. Rapid protein display profiling of cancer progression directly from human tissue using a protein biochip. Drug Dev. R. 49:34‐42.
   Paweletz, C.P., Charboneau, L., Bichsel, V.E., Simone, N.L., Chen, T., Gillespie, J.W., Emmert‐Buck, M.R., Roth, M.J., Petricoin, III E.F., and Liotta, L.A. 2001. Reverse phase protein microarrays which capture disease progression show activation of pro‐survival pathways at the cancer invasion front. Oncogene 20:1981‐1989.
   Schutze, K. and Lahr, G. 1998. Identification of expressed genes by laser‐mediated manipulation of single cells. Nature Biotechnol. 16:737‐742.
   Simone, N.L., Remaley, A.T., Charboneau, L., Petricoin, E.F., Glickman, J.W., Emmert‐Buck, M.R., Fleisher, T.A., and Liotta, L.A. 2000. Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection. Am. J. Pathol. 156:445‐452.
   von Eggeling, F., Davies, H., Lomas, L., Fiedler, W., Junker, K., Claussen, U., and Ernst, G. 2000. Tissue‐specific microdissection coupled with ProteinChip array technologies: Applications in cancer research. Biotechniques 29:1066‐1070.
   Westphal, G., Burgemeister, R., Friedemann, G., Wellmann, A., Wernert, N., Wollscheid, V., Becker, B., Vogt, T., Knuchel, R., Stolz, W., and Schutze, K. 2002. Noncontact laser catapulting: A basic procedure for functional genomics and proteomics. Methods Enzymol. 356:80‐97.
   Wright, Jr. G.L.. Cazares, L.H., Leung, S.‐M., Nasim, S., Adam, B.L., Yip, T.‐T., Schellhammer, P.F., Gong, L., and Vlahou, A. 1999. ProteinChip surface enhanced laser desorption/ionization (SELDI) mass spectrometry: A novel protein biochip technology for detection of prostate cancer biomarkers in complex protein mixtures. Prostate Cancer Prostatic Dis. 2:264‐276.
Key References
  Conn, P.M. (ed.). 2002. Laser Capture Microscopy and Microdissection. In Methods in Enzymology, Vol. 356. Academic Press, San Diego, Ca.
  A whole volume devoted to all aspects of this technology, from a description of the different systems through to protocols and applications for laser‐based cell selection for subsequent analysis of either proteins or nucleic acids.
Internet Resources
  http://www.arctur.com/
  The homepage for Arcturus. This Web site and the sites below are the homepages of the companies supplying the different laser‐based microdissection systems and the SELDI‐MS. Their listing does not constitute an endorsement of their products by the authors but indicates where further technical information can be obtained. Some of the sites provide limited protocols, links to other relevant sites, and also listings of relevant publications.
  http://www.palm.spacenet.de/
  The homepage for PALM.
  http://www.leica‐microsystems.com/
  The homepage for Leica.
  http://www.ciphergen.com/
  The homepage for Ciphergen.
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