Laser Capture Microdissection and Two-Dimensional Polyacrylamide Gel Electrophoresis
2002; Elsevier BV; Volume: 160; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64904-8
ISSN1525-2191
AutoresRachel A. Craven, Nick Totty, Patricia Harnden, Peter J. Selby, Rosamonde E. Banks,
Tópico(s)Advanced Proteomics Techniques and Applications
ResumoLaser capture microdissection (LCM) is now well established as a tool for facilitating the enrichment of cells of interest from tissue sections, overcoming the problem of tissue heterogeneity. LCM has been used extensively in combination with analysis at the DNA and RNA levels, but only a small number of studies have employed LCM with subsequent protein analysis, albeit with promising results. This study focuses on the potential of LCM in combination with two-dimensional polyacrylamide gel electrophoresis. The effects of tissue section preparation and sample type were evaluated to fully determine the suitability of using LCM in global protein profiling. The effects of several histochemical stains (hematoxylin and eosin, methyl green and toluidine blue) and immunolabeling on subsequent two-dimensional polyacrylamide gel electrophoresis were investigated. Quantitative analysis was performed to establish the extent of changes in the relative intensity of protein species and their reproducibility. All staining protocols tested were found to be compatible with protein analysis although there was variation in protein recovery and the quality of the protein profiles obtained. LCM of renal and cervix samples indicated that protein yield after dissection was acceptable, although the extent of enrichment and dissection time was tissue-dependent, which may preclude the use of this approach with some tissue types. These results indicate that LCM has potential as a tool in proteomic research. Laser capture microdissection (LCM) is now well established as a tool for facilitating the enrichment of cells of interest from tissue sections, overcoming the problem of tissue heterogeneity. LCM has been used extensively in combination with analysis at the DNA and RNA levels, but only a small number of studies have employed LCM with subsequent protein analysis, albeit with promising results. This study focuses on the potential of LCM in combination with two-dimensional polyacrylamide gel electrophoresis. The effects of tissue section preparation and sample type were evaluated to fully determine the suitability of using LCM in global protein profiling. The effects of several histochemical stains (hematoxylin and eosin, methyl green and toluidine blue) and immunolabeling on subsequent two-dimensional polyacrylamide gel electrophoresis were investigated. Quantitative analysis was performed to establish the extent of changes in the relative intensity of protein species and their reproducibility. All staining protocols tested were found to be compatible with protein analysis although there was variation in protein recovery and the quality of the protein profiles obtained. LCM of renal and cervix samples indicated that protein yield after dissection was acceptable, although the extent of enrichment and dissection time was tissue-dependent, which may preclude the use of this approach with some tissue types. These results indicate that LCM has potential as a tool in proteomic research. Tissue heterogeneity and the consequent need for enrichment before sample analysis presents a major problem in the study of disease. Several strategies have been used to facilitate selective purification of relevant cell types. Antibody-based approaches have frequently been used,1Page MJ Amess B Townsend RR Parekh R Herath A Brusten L Zvelebil MJ Stein RC Waterfield MD Davies SC O'Hare MJ Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties.Proc Natl Acad Sci USA. 1999; 96: 12589-12594Crossref PubMed Scopus (185) Google Scholar, 2Reymond MA Sanchez JC Hughes GJ Günther K Riese J Tortola S Peinado MA Kirchner T Hohenberger W Hochstrasser DF Köckerling F Standardized characterization of gene expression in human colorectal epithelium by two-dimensional electrophoresis.Electrophoresis. 1997; 18: 2842-2848Crossref PubMed Scopus (57) Google Scholar, 3Sarto C Marocchi A Sanchez JC Giannone D Frutiger S Golaz O Wilkins MR Doro G Cappellano F Hughes G Hochstrasser DF Mocarelli P Renal cell carcinoma and normal kidney protein expression.Electrophoresis. 1997; 18: 599-604Crossref PubMed Scopus (131) Google Scholar but often require the use of short-term cell culture or enzymatic digestion to produce a single cell suspension as a starting material which may introduce in vitro artifacts. A number of manual and laser-assisted microdissection techniques have also been used4Sirivatanauksorn Y Drury R Crnogorac-Jurcevic T Sirivatanauksorn V Lemoine NR Laser-assisted microdissection: applications in molecular pathology.J Pathol. 1999; 189: 150-154Crossref PubMed Scopus (51) Google Scholar, 5Fend F Raffeld M Laser capture microdissection in pathology.J Clin Pathol. 2000; 53: 666-672Crossref PubMed Scopus (187) Google Scholar, 6Emmert-Buck MR Bonner RF Smith PD Chuaqui RF Zhuang Z Goldstein SR Weiss RA Liotta LA Laser capture microdissection.Science. 1996; 274: 998-1001Crossref PubMed Scopus (2113) Google Scholar, 7Gillespie JW Ahram M Best CJ Swalwell JI Krizman DB Petricoin EF Liotta LA Emmert-Buck MR The role of tissue microdissection in cancer research.Cancer J. 2001; 7: 32-39PubMed Google Scholar, 8Schütze K Lahr G Identification of expressed genes by laser-mediated manipulation of single cells.Nature Biotechnol. 1998; 16: 737-742Crossref Scopus (371) Google Scholar with laser capture microdissection (LCM) emerging as one of the methods of choice. The fast and precise dissection possible with LCM, combined with the ability to readily confirm the nature of the captured material are obvious advantages of this approach. As with other microscope-based dissection techniques however, LCM is dependent on previous fixation and staining of tissue sections and consequently there is a risk of artifacts. The effects of sample processing for LCM on nucleic acids have been thoroughly investigated9Burton MP Schneider BG Brown R Escamilla-Ponce N Gulley ML Comparison of histologic stains for use in PCR analysis of microdissected, paraffin-embedded tissues.Biotechniques. 1998; 24: 86-92PubMed Google Scholar, 10To MD Done SJ Redston M Andrulis IL Analysis of mRNA from microdissected frozen tissue sections without RNA isolation.Am J Pathol. 1998; 153: 47-51Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 11Fend F Emmert-Buck MR Chuaqui R Cole K Lee J Liotta LA Raffeld M Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis.Am J Pathol. 1999; 154: 61-66Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 12Goldsworthy SM Stockton PS Trempus CS Foley JF Maronpot RR Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue.Mol Carcinog. 1999; 25: 86-91Crossref PubMed Scopus (291) Google Scholar and a large number of studies have described analysis of DNA and RNA extracted from laser-captured material.5Fend F Raffeld M Laser capture microdissection in pathology.J Clin Pathol. 2000; 53: 666-672Crossref PubMed Scopus (187) Google Scholar These include global analyses of gene expression at the mRNA level using cDNA microarrays13Luo L Salunga RC Guo H Bittner A Joy KC Galindo JE Xiao H Rogers KE Wan JS Jackson MR Erlander MG Gene expression profiles of laser-captured adjacent neuronal subtypes.Nat Med. 1999; 5: 117-122Crossref PubMed Scopus (643) Google Scholar, 14Sgroi DC Teng S Robinson G LeVangie R Hudson Jr, JR Elkahloun AG In vivo gene expression profile analysis of human breast cancer progression.Cancer Res. 1999; 59: 5656-5661PubMed Google Scholar, 15Leethanakul C Patel V Gillespie J Pallente M Ensley JF Koontongkaew S Liotta LA Emmert-Buck M Gutkind JS Distinct pattern of expression of differentiation and growth-related genes in squamous cell carcinomas of the head and neck revealed by the use of laser capture microdissection and cDNA arrays.Oncogene. 2000; 19: 3220-3224Crossref PubMed Scopus (259) Google Scholar, 16Kitahara O Furukawa Y Tanaka T Kihara C Ono K Yanagawa R Nita ME Takagi T Nakamura Y Tsunoda T Alterations of gene expression during colorectal carcinogenesis revealed by cDNA microarrays after laser-capture microdissection of tumor tissues and normal epithelia.Cancer Res. 2001; 61: 3544-3549PubMed Google Scholar and construction of cDNA libraries.17Leethanakul C Patel V Gillespie J Shillitoe E Kellman RM Ensley JF Limwongse V Emmert-Buck MR Krizman DB Gutkind JS Gene expression profiles in squamous cell carcinomas of the oral cavity: use of laser capture microdissection for the construction and analysis of stage-specific cDNA libraries.Oral Oncol. 2000; 36: 474-483Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 18Emmert-Buck MR Strausberg RL Krizman DB Bonaldo MF Bonner RF Bostwick DG Brown MR Buetow KH Chuaqui RF Cole KA Duray PH Englert CR Gillespie JW Greenhut S Grouse L Hillier LW Katz KS Klausner RD Kuznetzov V Lash AE Lennon G Linehan WM Liotta LA Marra MA Munson PJ Ornstein DK Prabhu VV Prange C Schuler GD Soares MB Tolstoshev CM Vocke CD Waterston RH Molecular profiling of clinical tissue specimens: feasibility and applications.Am J Pathol. 2000; 156: 1109-1115Abstract Full Text Full Text PDF PubMed Scopus (71) Google ScholarProteomics provides a complementary approach to the study of gene expression, allowing additional information regarding the effects of post–translational modifications and post–transcriptional controls to be explored. Technological advances, particularly in two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry, have facilitated the study of gene expression at the protein level leading to the recent expansion of proteomics-based research.19Chambers G Lawrie L Cash P Murray GI Proteomics: a new approach to the study of disease.J Pathol. 2000; 192: 280-288Crossref PubMed Scopus (184) Google Scholar, 20Banks RE Dunn MJ Hochstrasser DF Sanchez JC Blackstock W Pappin DJ Selby PJ Proteomics: new perspectives, new biomedical opportunities.Lancet. 2000; 356: 1749-1756Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar This is well illustrated by examples from the field of tumor biology, with bladder cancer being one of the tumors most extensively studied.21Celis JE Wolf H Ostergaard M Bladder squamous cell carcinoma biomarkers derived from proteomics.Electrophoresis. 2000; 21: 2115-2121Crossref PubMed Scopus (95) Google Scholar The use of LCM in combination with protein analysis is now also increasing with study of specific proteins of interest by immunoassays22Simone NL Remaley AT Charboneau L Petricoin III EF Glickman JW Emmert-Buck MR Fleisher TA Liotta LA Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection.Am J Pathol. 2000; 156: 445-452Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 23Natkunam Y Rouse RV Zhu S Fisher C van De RM Immunoblot analysis of CD34 expression in histologically diverse neoplasms.Am J Pathol. 2000; 156: 21-27Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 24Ornstein DK Englert C Gillespie JW Paweletz CP Linehan WM Emmert-Buck MR Petricoin EF Characterization of intracellular prostate-specific antigen from laser capture microdissected benign and malignant prostatic epithelium.Clin Cancer Res. 2000; 6: 353-356PubMed Google Scholar, 25Paweletz CP Ornstein DK Roth MJ Bichsel VE Gillespie JW Calvert VS Vocke CD Hewitt SM Duray PH Herring J Wang QH Hu N Linehan WM Taylor PR Liotta LA Emmert-Buck MR Petricoin EF Loss of annexin 1 correlates with early onset of tumorigenesis in esophageal and prostate carcinoma.Cancer Res. 2000; 60: 6293-6297PubMed Google Scholar as well as global profiling by 2D-PAGE26Banks RE Dunn MJ Forbes MA Stanley A Pappin D Naven T Gough M Harnden P Selby PJ The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis—preliminary findings.Electrophoresis. 1999; 20: 689-700Crossref PubMed Scopus (280) Google Scholar, 27Ornstein DK Gillespie JW Paweletz CP Duray PH Herring J Vocke CD Topalian SL Bostwick DG Linehan WM Petricoin III EF Emmert-Buck MR Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines.Electrophoresis. 2000; 21: 2235-2242Crossref PubMed Scopus (223) Google Scholar, 28Emmert-Buck MR Gillespie JW Paweletz CP Ornstein DK Basrur V Appella E Wang QH Huang J Hu N Taylor P Petricoin III EF An approach to proteomic analysis of human tumors.Mol Carcinog. 2000; 27: 158-165Crossref PubMed Scopus (188) Google Scholar, 29Lawrie LC Curran S McLeod HL Fothergill JE Murray GI Application of laser capture microdissection and proteomics in colon cancer.Mol Pathol. 2001; 54: 253-258Crossref PubMed Scopus (109) Google Scholar and surface-enhanced laser desorption/ionization mass spectrometry,30Wright GL Cazares LH Leung S-M Nasim S Adam B-L Yip T-T Schellhammer PF Gong L Vlahou A ProteinchipR 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. 1999; 2: 264-276Crossref PubMed Scopus (255) Google Scholar, 31Paweletz CP Gillespie JW Ornstein DK Simone NL Brown MR Cole KA Wang QH Huang J Hu N Yip T-T Rich WE Kohn EC Linehan WM Weber T Taylor P Emmert-Buck M Liotta LA Petricoin EF Rapid protein display profiling of cancer progression directly from human tissue using a protein biochip.Drug Dev Res. 2000; 49: 34-42Crossref Scopus (139) Google Scholar, 32von Eggeling F Davies H Lomas L Fiedler W Junker K Claussen U Ernst G Tissue-specific microdissection coupled with ProteinChip array technologies: applications in cancer research.Biotechniques. 2000; 29: 1066-1070PubMed Google Scholar being used to analyze captured material. However, the effects of tissue section preparation on the protein profile have not been thoroughly evaluated and the scope of LCM as a tool in proteomics research remains to be determined.Here we describe in detail the effects of hematoxylin and eosin (H&E) staining of frozen sections on subsequent 2D-PAGE. In addition we examine the use of alternative histochemical stains and a rapid immunolabeling protocol as alternative methods for sample processing. We also compare the protein recovery and enrichment obtained after dissection of two contrasting tissue types to evaluate the potential and limitations of LCM as a tool in global protein expression profiling.Materials and MethodsMaterialsGeneral chemicals were obtained from BDH (Poole, UK) or Sigma (Poole, UK) unless stated otherwise. Ammonium persulfate, Tris, and urea were from ICN (Basingstoke, UK), glycine from Genomic Solutions (Cambridge, UK), CHAPS from Calbiochem (Nottingham, UK), Pharmalyte pH 3–10 from Amersham Pharmacia (Little Chalfont, UK), Protogel acrylamide (30% acrylamide:0.8% bis-acrylamide) from Flowgen (Sittingbourne, UK), LMP agarose from Gibco Life Technologies (Paisley, UK), Complete protease inhibitor cocktail tablets and trypsin (modified, sequencing grade) from Roche (Lewes, UK), Pefabloc, hematoxylin and eosin (1% aqueous solution) from BDH, toluidine blue O and methyl green from Sigma, and trifluoroacetic acid from Pierce (Rockford, IL). Milli-Q grade water was used for all solutions.TissuesSamples of normal renal cortex, renal cell carcinoma (RCC), and normal cervix were obtained immediately after surgery and areas of tissue selected by a pathologist. Tissue was cut into blocks, washed briefly with ice-cold phosphate-buffered saline (pH 7.2) followed by ice-cold isotonic (0.25 mol/L) sucrose, embedded in OCT compound, snap-frozen in liquid nitrogen and stored at −80°C.Section Fixation/Staining and LCMEight-μm frozen tissue sections were cut onto ethanol-dipped glass slides using a Leica Cryocut 1800 microtome and slides placed on dry ice until subsequent fixation and staining. For experiments in which several sample processing conditions were examined in parallel, sections were cut alternately for each condition to minimize changes resulting from alterations in the composition of the tissue block.H&E staining was performed using a modified rapid protocol for LCM as previously described.26Banks RE Dunn MJ Forbes MA Stanley A Pappin D Naven T Gough M Harnden P Selby PJ The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis—preliminary findings.Electrophoresis. 1999; 20: 689-700Crossref PubMed Scopus (280) Google Scholar Slides were defrosted, fixed (70% ethanol for 1 minute), H&E stained (Mayer's hematoxylin for 30 seconds, Scott's tap water for 10 seconds, eosin for 10 seconds), and dehydrated (70% ethanol for 30 seconds, 100% ethanol for 1 minute, xylene for 2 × 5 minutes). H&E solutions contained Complete protease inhibitor cocktail. For processing experiments investigating the effect of different stages of the staining protocol on the protein profile, individual steps were omitted and replaced with mock incubations in water.For alternative histochemical stains rapid-staining protocols based on those available at the Arcturus web site (www.arctur.com) were adopted. Slides were defrosted, fixed (70% ethanol for 1 minute), stained (1% w/v aqueous methyl green for 1 minute or 0.025% w/v toluidine blue O in 50 mmol/L sodium phosphate buffer, pH5.5, for 5 seconds), and then dehydrated (70% ethanol for 30 seconds, 100% ethanol for 1 minute, and xylene for 2 × 5 minutes). All stains contained Complete protease inhibitor cocktail.Immunolabeling was performed using a rapid-staining method that used gold-conjugated secondary antibodies and silver enhancement as a detection system. This was chosen as it does not include an enzymatic step that could potentially induce protein modifications. In initial experiments, parameters such as time of incubation and reagent concentration were optimized to yield the final protocol. Slides were defrosted, fixed (acetone for 2 minutes or 70% v/v ethanol for 1 minute) and labeled (primary antibody for 5 minutes and gold-conjugated secondary antibody for 5 minutes). Labeling steps were performed in Tris-buffered saline (pH 7.6) containing Complete protease inhibitor cocktail and followed by brief washes with Tris-buffered saline. Gold particles were visualized by silver enhancement (British BioCell International, Cardiff, UK) according to the manufacturer's instructions. Mouse anti-human CD13 (clone WM-47; DAKO, Cambridge, UK) was diluted 1 in 25 for acetone fixation or 1 in 5 for ethanol fixation and gold conjugated F(ab)2 anti-mouse IgGs for light microscopy (British BioCell International) was diluted 1 in 25.Sections were dissected using a Pixcell II LCM system (Arcturus Engineering Inc., Mountain View, CA) with a 15-μm diameter laser beam being used for cervix and RCC samples and a 7.5-μm diameter laser beam for normal renal cortex.Preparation of Protein ExtractsIn experiments examining fixation and staining effects, tissue sections subjected to the different fixation, staining, or immunolabeling protocols were scraped into lysis buffer using a sterile disposable scalpel. Control samples were prepared from sections cut directly into lysis buffer. Laser-captured material was directly solubilized from successive caps into lysis buffer.Tissue was solubilized in a urea/thiourea-based lysis buffer33Rabilloud T Adessi C Giraudel A Lunardi J Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients.Electrophoresis. 1997; 18: 307-316Crossref PubMed Scopus (403) Google Scholar (7 mol/L urea, 2 mol/L thiourea, 4% w/v CHAPS, 1% w/v dithiothreitol, 0.8% Pharmalyte pH 3–10, 1 mg/ml Pefabloc) for 30 minutes at room temperature. Samples were vigorously mixed then centrifuged (42,000 × g, 1 hour, 15°C) and extracts stored at −80°C.Protein extracts were assayed using a modified Bradford method (Protein Assay; Bio-Rad, Hemel Hempstead, UK). It should be noted that protein recovery figures given for processing experiments would be affected by loss of material during scraping of sections, so although the values can be directly compared, in absolute terms they will be underestimations.2D-PAGEIsoelectric focusing was performed on 18-cm immobilized pH gradient strips (pH 3-10NL, Amersham Pharmacia) using an IPGphor (Amersham Pharmacia). Samples were applied by in-gel rehydration34Sanchez JC Rouge V Pisteur M Ravier F Tonella L Moosmayer M Wilkins MR Hochstrasser DF Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients.Electrophoresis. 1997; 18: 324-327Crossref PubMed Scopus (283) Google Scholar (30 V, 13 hours) using 7 mol/L urea, 2 mol/L thiourea, 4% w/v CHAPS, 0.46% w/v dithiothreitol, 0.2% Pharmalyte pH 3 to 10 with a trace of bromophenol blue as the reswell buffer with a total sample volume of 450 μl. Thirty μg protein was generally loaded for analytical gels except for LCM samples in which the load was that procured by a specific number of laser shots as indicated in the text. For preparative gels 300 to 600 μg of protein was loaded. Focusing was performed for 65 kVh (200 V 1 hour, 500 V 1 hour, 1000 V 1 hour, 1000 to 8000 V 1 hour, 8000 V to end).SDS-PAGE was performed using 10% T or 12% T gels with a 4% T stack using the ISO-DALT system (Amersham Pharmacia). Immobilized pH gradient strips were incubated in equilibration buffer (6 mol/L urea, 30% v/v glycerol, 2% w/v sodium dodecyl sulfate, 0.05 mol/L Tris-HCl, pH 6.8) containing 1% w/v dithiothreitol for 15 minutes followed by equilibration buffer containing 4% w/v iodoacetamide for 10 minutes and then rinsed with gel running buffer. Strips were placed on top of the second dimension gels and overlaid with 1% w/v low melting point (LMP) agarose in Tris-glycine gel running buffer (24 mmol/L Tris, 0.2 mol/L glycine, 0.1% w/v sodium dodecyl sulfate). Electrophoresis was performed at 15 to 20 mA per gel at 12.5°C.For analytical purposes, gels were fixed (50% v/v methanol, 10% v/v acetic acid) and proteins detected by silver staining using OWL silver stain (OWL Separation Systems, Portsmouth, NH). For subsequent protein sequencing, preparative gels were fixed (40% v/v ethanol, 10% v/v acetic acid) and silver stained using the PlusOne silver staining kit (Amersham Pharmacia) as described in the manufacturer's instructions but omitting glutaraldehyde from the sensitizing step and formaldehyde from the silver reaction and adding only 100 μl of formaldehyde to the developer.35Yan JX Wait R Berkelman T Harry RA Westbrook JA Wheeler CH Dunn MJ A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry.Electrophoresis. 2000; 21: 3666-3672Crossref PubMed Scopus (645) Google ScholarGels were scanned using a Personal Densitometer (Molecular Dynamics, Chesham, UK). Analysis was performed using Melanie 3 software (GeneBio, Geneva, Switzerland).Peptide Mass FingerprintingProtein spots were excised from the gel, destained in 15 mmol/L potassium ferricyanide (III) and 50 mmol/L sodium thiosulfate for 10 to 20 minutes then incubated in MilliQ-H2O for 2 × 20 minutes. Gel pieces were cut into small pieces and incubated with 25 mmol/L ammonium bicarbonate for 3 × 20 minutes and sonicated for 5 minutes in an ultrasonic bath. After centrifugation at 13,000 rpm in a microfuge for 5 minutes, gel pieces were dried by incubation with acetonitrile (3 × 15 minutes) during which they were vortexed regularly to prevent clumping. After removal of the last acetonitrile wash, tubes were left to air dry for 30 to 60 minutes then cooled on ice for 10 minutes. Ice-cold trypsin (33 μg/ml in 25 mmol/L of ammonium bicarbonate) was added to rehydrate the gel pieces and samples were incubated on ice for 30 minutes. A further twenty-five μl of ice-cold 25 mmol/L ammonium bicarbonate was added and samples were incubated overnight at 37°C. Supernatants were transferred into Slick Seal tubes (Bioquote, York, UK). Peptides were extracted twice with 50% v/v acetonitrile and 0.1% v/v trifluoroacetic acid for 15 minutes in an ultrasonic bath. These extracts were pooled and samples were dried in a centrifugal evaporator.Samples were analyzed using a T of Spec 2E MALDI-TOF mass spectrometer (Micromass, Manchester, UK). Peptide masses were screened against the NCBI database using MASCOT software.36Perkins DN Pappin DJ Creasy DM Cottrell JS Probability-based protein identification by searching sequence databases using mass spectrometry data.Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6711) Google Scholar Alternatively samples were subjected to nano-lc tandem mass spectrometry using a Q-TOF mass spectrometer (Micromass) and data collected and analyzed using MS-TAG or MS-PATTERN (/Protein Prospector).ResultsTissue Preparation for LCMH&E is the most commonly used general histochemical stain. Good protein recovery (60 to 80%) was obtained after H&E staining, and as previously described, there was no gross effect on the resulting protein profile of cervix or kidney cortex (Figure 1A).26Banks RE Dunn MJ Forbes MA Stanley A Pappin D Naven T Gough M Harnden P Selby PJ The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis—preliminary findings.Electrophoresis. 1999; 20: 689-700Crossref PubMed Scopus (280) Google Scholar Although the spot patterns were very similar qualitatively, there were a number of specific changes and quantitative analysis showed that a significant number of proteins changed their relative abundance after fixation and staining. Comparison of two independent processing experiments indicated that these changes were generally reproducible. The ratio of spot intensities in the control and processed samples was calculated for 580 spots in each experiment (Figure 1B). Comparison of these values showed a significant correlation (r = 0.700, P < 0.001), confirming that consistent protein profiles were obtained after processing. A second consequence of H&E staining that was also apparent was a general detrimental effect on protein focusing, especially at the basic end of the gel (see Figure 1, Figure 2). Dissection of the staining procedure by omitting each step in turn indicated that the specific protein changes could not be totally attributed to a particular stage. However, the effect on focusing resulted, at least in part, from eosin staining as samples prepared from hematoxylin-stained tissue sections (that is, with the omission of eosin) were not affected (Figure 2). This is unlikely to be a serious problem with the much smaller amounts of sample available after LCM.Figure 2The effect of eosin staining on 2D-PAGE. Two-dimensional gels of samples prepared from sections cut onto slides and stained with hematoxylin alone (left) or H&E (right) are shown. It should be noted that the effect of H&E staining on separation of proteins by 2D-PAGE was more pronounced in some experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The use of alternative histochemical stains was also investigated. Methyl green was found to be compatible with subsequent 2D-PAGE, with no effect on the quality of protein separation being apparent (Figure 3A). Again changes in the relative intensity of many proteins were evident after processing but there was acceptable correlation between two independent processing experiments. Toluidine blue also gave satisfactory results (Figure 3B), although a detrimental effect on protein recovery and focusing was clearly apparent and increased markedly with the relative intensity of staining of tissue sections. Generation of good quality protein profiles and subsequent gel analysis were therefore more problematic and the changes visible in the gel pattern were slightly less well conserved.Figure 3The effect of methyl green and toluidine blue staining on 2D-PAGE. Two-dimensional gels of samples prepared from sections cut directly into lysis buffer (left) or onto slides and processed (right) are shown for methyl green (A) and toluidine blue (B) staining. C: Scatter plots showing the conservation of relative change in intensity of protein species after processing are presented (for details see Figure 1).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Antibodies can be useful tools to identify specific subpopulations of cells. A rapid immunolabeling method developed using detection of gold-conjugated secondary antibodies by silver enhancement allowed the successful visualization of proximal tubules in normal kidney cortex sections after anti-CD13 labeling (Figure 4). Both acetone and ethanol fixation were investigated as the reactivity of many antibodies is dependent on choice of fixative. The overall yield of protein from acetone-fixed and immunolabeled sections was poor (20%) and although good separation of proteins by 2D-PAGE was achieved there was clearly a selective loss of proteins occurring during fixation and staining (Figure 5A). Ethanol fixation in combination with immunolabeling was more successful. Total protein recovery was improved compared to acetone fixation (40%) and changes in the protein profile were less pronounced (Figure 5B).Figure 4Immunolabeling of proximal tubules in normal kidney cortex. Normal kidney cortex sections were labeled with anti-CD13 antibodies using a rapid-staining protocol that uses silver enhancement of gold labeling as the detection system.View Large
Referência(s)