5

Analysis of Laser Capture Microdissected Cellsby 2-Dimensional Gel Electrophoresis

Daohai Zhang and Evelyn Siew-Chuan Koay

Summary

Laser capture microdissection (LCM) is a powerful tool for procuring near-purepopulations of targeted cell types from specific microscopic regions of tissue sections,by overcoming problems due to tissue heterogeneity and minimizing intermixture andcontamination by other cell types. The combination of LCM with various proteomictechnologies has enabled high-throughput molecular analysis of human tumors, andprovided critical tools in the search for novel disease markers and therapeutic targets. Asan example, we describe the application of LCM in dissecting the tumor cells in breastcancer for macromolecular extraction and subsequent protein separation by 2-dimensionalgel electrophoresis (2-D GE). The protocols and the key issues involved in preparingethanol-fixed paraffin-embedded tissue blocks and microscopic sections, microdissectingthe cells of interest using the PixCell II LCM system, extracting and separating the cellularproteins by 2-D GE, and preparing selective proteins for peptide mass analysis by massspectrometry, are discussed. The aim is to provide a practical guide in performing high-throughput microdissection of target cells and gel-based proteomics, which can be adaptedto research in cancer formation and growth.

Key Words: laser capture microdissection; 2-dimensional gel electrophoresis; breastcancer; proteomics; silver staining.

1. IntroductionCellular proteins (collectively known as “proteomes”) are less susceptible

than the transcriptome to experimental artifacts arising from the rigors of tissuecollection and processing, and advances in global protein expression analysis

From: Methods in Molecular Biology, vol. 428: Clinical Proteomics: Methods and ProtocolsEdited by: A. Vlahou © Humana Press, Totowa, NJ

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78 Zhang and Koay

(expression proteomics) have been used in mapping cellular pathways, identi-fying the molecular alterations associated with disease onset and progressionand searching for potential tumor markers or drug targets in human disease,especially in cancer. However, to obtain cell-specific protein profiles, homoge-neous or near-pure populations of the cells of interest, free from contaminationby adjacent cell types, are prerequisites. Laser capture microdissection (LCM)was developed to enable the procurement of near-pure populations of the targetcells with a greater speed and precision than is possible with manual dissectionmethods. LCM permits selective transfer of specific cell types, under directmicroscopic visualization, from complex tissues onto a polymer film that isactivated by laser pulses, whilst retaining their morphology. The homogeneityof encapsulated cells can be verified microscopically. With these inherentadvantages, LCM has become a valuable research tool and has been applied tocellular and molecular studies of various cancers, including breast (1,2), colon(3), and liver (4) cancers. It is equally efficacious in procuring cell populationsfrom both frozen tissues (3,4) and ethanol-fixed, paraffin-embedded tissues(1,5).

Protein profiles of the LCM-dissected cells can be obtained by two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) (6),16O/18O isotopic labeling (7), differential iodine radioisotope detection (2),isotope-coded affinity tag (iCAT) coupled with two-dimensional tandem massspectrometry (2-D LCMS/MS) (8), and mass spectrometry compatible silverstaining (1,9). Protein samples from LCM-dissected cells can also be appliedto reverse-protein arrays to analyze the key cellular signaling pathways andmetabolic networks (10,11). In this chapter, the in-house protocols used inthe authors’ laboratory for procuring near-pure populations of breast tumorcells from clinical samples, and for the extraction, isolation, and analysis oftheir protein profiles, are described. These include: (1) preparation of ethanol-fixed paraffin-embedded tissue blocks; (2) microdissection using the Pix IILCM System and cellular protein extraction; (3) protein separation by 2-D gelelectrophoresis (2-D GE), silver staining, and gel image analysis; and (4) prepa-ration of targeted proteins of interest for peptide mass analysis by tandem massspectrometry and identification of proteins of interest via database search.

2. Materials2.1. Histology—Tissue Block and Tissue Section Preparation

1. 70% (v/v), 80% (v/v), 95% (v/v), 100% ethanol2. Deionized or Milli-Q water (Millipore, Bedford, MA, USA)3. Hematoxylin solution, Mayer’s (Sigma, St. Louis, MO, USA)4. Eosin Y solution (Sigma)

Combining LCM with 2-D Gel Electrophoresis 79

5. Complete, mini protease inhibitor cocktail tablets (Roche Applied Science,Pleasanton, CA, USA)

6. Disposable microtome blades (Feather Safety Razor Co., Ltd., Osaka, Japan)7. Uncharged microscopic glass slides (Paul Marienfeld GmbH & Co, KG, Lauda-

Koenigshofen, Germany)8. Sakura Tissue-Tek® V.I.P.TM 5 Jr tissue processor (Sakura Finetek, Inc. Japan

Co., Ltd, Tokyo)9. Paraffin wax—Paraplast® tissue embedding medium; melting point 56-58°C,

store at room temperature (RT) (Structure Probe, Inc., West Chester, PA, USA)10. Xylenes, Reagent Grade (Sigma)11. Embedding molds—super metal base molds, 66mm × 54mm × 15mm (Surgipath

Medical Industries, Richmond, IL, USA)

2.2. Laser Capture Microdissection and Protein Sample Preparation

1. PixCell II LCM system (Arcturus Engineering, Mountain View, CA, USA)2. CapSure transparent plastic caps (Arcturus Engineering)3. Lysis buffer: 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1% Nonidet P (NP)-40,

0.5% (v/v) Triton X-100, 50 mM dithiothreitol (DTT), 40 mM Tris-HCl, pH 7.5,2 mM tributyl phosphine (TBP), and 1% (v/v) IPG buffer (pH 3–10). Store at RT.

4. PlusOne 2-D Clean-up Kit (GE Healthcare, San Francisco, CA, USA)5. Immobilized pH gradient (IPG) buffer (pH 3–10) (GE Healthcare)6. PlusOne 2-D Quantitation Kit (GE Healthcare)

2.3. Isoelectric Focusing (IEF) and Sodium DodecylSulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

1. EttanTM IPGphorTM IEF electrophoresis unit (GE Healthcare)2. Ceramic strip holders and EttanTM IPGphorTM Strip Holder Cleaning Solution

(GE Healthcare)3. ImmobilineTM IPG DryStrips (18 cm, pH 3–10, NL) (GE Healthcare)4. DryStrip Cover Fluid (GE Healthcare)5. Sample rehydration buffer: 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1%

(w/v) NP-40, 1% (v/v) IPG buffer, 50 mM DTT. DTT was added freshly to therehydration buffer prior to use. Store at RT.

6. Equilibration buffer A (prepare 10 ml for each strip): 6 M urea, 30% glycerol,2% SDS, 1% DTT, 50 mM Tris-HCl, pH 8.8. DTT is added to the stock solutionbefore use.

7. Equilibration buffer B (prepare 10 ml for each use strip): 6 M urea, 30% glycerol,2% SDS, 250 mg (2.5%, w/v) iodoacetamide (IAA), 50 mM Tris-HCl, pH 8.8.IAA is added to the stock solution before use.

8. 10% SDS-acrylamide gel: 33 ml acrylamide/bis (30% T, 5% C) (Bio-RadLaboratories, Hercules, CA, USA), 25 ml Tris (1.5 M, pH 8.8), 1 ml 10% (w/v)SDS, 0.5 ml 10% (w/v) ammonium persulfate (freshly prepared on the day ofuse), 35 μl TEMED (Bio-Rad). Make up to 100 ml with Milli-Q water.

80 Zhang and Koay

9. Water-saturated isobutanol: Shake equal volumes of Milli-Q water and isobu-tanol in a glass bottle and allow the mixture to separate. Transfer the top layerto a new bottle and store at RT.

10. Agarose sealing solution: Dissolve 0.5% low-melting-point agarose and 0.1%(w/v) bromophenol blue in 1× SDS-PAGE running buffer. Store at RT.

11. SDS-PAGE running buffer: 25 mM Tris, 198 mM glycine, 0.2% (w/v) SDS,pH 8.3

12. PROTEANTM II xi Cell system (Bio-Rad)

2.4. Silver Staining (see Note 1)

1. Fix solution: 5% acetic acid and 50% ethanol per 100 ml2. Sensitivity-enhancing solution: 30% (v/v) ethanol, 6.8% (w/v) sodium acetate,

100 μl of 2% (w/v) sodium thiosulphate per 100 ml3. Silver staining solution: 0.25% (w/v) silver nitrate4. Development solution: 2.5% (w/v) anhydrous potassium carbonate, 20 μl of 2%

(w/v) sodium thiosulphate per 100 ml, 40 μl of 37% formaldehyde per 100 ml.5. Stop solution: 4% (w/v) Tris and 2% (v/v) acetic acid per 100 ml6. Gel store (soak) solution: 1% (w/v) sodium acetate and 10% (v/v) methanol per

100 ml

2.5. Gel Image Analysis

1. Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA, USA)2. ImageMaster 2D Elite (Platinum) software (GE Healthcare)

2.6. In-gel Trypsin Digestion and Preparation for MS Analysis

1. Destaining solution: 30 mM potassium ferricyanide and 100 mM sodiumthiosulfate (1:1)

2. 25 mM sodium bicarbonate3. Dehydrating solution: 50 mM sodium bicarbonate and 50% (v/v) methanol per

100 ml4. SpeedVac centrifuge (TeleChem International, Inc., Sunnyvale, CA, USA)5. Digestion solution: 40 ng/μl trypsin sequencing grade (Promega, Madison, WI,

USA) in 20 mM ammonium bicarbonate solution6. Extraction solution (for hydrophobic peptides): 5% (v/v) trifluoracetic acid

(TFA) and 50% (v/v) acetonitrile (ACN) per 100 ml7. Peptide reconstitution solution: 0.1% (v/v) TFA8. ZipTip C18 columns (Millipore)9. Eluant: 70% (v/v) ACN and 0.1% TFA per 100 ml

10. Stainless steel MALDI-TOF sample target plates (Applied Biosystems,Framingham, MA, USA)

11. Alpha-cyano-4-hydroxycinnamic acid (�-CHCA) matrix, 3 mg/ml (Sigma)12. Applied Biosystems 4700 MALDI-TOF/TOF mass spectrometer

Combining LCM with 2-D Gel Electrophoresis 81

2.7. Database Search for Protein Identification

1. MASCOT software (Matrix Science, London, England)2. MS-Fit software (http://prospector.ucsf.edu)

3. MethodsThe methods described below have been successfully used in the authors’

laboratory for proteomics studies in human breast cancer specimens (1,9) andcan be applied to other cancer tissues as well. Breast tumors and matchednormal tissues were obtained from the Tissue Repository Unit of the NationalUniversity Hospital, Singapore, after approval by our Institutional ReviewBoard.

3.1. Preparation of Tissue Sections for LCM

In this step, frozen tissues can be directly transferred from the –80°C freezer,where they had been stored after surgical excision and trimming, to a pre-cooledtube containing 70% (v/v) ethanol and kept on ice. Ethanol-fixed paraffin-embedded tissue blocks should be prepared as quickly as possible, and thecompleted blocks stored at or below 4°C.

1. Fix the frozen tissue overnight in 70% ethanol at 4°C.2. Place each ethanol-fixed tissue piece, trimmed to appropriate dimensions, into

a pre-cooled cassette within the tissue processor and dehydrate according to thefollowing procedure: 30 min each in 70% and 80% ethanol at 40°C; 45 min in95% ethanol at 40°C (twice); 45 min in 100% ethanol at 40°C (twice), and 45 minin xylene at 40°C (twice) (see Note 2).

3. Embed the specimen in paraffin using embedding molds, with four changes ofparaffin after every 30-min interval.

4. Store the paraffin blocks at or below 4°C, if they were not to be processedimmediately for sectioning.

5. Put the block in a –20°C freezer for at least 1 h before cutting sections from it.6. Cut sections of 8 μm thickness using a standard microtome. Blades should be

changed regularly (see Note 3).7. Collect the tissue sections on uncharged microscopic glass slides, allow tissue

sections to be air dried, and store the cut sections at or below 4°C.

3.2. Staining of Paraffin-embedded Sections

The staining of sections for LCM is similar to that used in most histologylaboratories for morphological assessment. However, using minimal amount ofthe stain to visualize the tissue for microdissection will improve macromoleculerecovery (see Note 4). One tablet of protease inhibitor cocktail should be added

82 Zhang and Koay

to every 10 ml of each reagent (except xylene), and all reagents prepared usingdouble deionized water or Milli-Q® water. Staining should be performed asclose as possible to the scheduled LCM dissection.

1. Deparaffinize the sections in fresh xylene for 5 min, followed by another 5 minwith a fresh change of xylene.

2. Rehydrate for 15 s in each step of the following series: 100% ethanol, 95%ethanol, 75% ethanol, and deionized water.

3. Stain with Mayer’s Hematoxylin for 30 s.4. Rinse off excess stain with deionized water for 15 s; repeat rinse a second time.5. Dehydrate for 15 s in 70% ethanol.6. Stain with Eosin Y for 5 s.7. Dehydrate the sections for 15 s (twice) in 95% ethanol, 15 s (twice) in 100%

ethanol, and 60 s in xylene.8. Air-dry for approximately 2–5 min to allow xylene to evaporate completely (see

Note 5).9. The tissue is now ready for LCM (see Note 6).

3.3. Laser Capture Microdissection and Protein Sample Preparation

The PixCell II LCM system (Arcturus Engineering, Mountain View, CA,USA) is used for specific microdissection of tumor cells in our laboratory.Tissue sections are usually mounted on uncoated glass slides to provide supportfor the CapSure cap during microdissection. LCM utilizes an infrared laserintegrated into a standard microscope, and when the desired cells move intothe path of the light source, the investigator activates the laser, which inturn activates the membrane (a short laser pulse emitted heats the transparentmembrane to ∼90°C for 5 ms). This melts the membrane, with subsequentbinding and encapsulation of the cells of interest, segregating them from thesurrounding cells and connective tissues. Images of the tissues before and aftermicrodissection and of the captured cells on the cap can be visualized, thusmaintaining an accurate record of each dissection. The laser beam diametermay be adjusted from 7.5 to 30 μm to procure either single cells or groups ofcells, respectively.

1. Place the slide containing the prepared tissue on the microscope stage. Set thelaser parameters as follows: spot diameter at 15 μm, pulse duration at 5 ms, andpower at 50 mW.

2. Scan the tissue section to locate the desired cells. Dissect out the target cells ofinterest and capture all encapsulated cells from each section in quick successioninto one cap. Cells dissected from ∼2500 shots can be captured into one cap (seeNote 7). Figure 1 shows an example of tumor cells before and after microdis-section.

Combining LCM with 2-D Gel Electrophoresis 83

A B C

Fig. 1. Laser capture microdissection (LCM) of breast tumor cells. The tissue sectionon the uncharged glass slide was stained with hematoxylin and eosin and microdissectedwith the PixCell II LCM system (Arcturus Engineering). (A) section before LCM; (B)section after LCM; (C) microdissected cell.

3. Place the LCM cap on an Eppendorf tube containing 100 μl of lysis buffer withprotease inhibitor and invert the tube and vortex vigorously for 1 min.

4. Place the tube on ice for approximately 20 min and sonicate the microdissectedsample in a bath sonicator with 5 s pulses, in between 5-s intervals, for a durationof 1 min.

5. Replace the sample on ice immediately after 1-min sonication.6. Centrifuge the sample at 16,000 g for 20 min at 4°C and transfer the supernatant

to a new Eppendorf tube.7. Determine the protein concentration using the PlusOne 2D Quantitation kit (GE

Healthcare) and clean up the sample using the PlusOne 2-D cleanup kit (GEHealthcare), following the manufacturer’s instructions closely.

8. Dissolve the protein pellet in the appropriate volume of sample rehydration bufferand aliquot according to experimental plans for immediate and later usage. Storethe aliquotted samples at –80°C until analyzed (see Note 8).

3.4. First-dimension Gel Electrophoresis (Isoelectric Focusing)

1. Prepare the strip holder for the 18-cm IPG strip (see Note 9).2. Squeeze a few drops of Ettan™ IPGphor™ Strip Holder Cleaning Solution (GE

Healthcare) into the slot and clean thoroughly. Rinse with Milli-Q water and drycompletely.

3. Mix approximately 50 μl of the reconstituted protein samples (∼100–150 μg)with the appropriate volume of rehydration buffer. The total volume should be340 μl for one 18-cm IPG strip.

4. Transfer the entire volume of the diluted protein sample into the groove of theIPG strip holder.

5. Remove the cover from the IPG strip (18 cm, pH 3–10) and place the IPG stripin the holder such that the gel of the strip is in contact with the sample (i.e., gel

84 Zhang and Koay

side down). Try to remove any trapped air bubbles by lifting the strip up anddown from one side.

6. Overlay the IPG strip with 2–3 ml of DryStrip Cover Fluid to prevent ureacrystallization and evaporation, and replace the cover on the strip holder.

7. Rehydrate the IPG strip at 20 V for 12 h at 20°C.8. Perform IEF under the following conditions: 500 V for 1 h, 2000 V for 1 h,

4000 V for 1 h, and 8000 V for 6 h.9. Once focusing is complete, pour off the oil. The strips can be stored at –20°C for

several weeks, or immediately treated as described below (see Subheading 3.5).

3.5. IPG Strip Equilibration

1. Place the focused IPG strips in a container with 10 ml of equilibration buffer Aand shake for 15 min at RT (see Note 10).

2. Transfer the IPG strip to a container with 10 ml of equilibration buffer B andshake for 15 min at RT (see Note 10).

3. The equilibrated strips can then be processed for second-dimension gelelectrophoresis.

3.6. Second-dimensional SDS-PAGE

Prepare the SDS-polyacrylamide gels in advance, and make sure that thegels are well polymerized before performing the equilibration of IPG strips.The proteins have to be charged by equilibration with SDS, and be reducedand alkylated to avoid the formation of oligomers. In our laboratory, we usethe PROTEAN II xi Cell system (Bio-Rad) for SDS-PAGE.

1. Assemble the gel casting cassette as per the manufacturer’s instructions.2. Prepare 10% SDS-PAGE (see Note 10) and pour the solution slowly into the

cassette (two 16 cm × 20 cm glass plates sandwiched by 1.5-mm thick spacers)until the gel height is approximately 1 cm from the top.

3. Overlay the gel solution with 2 ml of water-saturated isobutanol. It is best topour 1 ml of water-saturated isobutanol from one side of the gel and 1 ml onthe other side. Do not pour it all along the gel meniscus.

4. Allow the gel to polymerize for at least 2 h.5. When polymerization is completed, remove the water-saturated isobutanol and

rinse with water again.6. With a pair of forceps, carefully place the equilibrated strip on top of the PAGE

gel, with the acidic side of the strip at left. Cover the strip with melted agarosesealing solution (see Note 11).

7. Assemble the electrophoresis unit (Bio-Rad) and perform electrophoresis at 15°Cas follows: 40 V for 15 min or until the blue dye enters the gel and then raisethe voltage to 125 V and run the gel overnight or until the blue dye migrates tothe bottom of the gel.

8. Switch off the main power and disassemble the gel cassette.

Combining LCM with 2-D Gel Electrophoresis 85

9. Place the gel in a glass container and wash the gel with Milli-Q water.10. Stain the gel using the mass spectrometry-compatible silver staining protocol

(see Subheading 3.7).

3.7. Silver Staining and Image Analysis

1. The silver staining protocol as described below is used in the authors’ laboratoryand is highly compatible with protein identification by MALDI-TOF MS andMALDI-TOF/TOF MS/MS. It should be noted that adequate washing with Milli-Q water is essential to reduce the risk of keratin contamination. All the solutionsmust be prepared with Milli-Q water, and all the chemical reagents should befiltered to remove any particles that may cause interference during MS analysis.All solutions prepared from solid chemicals should be freshly prepared beforeperforming silver staining. Fix the gel with fixing solution for at least 2 h,changing the solution afresh at hourly intervals.

2. Briefly wash with Milli-Q water, with constant shaking for about 15 min.3. Remove the wash and cover the gel with appropriate sensitivity-enhancing

solution and incubate for 1 h, with constant shaking.4. Wash the gel thoroughly with Milli-Q water for 6 × 15 min, with gentle shaking

and replacing with fresh Milli-Q water after each cycle (see Note 12).5. Stain the gel with silver staining solution for 30 min.6. Wash off excess stain from the gel with Milli-Q water (twice, for 2 × 1 min).7. Develop the gel for 5–30 min in a developing solution (see Note 13).8. Add Stop Solution and shake the gel for approximately 20 min to stop the

reaction.9. Wash the gel using Milli-Q water for 20 min; replace water and repeat the wash.

10. Scan the gel using Personal Densitometer SI, or store the gel in the gel soaksolution for analysis at a later time.

11. Capture the image using ImageMaster 2D Elite software (GE Healthcare). Theimage analysis includes spot detection, quantification and normalization of spotintensity to the background interferences, according to the instructions from thesoftware. An example of images showing the differences between the proteinprofiles of LCM-microdissected HER-2/neu positive and -negative tumor cellsis shown in Fig. 2.

12. Analyze the image using the software and identify spots that show signif-icant differences in spot intensities (see Note 14), reflecting differential proteinexpression in the two subtypes of breast cancer triggered by the presence orsuppression of HER-2/neu oncogene. Only those spots that show either morethan threefold or less than threefold change in signal intensity, consistentlyfrom three replicate sets of gels, are considered as demonstrating differentialprotein expression and selected for further analysis by MALDI-TOF MS/MS.The likelihood of any protein displaying less convincing evidence of differentialprotein expression being a potential biomarker for early detection of tumorgrowth or a therapeutic target for breast cancer treatment is low.

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NP000627

P06753-2

AAB49495

P07339

AAH025396 P04075

28

35

50

92

kDapI3 pI3

HER-2/neu-P HER-2/neu-N10 10

NP004095

NP001531

Fig. 2. Silver-stained protein profiles of LCM-dissected cells. Protein samples fromHER-2/neu positive and -negative cells are separated by using IPG®( strips (18 cm,pH 3–10 NL) and homogeneous SDS-PAGE (10%), and then stained with silvernitrate. Silver-stained gels were scanned using the Personal Densitometer SI (MolecularDynamics) and differentially expressed protein spots were analyzed by ImageMaster2-D Elite software (GE Healthcare). The Accession Numbers indicate the proteinID identified by MALDI-TOF/TOF tandem mass spectrometry and NCBInr databasesearch using Mascot software (Matrix Science, London, UK).

3.8. Trypsin Digestion and Preparation of Peptides for MassSpectrometric Analysis

1. Excise the silver-stained protein spots showing significant differential proteinexpression, as mentioned above, one at a time, taking care not to include adjacentproteins in vicinity, and transfer to individual tubes.

2. Wash with 100 μl of Milli-Q water for 5 min.3. Add 50 μl of the destaining solution into the tubes, and about 20 min on a

platform shaker at RT until the gels become clear in color.4. Remove the solution carefully and wash with 100 μl of Milli-Q water.5. Incubate the gel pieces with 25 mM sodium bicarbonate for 20 min, and then

cut them into smaller pieces with the tip of the transfer pipette. Avoid carryoverand contamination during repetitive work on consecutive samples.

6. Rinse the gel pieces with Milli-Q water, discard the wash after pulsing downthe gel pieces, and repeat the washing process three times.

7. Add 100 μl of dehydrating solution and incubate for 20 min at RT.8. Dry the gel pieces in a SpeedVac centrifuge.9. Re-swell the dried gel pieces with 10–20 μl of Digestion Solution and leave

overnight at 37°C to ensure complete digestion.10. Extract the resultant hydrophilic peptides first with 10 μl of Milli-Q water for 1 h.

Combining LCM with 2-D Gel Electrophoresis 87

11. Then extract the hydrophobic peptides with Extraction Solution for 2 h.12. Pool the extracted hydrophilic and hydrophobic peptides and dry the peptide

mixture using the SpeedVac centrifuge.13. Redissolve the dried peptides in 10 μl of 0.1% (v/v) TFA.14. Desalt the sample with ZipTip C18 columns (Millipore) and elute the treated

and purified peptides with 2.5 μl of Eluant.15. Mix 0.5 μl of the sample eluate with 0.5 μl of CHCA matrix (3 mg/ml) and spot

the mixture onto the stainless steel MALDI-TOF sample target plates.16. The pretreated peptide samples must be stored on ice during transfer to the

core facility for mass spectrometric analysis. In our laboratory, peptide massspectra are obtained by the Applied Biosystems 4700 Proteomics AnalyzerMALDI-TOF/TOF mass spectrometer, set in the positive ion reflector mode.The subsequent MS/MS analyses are performed in a data-dependent manner,and the 10 most abundant ions fulfilling certain preset criteria are subjected tohigh-energy CID analysis. The collision energy is set to 1 keV, and nitrogen isused as the collision gas.

3.9. Database Search to Match Protein Identities

Database searches were conducted using the MASCOT search engine(http://www.matrixscience.com). For database search, known contaminationpeaks, such as keratin and autoproteolysis peaks, were removed prior todatabase search. Protein identification was performed using the MASCOTsoftware (Matrix Science, London, UK), and all tandem mass spectra weresearched against the NCBInr database, with mass accuracy of within 200 ppmfor mass measurement, and within 0.5 Da for MS/MS tolerance window.Searches were performed without constraining the protein molecular weight(Mr) or isoelectric point (pI) and species, and allowing for carbamidomethy-lation of cysteine and partial oxidation of methionine residues. Up to one missedtryptic cleavage was considered for all tryptic-mass searches. Protein scoresgreater than 75 are considered to be significant (p < 0.05).

3.10. Experimental Example: Differential Protein Profilesbetween HER-2/neu Positive and -Negative Breast Tumors

We dissected the tumor cells from two different subtypes of breast tumorsand compared their protein profiles, based on the protocols described above.Figure 2 shows the LCM-dissected tumor cell protein patterns visualized bysilver staining. It should be noted that pooled protein samples from differentcases of the same tumor subtypes were used for 2-D GE. This gel-basedprotein visualization technique requires high amount of proteins, and thusmore sensitive detecting reagents and protein identification strategies had tobe developed to produce meaningful results (see Notes 15 and 16). Using

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the silver-staining protocol, we identified 500–600 protein spots in the proteinprofiles generated by coupling LCM and 2-D GE. Protein spots of interest wouldbe excavated and digested with trypsin (Promega), desalted with ZipTipc18

(Millipore), and analyzed using MALDI-TOF/TOF tandem mass spectrometry.Protein identities, as shown in Fig. 2, are obtained by searching the NCBInrdatabases using the MASCOT software (Matrix Science).

4. Notes1. All the chemical solutions should be filtered by passing them through filter paper

(Cat No. 1001 150, Whatman®, Whatman International Limited, SpringfieldMill, Maidstone, Kent, England) to minimize precipitates occurring onto thegels during silver staining.

2. Tissue processors in standard histopathology laboratories generally includeformalin fixation as the first step in the paraffin infiltration procedure. It isimportant to avoid these steps when processing tissues intended for moleculargene and proteome profiling.

3. Consistent LCM transfers have been demonstrated from 5–10 μm thick paraffin-embedded tissue sections. For a successful LCM transfer, the strength of the bondbetween polymer film and targeted tissue must be stronger than that between thetissue and the underlying glass slide. Therefore, for most tissue types, sectionsshould be collected with uncharged glass slides. To prevent cross-contaminationwhile sectioning, residual paraffin and tissue fragments should be wiped offfrom the area of the sectioning blade with xylenes between consecutive slides.If possible, a fresh microtome blade should be used to section a different block.

4. In our hands, hematoxylin and eosin are best reduced to 10% of their standardconcentrations used for routine histomorphological work, when applied to slidesprepared for LCM. Breast tumor cells can be clearly visualized and identifiedfrom other cell types, without influencing the procurement of tumor cells byLCM, with this modification. Minimum staining also improves macromolecularrecovery during cellular protein extraction.

5. Complete dehydration and air drying of sections are the main factors influencingthe efficiency of LCM. Prolonged air drying or presence of moisture in thesections appears to inhibit, at least partially, the transfer of cells to the plasticfirm.

6. If the investigators have less experience in checking cancer tissue sections,we strongly recommend that investigators consult with the pathologists in theirinstitutions to get assistance in identifying the target cell types that will bemicrodissected using LCM. It is essential to avoid contamination of other celltypes, or dissecting the wrong cells.

7. During microdissection, make sure that there are no irregularities on the tissuesurface in or near the area to be microdissected. It should also be noted thatwrinkles can elevate the LCM cap away from the tissue surface and decrease the

Combining LCM with 2-D Gel Electrophoresis 89

membrane contact during laser activation. Use an adhesive pad after microdis-section to remove cells that may have attached non-specifically to the LCMcap. A cap-alone control is recommended for each experiment to ensure thatnon-specific transfer is not occurring during microdissection. The cap should beprocessed together with other tissue-containing caps and serves as a negativecontrol. For protein separation by 2-D GE, 20 to 30 sections from each tissuesample are dissected, depending on the percentage of targets cells in the fullsections. Generally, 2300–2700 laser pulse shots are used for each cup. Cellsfrom at least 50,000 shots (spot diameter is 15 μm) are required for each18-cm gel.

8. Up to 15 mg of proteins can be solubilized with 500 μl of the sample rehydrationbuffer, but with our breast tumor tissue samples, we usually reconstitute 1–2 mgof extracted proteins in 500 μl, or 2–4 mg/ml. It is recommended that thereconstituted proteins be stored in appropriate aliquots, and that only the requirednumber of aliquots needed for the experiment at hand be removed at any time,to avoid repeated freezing and thawing the peptides, which will lead to sampledeterioration.

9. IEF is performed using Ettan™ IPGphor™ IEF electrophoresis unit. Rehydrationloading of protein samples is used in the authors’ laboratory. The IPG strips forfirst-dimensional separation are commercially available, and can be procuredfrom GE Healthcare and other suppliers. IPG strips with various pH gradients anddimensions are available. They are used for protein separation with appropriateresolution needed. The strips should be kept frozen at –20°C, and thawed justbefore use. The IEF conditions are dependent on the pH range. Reference to themanufacturer’s protocol is recommended. For alkali pH loading, cup loadingis a must, and DTT in the rehydration buffer should be replaced by otherreducing agents, such as hydroxyethyl-disulfide (HED) reagent (Destreak, GEHealthcare).

10. It is essential to equilibrate the strips before being applied for the second-dimension gel electrophoresis (2-D SDS-PAGE). DTT added to buffer A willreduce the disulfide bonds whereas IAA in buffer B will alkylate the formedsulfydryl groups of proteins. This is to prevent re-oxidation of sulfydryl groupsand streaking of spots during 2-D SDS-PAGE. Further, the presence of SDSmakes the proteins negatively charged and suitably primed for SDS-PAGE. Usethe best quality SDS available for sample and running buffers that include SDSin their formulation. We recommend C12 Grade SDS from Pierce (Rockford, IL,USA).

11. When placing the strips on top of the gel, ensure that the plastic backing of thestrips is in contact with the glass wall. If necessary, the strips can be trimmedproperly. When adding agarose sealing solution, make sure that there are no airbubbles trapped between the IEF strip and 2-D gel.

12. Wash the gels thoroughly and repeatedly, as recommended, prior to the devel-opment step and during the development step itself, to get clear stained gels.During the development of the gels, formaldehyde should be added prior to use,

90 Zhang and Koay

and the suggested concentration should be followed strictly to avoid interferenceduring MALDI-TOF analysis. During the developing stage, the gel should beconstantly shaken to reduce the background.

13. The developing time depends on the total amount of protein that is used for2-D separation. With a higher amount of protein, a shorter developing time canbe used, without compromising the aim of visualizing the maximum number ofprotein spots.

14. It is important to manually verify spot detection and matching, as the variationsin gel resolution, staining, gel background, and automatic image analysis maynot correctly define the spot contours in every case. This variability and thecomplexity of 2-D gel patterns hinder the accurate matching of analogous spotsin different gels.

15. In our experience, approximately 500 to 600 distinct proteins from the dissectedbreast tumor cells can be visualized on 2D-PAGE stained with silver. On average,we can extract approximately 4–6 μg of total cellular proteins from 2500 laserpulses. Our experience is that silver staining of LCM-dissected cell proteins is asufficiently sensitive tool for isolating and identifying the dysregulated cellularproteins of high or moderate abundance. However, for the dysregulated proteinsof low abundance, the lower detection limit of this technology would have tobe enhanced by other techniques such as 125-iodine labeling or biotinylationand fluorescent dye labeling. In addition, the use of scanning immunoblottingwith class-specific antibodies, for example, would allow sensitive detection ofspecific subsets of proteins, e.g., all known proteins involved with cell-cycleregulation.

16. Protein identification by MALDI-TOF, LC-MS/MS, or other techniques is alsolimited by the requirement of a minimal protein input amount, which is often notattainable from certain types of biopsy samples. A useful strategy to improveprotein identification is to produce parallel “diagnostic” fingerprints derivedfrom microdissected cells and “sequencing” the fingerprints generated from thewhole tissue section from each case. Alignment of the diagnostic and sequencing2D gels permits determination of the proteins of interest for subsequent massspectrometry or N-terminal sequence analysis.

AcknowledgmentsThe Tumor Repository of the National University Hospital, Singapore,

provided the clinical breast cancer frozen tissues for LCM. The use of thePixCell II LCM system was courtesy of the Department of Pathology, YongLoo Lin School of Medicine, National University of Singapore (NUS). Thiswork was supported by an Academic Research Fund from the NUS (Grant No.R-179-000-032) to the authors.

Combining LCM with 2-D Gel Electrophoresis 91

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