itraq-facilitated proteomic analysis of human prostate cancer cells identifies proteins associated...

iTRAQ-Facilitated Proteomic Analysis of Human Prostate Cancer

Cells Identifies Proteins Associated with Progression

Adam Glen,† Chee S. Gan,† Freddie C. Hamdy,† Colby L. Eaton,† Simon S. Cross,‡

James W. F. Catto,† Phillip C. Wright,§ and Ishtiaq Rehman*,†

Academic Urology Unit, Section of Oncology, University of Sheffield, Floor K, Royal Hallamshire Hospital,Glossop Road, Sheffield, S10 2JF, U.K., Academic Unit of Pathology, Floor E, Medical School, Beech Hill Road,

Sheffield S10 2RX, U.K., and Biological and Environmental Systems Group, Department of Chemical andProcess Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD, U.K.

Received June 15, 2007

The unpredictable behavior of prostate cancer presents a major clinical challenge during patientmanagement. In order to gain an insight into the molecular mechanisms associated with prostate cancerprogression, we employed the shot-gun proteomic approach of isobaric tags for relative and absolutequantitation (iTRAQ), followed by 2D-LC-MS/MS, using the poorly metastatic LNCaP cell line and itshighly metastatic variant LNCaP-LN3 cell line as a model. A total number of 280 unique proteins wereidentified (g95% confidence), and relative expression data was obtained for 176 of these. Ten proteinswere found to be significantly up-regulated (g1.50 fold), while 4 proteins were significantly down-regulated (g-1.50 fold), in LNCaP-LN3 cells. Differential expression of brain creatine kinase (CKBB),soluble catechol-O-methyltransferase (S-COMT), tumor rejection antigen (gp96), and glucose regulatedprotein, 78 kDa (grp78), was confirmed by Western blotting or independent 2D-PAGE analysis.Additionally, iTRAQ analysis identified absence of the lactate dehydrogenase-B (LDH-B) subunit inLNCaP-LN3 cells, confirming our published data. The clinical relevance of gp96 was assessed byimmunohistochemistry using prostate tissues from benign (n ) 95), malignant (n ) 66), and metastaticcases (n ) 3). Benign epithelium showed absent/weak gp96 expression in the basal cells, in contrast tothe moderate/strong expression seen in malignant epithelium. Furthermore, there was a statisticallysignificant difference in the intensity of gp96 expression between benign and malignant cases (p <0.0005, Mann–Whitney U). Our study is the first to report the application of iTRAQ technology and itspotential for the global proteomic profiling of prostate cancer cells, including the identification of absentprotein expression.

Keywords: benign hyperplasia • shot-gun proteomics • 2D-PAGE • immunochemistry • tumor rejectionantigen (gp96)

Introduction

Prostate cancer is the most common cancer diagnosis in menand the second most common cause of cancer-related deathsin the USA and Europe.1 It has been estimated that in the USAalone over 200 000 men will be diagnosed with the disease in2007, causing over 27 000 deaths.2 Although screening forprostate cancer, based on serum prostate specific antigen (PSA)measurements, has led to earlier disease diagnosis, the chal-lenges facing clinicians now are to determine which patientswill progress to develop an aggressive disease, thus requiringearly intervention, from those patients with a clinically localizedand indolent disease, requiring little or no treatment.3,4 Thus,our ability to effectively manage patients with prostate cancer

rests upon a better understanding of the molecular processesunderlying disease development and progression.

In an effort to understand the mechanisms of prostate cancerprogression, Pettaway et al. (1996) developed a number of invivo models for prostate cancer progression, using previouslyestablished human prostate cancer cell lines, such as theLNCaP cell line which was originally derived from a patientwith metastatic prostate cancer in the lymph-node.5,6 Bysuccessive orthotopic implantation of LNCaP cells into nudemice, several variant cell lines were generated, each withdifferent growth and metastatic properties. For instance, theLNCaP-LN3 cells gave a significantly higher incidence ofregional lymph-node metastases and produced larger tumorscompared with the parental LNCaP cells.5 These isogeneticmetastatic variant cell lines serve as useful tools for studies intothe biology of prostate cancer progression.

The molecular events associated with prostate cancer pro-gression are poorly understood.7 Previous studies aimed atidentifying candidates associated with prostate cancer progres-

* Corresponding author. Tel: + 44 (0) 114 271 1798. Fax: + 44 (0) 114 2712268. E-mail: [email protected].

† Section of Oncology, University of Sheffield.‡ Academic Unit of Pathology, Floor E, Medical School.§ Department of Chemical and Process Engineering, University of Sheffield.

10.1021/pr070378x CCC: $40.75 2008 American Chemical Society Journal of Proteome Research 2008, 7, 897–907 897Published on Web 01/31/2008

sion have employed a number of genomic and proteomic basedapproaches, including 2D-PAGE, SILAC, and SELDI-TOF.8 Forinstance, Liu et al. compared the protein profiles of a highlymetastatic human prostate cancer cell line M12 with its poorlytumorigenic variant M12(F6), using 2D-PAGE, and identifiedreduced levels of vimentin.9 SELDI-TOF has been used to studyproteomic pattern changes in Dunning rat prostate cancer cellsof varying metastatic potentials and was able to correlate theprotein profiles with differences in metastatic potential.10 Astudy by Everley et al. employed SILAC to characterize dif-ferential protein expression levels between PC3 M and PC3M-LN4 prostate cancer cells with low and high metastaticpotentials, respectively, and identified a total of 82 differentiallyexpressed proteins thought to be involved in prostate cancerprogression.11 We have previously employed 2D-PAGE followedby tandem mass spectrometry to identify loss of lactatedehydrogenase-B (LDH-B), in the highly metastatic LNCaP-LN3cells in addition to human prostate cancer tissues.12 Morerecently, a high-throughput immunoblotting approach wasused to identify candidates associated with prostate cancerprogression.13

Among the emerging proteomic technologies, iTRAQ (iso-baric tags for relative and absolute quantitation), is a shot-gunbased technique which allows the concurrent identification andrelative quantification of hundreds of proteins in up to (andsoon 8) different biological samples in a single experiment.14,15

The iTRAQ technology has many advantages over other pro-teomic techniques, such as being relatively high throughputdue to sample multiplexing. In addition, during the tandemmass spectrometric analysis, more than one peptide represent-ing the same protein may be identified, which providesincreased confidence in both the identification and quantifica-tion of the protein. To date, the iTRAQ technique has beenapplied to the study of a range of biological samples includingbacteria, yeast, human tissues, cells, and fluids in an effort toidentify and quantify the proteins in these samples.16 Further-more, the technique has been shown to be suitable for theidentification of low abundance proteins such as transcriptionfactors.16

Here we have employed an iTRAQ workflow to identify andquantify proteins associated with prostate cancer progression,using the poorly metastatic LNCaP cells and its highly meta-static variant cells LNCaP-LN3 as a model. Furthermore, someof the differentially expressed candidates were verified byWestern blotting or independent 2D-PAGE followed by LC-ESI-MS/MS analyses.

Materials and Methods

Cell Lines and Culture. The human prostate cancer cell lineLNCaP was obtained from the American type Culture Collection(Manassas, VA). LNCaP-LN3 cells were kindly provided by Dr.Curtis Pettaway (University of Texas, M.D. Anderson CancerCentre), following completion of the relevant Material TransferAgreements.5 Cells were maintained in RPMI-1640 mediasupplemented with 10% fetal calf serum, glutamine, vitamins,nonessential amino acids, and antibiotics (Penicillin/ Strepto-mycin) (Gibco-BRL, Paisley, United Kingdom). Cells wereconfirmed to be Mycoplasma free prior to analysis, using theEZ-PCR test kit (Geneflow, Staffordshire, UK).

Patient Material. Paraffin-embedded tissues obtained byradical prostatectomy, cystoprostatectomy, or prostatic resec-tion from patients attending the Urology Clinic of the RoyalHallamshire Hospital, Sheffield (UK), were arranged into tissue

microarrays (TMAs). Three cases of metastatic prostate cancerin bone were collected by 8 mm trephine under generalanesthesia.

TMAs were comprised of 66 cases of prostatic adenocarci-noma of various Gleason grades (1–5): 60 cases of nonmalignanttissue (mostly matched), 35 cases of benign prostatic hyper-plasia (BPH), 6 cases of high-grade prostatic intraepithelialneoplasia (PIN) lesions. Prior local ethics committee approvaland informed patient consent were obtained in all cases.

Two-Dimensional Polyacrylamide Gel Electrophoresis. Pro-tein fractions were prepared using the native membraneprotein extraction kit (Calbiochem, Nottingham, UK), accordingto the manufacturer’s instructions with the addition of COM-PLETE EDTA-free protease inhibitor mixture (Roche Diagnos-tics, Sussex, UK). Protein concentration was determined usingthe BCA protein assay kit (Sigma, Dorset, UK), according tothe supplied instructions.

Two-dimensional PAGE analyses of the extracted proteinswere performed as previously described.17,18 For analytical 2D-PAGE gels, 100 µg of extracted protein was made up to 300 µLwith reswell buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mMDTT, and 0.2% v/v ampholyte, pH 4–7) (Bio-Rad, Hertfordshire,UK). For preparative gels, 800 µg of protein was used. Each300 µL sample was applied to a 17 cm, pH 4–7, immobilizedpH gradient (IPG) gel strip (Bio-Rad). The IPG strips were thenactively rehydrated (50 V for 16 h), using the Protean isoelectricfocusing (IEF) cell (Bio-Rad). IEF conditions were: 250 V for 15min, linear ramping from 250 to 10 000 V over 3 h, followedby 10 000 for 60 000 V/h. The strips were then equilibrated for10 min in equilibration buffer (0.375 M Tris, pH 8.8, containing6 M urea, 2% w/v SDS, 20% v/v glycerol), containing 2% w/vDTT followed by 10 min in equilibration buffer containing 2.5%w/v iodoacetamide. The strips were then loaded onto 20 × 18cm, 10–12% SDS-PAGE gels, and electrophoresis was performedusing a Protean II xi electrophoresis cell (Bio-Rad). Analyticaland preparative 2D-PAGE gels were stained with silver stain(Autogen Bioclear, Wiltshire, UK) or Coomassie G-250 stain(Bio-Rad, Hertfordshire, UK), respectively, according to themanufacturer’s instructions. Molecular weight and pI valuesof proteins were estimated with 2D-standards (Bio-Rad, Hert-fordshire, UK). Analytical gels were scanned using a Bio-RadGS-700 scanner and analyzed by the manual zoom method,consisting of digital enlargement of each sub-region of the gelfollowed by examination of the gels by two independentreviewers, using a range of brightness/contrast settings.18,19 Toincrease the confidence of differential protein expression, 2D-PAGE gels were performed at least 4 times for each cell lineand with varying protein loads. For the purpose of this study,only those protein expression differences seen as reproducible>2 fold or >-2 fold regulation were considered as candidates.Any differentially expressed proteins were manually excisedfrom the gel and identified by in-gel trypsin digestion followedby LC-ESI-MS/MS analyses, carried out at the Centre for StemCell Biology, University of Sheffield, Sheffield (UK), as describedpreviously.20

iTRAQ Protein Sample Preparation. Protein extraction wasperformed according to previously published methods.21 Briefly,adherent cells were detached using trypsin then washed twicein PBS. Pelleted cells (8 × 106) were homogenized in 0.5 mL ofPBS containing a cocktail of protease inhibitors (Roche Diag-nostics, Sussex, UK). The cells were mechanically homogenizedusing a hand-held homogenizer in a 1.5 mL microcentrifugetube kept at 4 °C. The disrupted cells were centrifuged at

research articles Glen et al.

898 Journal of Proteome Research • Vol. 7, No. 3, 2008

10 000g for 30 min at 4 °C to pellet debris. The proteinconcentration of the supernatants was determined by the BCAprotein assay kit (Sigma, Dorset, UK) and stored in aliquots at-80 °C until use.

Isobaric Labeling. Two-hundred micrograms of protein fromeach (LNCaP and LNCaP-LN3) phenotype was subjected toacetone precipitation overnight before re-suspending into 20µL of 500 mM triethylammonium bicarbonate (TEAB). Subse-quently, the re-suspended proteins were reduced, alkylated,and digested with trypsin according to the manufacturer’sprotocol (Applied Biosystems, Framingham, MA, USA). Sampleswere iTRAQ labeled in duplicate as follows (100 µg each perlabel): LNCaP, 114; LNCaP, 115; LNCaP-LN3, 116; LNCaP-LN3,117. The labeled peptide samples were then pooled and driedin a vacuum concentrator prior to SCX fractionation.

SCX Fractionation. Dried labeled peptides were re-sus-pended in 200 µL of buffer A and fractioned using a PolySUL-FOETHYL A column (PolyLC, Columbia, MD, USA) of 5 µmparticle size of 200 mm length × 2.1 mm id, 200 Å pore size,on a BioLC HPLC unit (Dionex, Surrey, UK) with a constantflow rate of 0.2 mL/min and an injection volume of 200 µL.Buffer A consisted of 10 mM KH2PO4 and 25% acetonitrile, pH3.0, and buffer B consisted of 10 mM KH2PO4, 25% acetonitrile,and 500 mM KCl, pH 3.0. The 60-min gradient consisted of100% A for 5 min, 5% to 30% B for 40 min, 30% to 100% B for5 min, 100% B for 5 min, and finally 100% A for 5 min. Thechromatogram was monitored through a UV Detector UVD170Uand Chromeleon Software, version 6.50 (Dionex/LC Packings,The Netherlands). The UV wavelengths were set at 280, 254,and 214 nm. Fractions were collected every minute using a FoxyJr. Fraction Collector (Dionex) and later were pooled togetheraccording to variations in peak intensity. A total of 29 SCXfractions were pooled for subsequent nano-LC-MS/MS analysis.Pooled fractions were dried in a vacuum concentrator andstored at -20 °C prior to mass spectrometric analysis.

Mass Spectrometric Analysis. Each dried SCX iTRAQ-labeledpeptide fraction was re-dissolved in 100 µL of 0.1% formic acidand 3% acetonitrile, and then 20 µL of sample was injectedinto the nano-LC-ESI-MS/MS system for analysis. Mass spec-trometry was performed using a Q-Star XL Hybrid ESI Quad-rupole time-of-flight tandem mass spectrometer, ESI-qQ-TOF-MS/MS (Applied Biosystems; MDS-Sciex), coupled to an onlinecapillary liquid chromatography system (Famos, Switchos, andUltimate from Dionex/LC Packings, Amsterdam, The Nether-lands) as described elsewhere.22 The peptide mixture wasseparated on a PepMap C-18 RP capillary column (LC Pack-ings), with a constant flow rate of 0.3 µL/min. The LC gradientstarted with 3% buffer B (0.1% formic acid in 97% acetonitrile)and 97% buffer A (0.1% formic acid in 3% acetonitrile) for 3min, followed by 3% to 30% buffer B for 90 min, then 90%buffer B for 7 min, and finally 3% buffer B for 8 min. The massspectrometer was set to perform data acquisition in the positiveion mode, with a selected mass range of 300–2000 m/z. Peptideswith +2 to +4 charge states were selected for tandem massspectrometry, and the time of summation of MS/MS events wasset to 3 s. The two most abundantly charged peptides above a 5count threshold were selected for MS/MS and dynamicallyexcluded for 60 s with (50 mmu mass tolerance.

For mass spectrometric analysis of the proteins excised from2D-PAGE gels, the gel pieces were rinsed with water thendestained using Coomassie destaining solution containing 40%(v/v) acetonitrile (VWR International Ltd., Leicester, UK), in 200mM ammonium bicarbonate.20 The gel pieces were next

incubated with acetonitrile for 15 min and dried in a vacuumcentrifuge. The dried proteins were digested with 20 ng/µL ofsequencing grade Trypsin (Promega, Southampton, UK), in 50mM ammonium bicarbonate for 12 h at 37 °C. Followingdigestion, the peptides were extracted sequentially three timesby incubation with peptide extraction solution consisting of25 mM ammonium bicarbonate (10 min, room temperature),5% formic acid (15 min, 37 °C), and acetonitrile (15 min, 37°C), followed by centrifugation and removal of the supernatantat each step. The original supernatant and the supernatantsfrom the sequential extractions were combined and dried in avacuum centrifuge. The dried peptides were dissolved in 7 µLof 0.1% (v/v) formic acid in 3% (v/v) acetonitrile in water.Samples were centrifuged for 5 min at 12 000g, and thesupernatants were subject to LC-ESI-MS/MS. Liquid chromato-graphic separations of the tryptic digests were performed usinga reverse phase CapLC system (Waters, Manchester, UK).Peptides were delivered to a PepMap C18 microguard column(300 µm internal diameter × 1 mm) (LC-Packings Dionex,Leeds, UK) at 5 µL/min. A splitter then reduced the flow rateto approximately 200 nL/min, and peptides were transferredto the analytical column (PepMap C18; 75 µm internal diameter× 15 cm column) (LC-Packings, Dionex). The peptides wereeluted with a linear gradient from 0 to 80% buffer B (0.1%formic acid in 95% (v/v) acetonitrile in water) over 60 min. Thecolumn eluent was sprayed directly into the nano-ESI sourceof a Q-TOF micro (Waters). Spray voltage was set to 3000 V,and source temperature was 80 °C. An initial MS scan wasperformed, and selection of ions for collision induced dissocia-tion (CID) was automated by Masslynx sofware, version 4.0 sp1(Waters). CID selection criteria were set for 2+ and 3+ ionswithin the range of 100–1500 m/z above 10 ion counts.

Protein Identification and Database Searches. Proteinidentification and quantification for iTRAQ experiments wascarried out using the ProteinPilot software v2.0 (AppliedBiosystems; MDS-Sciex).23,24 The search was performed againstan IPI human database (version 3.28) downloaded from theEBI Web site (http://www.ebi.ac.uk/IPI/IPIhelp.html).25 A con-catenated target-decoy database search strategy was alsoemployed to estimate the rate of false positives.26 The Paragonalgorithm in ProteinPilot software was used as the defaultsearch program with trypsin as the digestion agent and cysteinemodification of methyl methanethiosulfonate.23 The search alsoincluded the possibility of more than 80 biological modifica-tions and amino acid substitutions of up to two substitutionsper peptide using the BLOSUM 62 matrix. Only proteinsidentified with at least 95% confidence, or a ProtScore of 1.3,were reported. The results obtained from ProteinPilot v2.0software were exported to Microsoft Excel and Microsoft Accessfor further analysis.

Peptide mass spectra obtained following 2D-PAGE-LC-ESI-MS/MS were searched against the Mass Spectrometry Database(MSDB) in a sequence query search using the MASCOT 2.0software (www.matrixscience.com). Peptide tolerance was setto 0.5 Da, and the MS/MS tolerance was 0.3 Da. The searchparameters were as follows: Tryptic digest was assumed to havea maximum of one missed cleavage; cysteine residues modifiedby carbamidomethylation and methionine residues modifiedby oxidization were considered; the taxonomy group searchedwas Homo sapiens. Proteins were considered a significantmatch (P < 0.05) when probability-based MOWSE scores were>63.

iTRAQ Analysis of Human Prostate Cancer research articles

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Classification of iTRAQ Identified Proteins. Proteins identi-fied by iTRAQ analyses were classed into their biologicalprocess and molecular functions, by inputting their proteinidentification numbers into the PANTHER classification sys-tem.27

Western Blotting. For Western blot analysis, total proteinwas extracted from prostate cancer cells using the mammaliancell lysis kit (Sigma, Dorset, UK), according to the manufac-turer’s instructions. Protein extracts (12–35 µg/well) were runon 10–16% SDS-Tris-glycine gels and blotted onto immobilonPVDF transfer membranes for 1 h at 65 V. Blots were blockedusing 5% nonfat milk for 1 h, then incubated overnight at 4 °Cwith the appropriate dilution of antibody (as below). Afterincubation with the appropriate Horseradish peroxidise (HRP)-conjugated secondary antibody, the membranes were devel-oped using the ECL Advance Western blotting detection kit (GEHealthcare, Buckinghamshire, UK), following X-ray film expo-sure. Primary antibodies, dilutions used, and catalogue num-bers were: rabbit polyclonal anti-CKBB (1/500) (Abcam, ab38212,Cambridge, UK); goat polyclonal anti-COMT (1/500) (Abcam,ab36144, Cambridge, UK); rat monoclonal anti-gp96 (1/1000)(Stressgen, spa-850, Victoria, Canada); goat polyclonal anti-grp78 (1/300) (Santa Cruz, sc-1050 (NC-20), Heidelberg, Ger-many); mouse monoclonal anti-GAPDH (1/5000) (Abcam,ab9482, Cambridge, UK); rabbit polyclonal anti-actin (1/2000)(Sigma-Aldrich, A5060, Dorset, UK). Secondary antibodies usedwere all HRP-conjugated: rabbit anti-goat (Dako, P0449, Den-mark), donkey anti-rabbit (Amersham, NA931V, Bucks, UK),rabbit anti-rat (Dako, P0450, Denmark), and Donkey anti-mouse (Amersham, NA934V, Bucks, UK). Molecular weightmarkers used were the Dual color precision plus markers (Bio-Rad, Hertfordshire, UK).

Immunohistochemistry. Immunohistochemistry was per-formed essentially as previously described.28 For gp96 immu-nostaining, sections were blocked for 30 min using 1 × Casein(Vector laboratories, Peterborough, UK) in 0.5 M TBS, 2 mMCaCl2, 0.05% Tween 20, pH 7.5, then incubated for 1 h with ratmonoclonal anti-human gp96 specific antibody (Stressgen, spa-850, Victoria, Canada) at a 1/100 dilution in ¼ × Casein in 0.5M TBS, 2 mM CaCl2, 0.012% Tween 20, pH 7.5. The secondaryantibody was biotinylated rabbit anti-rat used at 1/300 dilution(Vector Laboratories, Peterborough, UK). Following the PBSwash steps, preformed Avidin:Biotin enzyme Complex (ABC)from the Vectastain ABC staining kit (Vector Laboratories,Peterborough, UK) was added, and the brown color wasdeveloped using 3,3′-diaminobenzidine (DAB) as the chro-mogen. Negative controls included omission of the primaryantibody. Immunostaining intensity was assessed by a boardcertified Histopathologist (S.S.C), where 0 ) absent staining, 1) weak, 2 ) moderate, and 3 ) strong.

Cellulose Acetate Electrophoresis. Zymography for LDHisoenzymes was performed on cellulose acetate membranes(Helena Biosciences, Titan III (94 × 76 mm)), using Tris-Glycinebuffer according to the supplied instructions. Cells (6 × 106)were mechanically homogenized using a hand-held homo-genizer in a 1.5 mL microcentrifuge tube kept at 4 °C andcentrifuged at 10 000g for 30 min at 4 °C. The resultingsupernatants were applied to the Cathode end of the mem-brane, using the Super Z-12 applicator (Helena, Biosciences),and electrophoresis was performed for 12 min at 300 V. TheLDH bands were visualized using the LD Vis isoenzyme reagentaccording to supplied instructions (Helena Biosciences).

Results

Peptide Selection Criteria for Relative Quantitation. Toallow analytical replicate measurements, the experiment wasdone in a double duplex fashion (LNCaP cells labeled with 114and 115, whereas LNCaP-LN3 cells were labeled with 116 and117). A total of 280 proteins were identified with g95%confidence from the 29 773 possible identified peptides. Morethan 90% of the identified proteins were identified with at leasttwo MS/MS spectra. Using the concatenated target-decoydatabase search strategy as detailed by Elias and Gygi (2007),26

a 0% rate of false positives was estimated, which furtherstrengthened the reliability of our data.

Based on the peptide selection criteria pre-set in ProteinPilotv2.0 software, all peptides were used for quantitation, exceptpeptides without an iTRAQ modification or reporter ions (i.e.,no quantitation): peptides with a low intensity ratio, i.e., a totalarea count less than 40; shared peptides between similarprotein isoforms; peptides derived from the same MS/MSspectrum window; and peptides with a confidence below 0.5%.Based on all these criteria, 202 out of 280 total proteins (72%)gave at least one relative quantitative ratio in either 115:114,116:114, or 117:114 from approximately 2995 peptides. How-ever, it was unconvincing if the relative quantitative ratio wasderived from a peptide with 1% or even 50% confidence.Furthermore, peptides with a quantitation ratio of 9999, 0, orblank can sometimes be a true observation as a result ofintrinsic biological effects.

The estimation (and calculation) of the relative quantitativeratio from an iTRAQ experiment was reported earlier.29 Onlypeptides above or equal to a 70% confidence were taken intoconsideration in addition to the default peptide selectioncriteria. From manual inspection, none of the peptides showeda quantitative value of 0 and 9999, and all the blank valueswere either unsustained or statistically insignificant as a resultof intrinsic biological effects. After a re-evaluation, only 176out of 280 total proteins (65%) gave at least one relativequantitative ratio from approximately 1747 peptides (Table S1in the Supporting Information). Further analysis also revealedthat among these 176 quantified proteins 112 proteins derivedtheir quantitative ratio from at least two or more MS/MSspectra, whereas 64 proteins were quantified based on singleMS/MS spectra (Table S2 in the Supporting Information).

Replicate Analysis and Cutoff Point Estimation. Since theiTRAQ experiment was done in a double duplex manner, it waspossible to estimate the cut-off point to define significantdifferential protein regulation. The theoretical relative quantita-tive ratio between the 115:114 (LNCaP replicate) duplex andthe 116:117 (LNCaP-LN3 replicate) duplex should be unity, asdescribed elsewhere.29 However, due to nuisances, not allrelative protein ratios are likely to agree with the theoreticalvalue. Therefore, any deviation from the theoretical value canbe accurately estimated to define a cut-off point. Based on 161and 169 relative protein abundance ratios from the LNCaP andLNCaP-LN3 replicates, respectively, an average variation of 18%((0.18) was measured.

However, if the cut-off point was set at an 18% averagevariation, only 67% of proteins would fall within this variationrange; i.e., a 67% accuracy was measured. As the cut-off pointwas increased from 18% to 50%, the percentage of accuracyincreased from 67% to 92% (Figure 1). Therefore, the cut-offpoint used in this context was defined at 50% ((0.50). Anyrelative protein quantitation change above or below (1.50-fold



would thus be considered as significantly up- or down-regulated. We have demonstrated and validated the utility ofthis approach across the three domains of life previously.29

Significant Differentially Regulated Proteins. Of the 280identified proteins, 10 were significantly differentially up-regulated and 4 were significantly down-regulated by more thanor equal to 50% (relative linear protein ratio g (1.50 fold)(Table 1). These protein candidates were filtered and inspectedmanually to ensure their credibility. First of all, their averagerelative ratio (between 115:114 and 116:114) must be more than(1.50-fold. However if the log mean standard deviation (SD)between these values was greater than 0.05, they were removed.At least two or more unique distinct peptides were required,and they must be quantified by at least g2 MS/MS spectra.Lastly, the relative ratio of 115:114 (LNCaP replicate) must nothave a variation greater than the recommended cutoff of (50%.

A large number of proteins (32%) were confidently identifiedwith more than two MS/MS spectra yet not quantified due to

statistically insignificant quantitation derived from the reporterions. Comparatively, some of the proteins (16%) were quanti-fied based on a single peptide but identified with multiple MS/MS spectra (refer to Table S1 in Supporting Information).However, the relative quantitation of these (single peptidequantitation) proteins requires further validation before draw-ing any firm conclusions.

Protein Classifications. Data analysis on the 280 uniqueproteins identified by iTRAQ was performed using the PAN-THER classification system to class each protein into itsrespective biological process.27 The total number of proteinsidentified by iTRAQ was found to represent a total of 23biological processes (Figure 2). The top five biological processcategories were: protein metabolism (28.0%), nucleic acidmetabolism (14.8%), unclassified biological processes (8.8%),immunity and defense (7.0%), and carbohydrate metabolism(6.0%). Of the 14 significantly differentially expressed proteins,some of these were classed into biological processes previouslyassociated with cancer progression, such as carbohydratemetabolism, cell cycle, cell structure and motility, cell prolifera-tion, and differentiation.30

Differentially Expressed Candidates Identified by 2D-PAGE and LC-ESI-MS/MS. Following comparisons of the 2D-PAGE silver stained gels of LNCaP and LNCaP-LN3 cells, fiveproteins were found to be reproducibly down-regulated (g-2fold), in LNCaP-LN3 cells (Figure 3). Subsequent LC-ESI-MS/MS analyses significantly identified these (p < 0.05) as beingperoxiredoxin-2 (PRDX2) (P32119); adenine phosphoribosyl-transferase (APRT) (P07741); glyoxalase 1 (GLO1) (Q04760);catechol-O-methyltransferase (COMT) (CAG30308); and Rho-GDP dissociation inhibitor 1 (Rho-GDI 1) (P52565), (Table 2).

Validation of iTRAQ Identified Candidates. Western blotanalysis for brain creatine kinase (CKBB), tumor rejection

Figure 1. Frequency distribution (bars) from both LNCaP andLNCaP-LN3 duplex replicates across different ranges of varia-tions. The cumulative percentage of proteins (lines) is definedas the cumulative number of proteins falling within the definedvariation range against the total number of proteins.

Table 1. Significant Differentially Up-Regulated (g1.50-Fold) and Down-Regulated (g–1.50-Fold) Proteins Identified by iTRAQ inLNCaP and LNCaP-LN3 Cells

IPI accessions protein names % coverageano. of

peptidesbno. of

MS/MSb

average LNCaP-LN3:LNCaPlog meanc

standarddeviationc

average LNCaP-LN3:LNCaPlinear ratio

Significant Up-Regulated ProteinsIPI00644989.2 Protein disulfide-isomerase-associated

6 precursor/ Erp566.6 2 3 0.358 0.014 2.28

IPI00020599.1 Calreticulin precursor 42.4 3 13 0.281 0.008 1.91IPI00025252.1 Glucose regulated protein,

58 kDa (grp58)65.1 6 32 0.271 0.010 1.87

IPI00549248.4 Isoform 1 of Nucleophosmin 76.9 7 28 0.236 0.013 1.72IPI00003362.2 Glucose regulated protein,

78 kDa (grp78)84.9 8 32 0.233 0.028 1.71

IPI00744692.1 Transaldolase 73.0 4 9 0.217 0.018 1.65IPI00783736.1 Parathymosin (15 kDa) protein 58.6 4 13 0.215 0.032 1.64IPI00027230.3 Tumor rejection antigen

(gp96)/ grp9460.3 2 4 0.211 0.028 1.63

IPI00291006.1 Malate dehydrogenase,mitochondrial precursor

71.9 5 25 0.187 0.048 1.54

IPI00788802.1 Transketolase variant (Fragment) 75.3 7 35 0.185 0.037 1.53

Significant Down-Regulated ProteinsIPI00013452.8 glutamyl-prolyl tRNA synthetase 55.6 3 5 -0.193 0.035 0.64IPI00745729.1 53 kDa protein 69.2 3 7 -0.195 0.043 0.64IPI00419373.1 Isoform 1 of Heterogeneous nuclear

ribonucleoprotein A375.1 3 8 -0.225 0.010 0.60

IPI00022977.1 Brain Creatine kinase (CKBB) 82.4 12 65 -0.268 0.025 0.54

a The percentage protein coverage was extracted from ProteinPilot v2.0 software, where it was based on all the observable peptides, including lowconfidence peptides. b The numbers of MS/MS experiments and peptides observed were determined only for those peptides with g70% confidence. c Theestimation of the relative protein ratio and the standard deviation was calculated in log10 space.



antigen (gp96), and glucose regulated protein, 78 kDa (grp78),gave bands at the expected sizes and confirmed differentialexpression in all cases, in agreement with our iTRAQ data(Figure 4). Although the relative protein ratio for COMT (-1.48-fold), fell just below the cutoff at -1.50-fold (Table S1, Sup-porting Information), this was selected for further validationbased on its differential expression seen in our independent2D-PAGE analysis (Figure 3 and Table 2). Because of the high

sequence homology between the membrane-bound-COMT(MB-COMT) and soluble-COMT (S-COMT) isoforms,31 tandemmass spectrometry analyses performed during 2D-PAGE LC-ESI-MS/MS and iTRAQ were unable to reliably distinguishbetween these two isoforms. However, subsequent Western blotanalysis using antibodies reactive to both MB-COMT andS-COMT gave a single band at 25 KDa, confirming down-regulation of the S-COMT isoform in LNCaP-LN3 cells (Figure4). In all cases, although Western blot analysis confirmed thedirection of change observed in iTRAQ, the fold changes werenot consistent with the ratios seen in iTRAQ. This discrepancyhas been reported by other groups.32

Following 2D-PAGE LC-ESI-MS/MS analyses, we also identi-fied significant down-regulation (>-2.00-fold) of PRDX2, APRT,GLO1, and Rho-GDI 1 in LNCaP-LN3 cells (Table 2 and Figure3). Down-regulation of GLO1 and PRDX2 in LNCaP-LN3 cellswas also suggested by the iTRAQ analysis, although thedifference fell below significance (Table S1, Supporting Infor-mation). However, Rho-GDI 1 and APRT were not identifiedby iTRAQ.

In addition, iTRAQ analysis showed an absence of the lactatedehydrogenase-B (LDH-B) subunit in LNCaP-LN3 cells, whereasthe LDH-A subunit was seen to be expressed (Figure 5 andTable S1, Supporting Information). This observation sup-ports the data from our previous study,12 showing absence ofthe LDH-B subunit in LNCaP-LN3 cells, and was furtherconfirmed by LDH isoenzyme zymography using celluloseacetate electrophoresis (Figure 6).

Clinical Relevance of the gp96 in Tumor Progression. Theclinical relevance of gp96 was assessed by immunohistochem-istry using human prostate tissues. In BPH and nonmalignantepithelium (total n ) 95 cases), gp96 expression was seen tobe absent (19%) or weak (81%) in the basal cell layer, whileluminal cells showed absent expression (Figure 7). In contrast,54% (36/66) of adenocarcinomas showed moderate/ strongexpression of gp96. One of the three metastatic lesions showedstrong expression. The difference in staining intensity betweenBPH/nonmalignant epithelium and malignant epithelium wasstatistically significant (p < 0.0005, Mann–Whitney U). Over-expression of gp96 was seen in 4/6 PIN lesions. The stainingintensity was not associated with Gleason grade.

Figure 2. Pie chart showing the various biological processes as a percentage of the 280 proteins identified by iTRAQ according to thePANTHER classification system.

Figure 3. Representative whole 2D-PAGE images of LNCaP andLNCaP-LN3 cells stained using silver stain, showing proteinsexpressed over the pH range of 4–7 and molecular mass of∼10–100 KDa. The boxed region of the gels shows the locationsof the five differentially down-regulated proteins, which wereexcised from the gel and identified by LC-ESI-MS/MS analyses(Table 2). Abbreviations are: peroxiredoxin-2 (PRDX2), adeninephosphoribosyltransferase (APRT), glyoxalase 1 (GLO1), cat-echol-O-methyltransferase (COMT), Rho-GDP dissociation inhibi-tor 1 (Rho-GDI 1). Protein spots marked by arrowheads (black)were used as controls and showed no significant differentialexpression between the two cell lines.



Discussion

In an effort to identify proteins associated with prostatecancer progression, we employed the iTRAQ shot-gun pro-teomic approach, using the poorly metastatic LNCaP cell lineand its highly metastatic variant LNCaP-LN3 cell line as amodel. Overall 280 unique proteins were identified with g95%confidence. Using the PANTHER classification system, the topfive biological process categories were protein metabolism,nucleic acid metabolism, unclassified biological processes,immunity and defense, and carbohydrate metabolism. Fur-thermore, these classes of proteins have previously beenidentified using iTRAQ in a search for markers of endometrialcancer.21 Of the 14 significantly differentially expressed pro-teins, some of these classed to biological processes previouslyassociated with cancer progression, such as carbohydratemetabolism, cell cycle, cell structure and motility, cell prolifera-tion, and differentiation.30 Additionally, we were able to identifyputative low abundance proteins such as transcription factorsconfirming the high sensitivity of iTRAQ.16

Based on the replicate analysis, an average variation of 18%was measured. This value was slightly higher than our previ-ously reported technical average variation at 11%.29 However,the (50% differential expression change cut-off point used hereshould be able to incorporate most of the structural andbiological nuisances, as detailed elsewhere.29

It is worth mentioning that a large number of identifiedproteins (37%) were without a significant quantitative ratio.

This was likely to be due to the stringent criteria used duringthe estimation of the iTRAQ quantitation ratio. Based onpeptide selection criteria outlined in this study, many peptides(42% of all the identified peptides) with a quantitation valuewere ignored since the confidence level was set at 70%compared to the recommended/default level of 1% in theProteinPilot software. This step eliminated a large number ofpotentially true peptides (or proteins) and reduced the totalnumber of proteins being identified and quantified to 176 (from202), out of the 280 total proteins identified. Since we wereparticularly interested in protein candidates associated withprostate cancer progression, we applied a stringent set of rulesto narrow down the potential candidates.

Furthermore, as a consequence of the autoselection criteriain ESI-MS/MS, different selected peptides fragment differently.Depending on the fragmentation efficiency of each individualpeptide, the degree of dissociation between the reporter ionand the main peptide backbone varies upon collision. Theresulting low quality fragmented reporter ions, particularlythose yielding peak areas less than 40, were ignored. This alsocaused a large number of peptides/proteins being identifiedto be without a reliable quantitative value/ratio. However, inthis study, the experimental design of isobaric labeling wasmodified such that it would enhance the labeling efficiency.The organic content (ethanol) was increased to twice as muchas recommended in the manufacturer’s protocol, and thelabeling incubation time was lengthened to 2 h. Since iTRAQreagents degrade faster in aqueous solution, the higher con-centration of ethanol should theoretically increase the reagents′stability.

Some of the differentially expressed candidates identified byiTRAQ have previously been associated with prostate cancerprogression such as grp78, gp96 (also known as grp94), andLDH-B,12,33–35 whereas others such as S-COMT, grp58, andCKBB appear to be novel associations. Of the up-regulatedproteins, increased grp78 expression has previously beenreported in metastatic prostate cancer in bone,34 and increasedexpression has been shown in LNCaP-LN3 cells compared withLNCaP cells.35 Furthermore, increased grp78 expression hasbeen associated with the development of androgen-indepen-dent prostate cancer.33 A possible mechanism for this mayinvolve an inhibition of apoptosis due to the ability of grp78to interact with intermediates of the apoptotic pathway.36 Thus,an antiapoptotic role of grp78 could facilitate prostate cancerprogression. Of further relevance to our findings, elevated grp78expression has recently been associated with increased lymph-node metastasis and poor prognosis in patients with gastriccancer.37 Another up-regulated protein, gp96, is a glycoproteinbelonging to the heat shock protein family which has a role inprotein homeostasis, cell differentiation, and development andhas also been shown to play a role in eliciting antitumorimmunity.38,39 Although gp96 expression has not been associ-ated with prostate cancer progression using patient material

Table 2. Down-Regulated Proteins in LNCaP-LN3 Cells Identified by 2D-PAGE followed by LC-ESI-MS/MS Analysis

protein name abbrev.accessionnumber

theo.mass (Da) calcd pI

peptidesmatched

sequencecov. (%)

mowsescore

Peroxiredoxin 2 PRDX2 P32119 21747 5.67 8 37 331Adenine Phosphoribosyltransferase APRT P07741 19464 5.79 4 32 174Glyoxalase 1 GLO1 Q04760 20575 5.25 9 31 149Catechol O-methyltransferase COMT CAG30308 30018 5.26 5 23 253Rho-GDP dissociation inhibitor 1 Rho-GDI 1 P52565 23193 5.02 3 21 95

Figure 4. Western blot analyses confirming the identity anddifferential expression of selected candidates identified by iTRAQ.CKBB and S-COMT can be seen to be down-regulated in LNCaP-LN3 cells, whereas grp78 and gp96 can be seen to be up-regulated. The expression of Actin and GAPDH used as controlswas unchanged.



as demonstrated in the present study, a previous study35 hasshown increased expression levels in LNCaP-LN3 cells com-

Figure 5. MS/MS spectrum of (A) LDH-A (peptide sequence: ATLKDQLIYNLLKEEQTPQNK) and (B) LDH-B (peptide sequence:SADTLWDIQKDLKDL) subunits in LNCaP and LNCaP-LN3 cells, labeled in duplicate. LNCaP cells were labeled with the 114 and 115tags, and LNCaP-LN3 cells were labeled with the 116 and 117 tags. Peak area at the low mass/charge (m/z) end (inset) showing arelative abundance of the LDH-A subunit in both LNCaP and LNCaP-LN3 cells, but a complete absence of the LDH-B subunit in LNCaP-LN3 cells, as seen by the absence of peaks at 116 and 117 m/z.

Figure 6. Zymogram of LDH isoenzyme patterns determined bycellulose acetate electrophoresis. Note the absence of the LDH-Bsubunit in LNCaP-LN3 cells but expression of the LDH-A subunit.In mammalian cells, two genes code for two LDH subunits, LDH-Aand LDH-B, which associate as tetramers to give rise to fivetetrameric forms of LDH isoenzymes: LDH-A4 (LDH-5), LDH-A3B1(LDH-4), LDH-A2B2 (LDH-3), LDH-A1B3 (LDH-2), and LDH-B4(LDH-1). Note the presence of all five isoenzymes in LNCaP cells;however, loss of the LDH-B subunit eliminates the expressionof isoenzymes LDH-1 to four in LNCaP-LN3 cells. LNCaP-LN3 cellscan be seen to express solely the LHD-5 isoenzyme.

Figure 7. Immunoexpression of gp96 in prostate tissues. Gp96expression is seen to be weak in the basal cells of BPH epithelium(A), while moderate/strong expression can be seen in high-gradePIN (B) and in a Gleason grade 3 cancer (C) including a case ofmetastatic prostate cancer in bone (D). There was a statisticallysignificant increased expression of gp96 in cancer compared withthe BPH tissue (p < 0.0005, Mann–Whitney U).



pared with LNCaP cells, in agreement with our findings.Furthermore, the molecule has been shown to undergo alter-ations in glycosylation associated with increased malignantbehavior.40 Thus, our finding of significantly increased gp96immunoexpression in prostate cancer and precursor lesionssuggests that in addition to altered glycosylation, up-regulationof gp96 may also be involved in cancer progression and is likelyto be involved at an early stage.

Of the down-regulated proteins, S-COMT is a soluble formof the catechol-O-methyltransferase enzyme, which normallyfunctions to inactivate the activity of a number of biologicalactive and toxic catechols, including neurotransmitters andhormones, by the transfer of a methyl group from the methyldonor S-adenosyl-L-methionine to the hydroxyl moiety of thecatechol ring of a substrate.31 Although a role for S-COMT isnot known in prostate cancer progression, elevated levels ofS-COMT have been reported in mammary carcinoma.31 An-other down-regulated protein was CKBB, an enzyme involvedin energy transduction pathways which catalyzes the formationof ATP from ADP. Although there are no reports on theinvolvement of CKBB in prostate cancer progression, reducedexpression has been reported in colon cancer compared withmatched adjacent normal colon tissues.41 Thus, our finding ofreduced levels of CKBB in LNCaP-LN3 cells suggests a novelassociation for CKBB with prostate cancer which needs to beinvestigated further.

Of the five down-regulated proteins identified by 2D-PAGEfollowed by LC-ESI-MS/MS analyses, three of these (COMT,GLO1, and PRDX2) were also identified in the iTRAQ analyses,whereas Rho-GDI 1 and APRT were not identified by iTRAQ.This difference could be due to a number of reasons relatingto the different methods used for protein extraction or thatcertain proteins are unsuitable for iTRAQ analyses. A previousstudy comparing DIGE, iTRAQ, and ICAT methods has showna limited overlap among the proteins identified by these threemethods, suggesting that these methods are complementaryin nature.42 Thus, our findings support the conclusions of thisprevious study. Nevertheless, Rho-GDI 1, PRDX2, and APRTidentified by 2D-PAGE LC-ESI-MS/MS may play importantroles in prostate cancer progression which need to be inves-tigated further.

Although the 2D-PAGE technique provides a visual repre-sentation of the proteins and is able to identify putative post-translational modifications, the technique suffers from a num-ber of limitations such as that it is relatively labor intensive,requires a relatively large amount of protein, and lacks repro-ducibility.8 In addition, there are certain types of proteins thatare not well represented by 2D-PAGE including low-molecularmass proteins (<20 KDa) and those with extreme isoelectricpoints (e.g., pI <4 and >9) or extreme molecular weights. Theselimitations are to some extent overcome by iTRAQ in that it isrelatively less labor intensive and does not rely on proteinsolubility or pI. Furthermore, the relative protein quantitationmeasurements at the MS/MS level using isobaric tags haveproven to be very reliable,29 in contrast to 2D-PAGE, whichrelies on the measurements of staining intensities with stainsthat have a limited dynamic range. Nevertheless, in the presentstudy, we have utilized both methods and shown that they arecomplementary.

Previously, using 2D-PAGE analysis followed by tandem massspectrometry of differentially expressed proteins, we haveshown an absence of the LDH-B subunit in LNCaP-LN3 cells,whereas LNCaP cells express both LDH-A and LDH-B sub-

units.12 Furthermore, the mechanism underlying absent LDH-Bexpression was shown to involve promoter hypermethylation.12

Our finding of absent LDH-B subunit expression, but expres-sion of the LDH-A subunit by LNCaP-LN3 cells using iTRAQ,is in agreement with our previous study. Furthermore, ourunpublished study using iTRAQ has shown relative absentexpression of Vimentin in LNCaP cells and was verified byWestern blotting, thus confirming the validity of iTRAQ for theidentification of absent protein expression. However, for LDH-B, not all of the 11 identified MS/MS spectra (above 70%confidence) yielded statistically significant quantitation forLNCaP cells, as their total area counts were less than 40.Nonetheless, to our knowledge, this is the first report of iTRAQfor the detection of absent protein expression and is particularlyimportant since it has been reported that shot-gun proteomicapproaches have an inability to quantify zero protein expres-sion levels.16 Thus, it is important to bear in mind that it isnot possible to rely completely on the available bioinformaticssoftware to detect zero protein expression and that manualinspection of the MS/MS data is crucial.

Conclusions

Using the iTRAQ shot-gun proteomic approach on variantmetastatic prostate cancer cells, we have identified 14 candi-dates to be significantly differentially expressed between thepoorly metastatic LNCaP cells and highly metastatic LNCaP-LN3 cells, among a background of 280 unique proteins. Thesedifferentially expressed proteins represent diverse biologicalprocesses previously associated with cancer progression.30,33,37

Although the clinical relevance of one of the candidates, gp96,was demonstrated using prostate tissue material, validation ofthe other candidates is warranted using a large case-mix cohortof patient material with long-term follow up data. Furthermore,these candidates either individually or when studied as a panelmight provide important prognostic information, in order totailor patient management according to individual patientneeds. Further studies aimed at understanding the biologicalroles of the identified candidates may provide importantinsights into the mechanisms of prostate cancer progressionand may also serve as targets for novel drug therapies. Finally,our study provides proof of principle for the application ofiTRAQ in the study of human prostate cancer progression andin the identification of candidate biomarkers, including theidentification of relative absent protein expression. Our ap-proach could readily be translated to other cancer systems.

Abbreviations: iTRAQ, isobaric tags for relative and absolutequantitation; TMA, tissue microarray; PIN, prostatic intraepi-thelial neoplasia; BPH, benign prostatic hyperplasia; COMT,catechol-O-methyltransferase isoform; CKBB, brain creatinekinase; LDH, lactate dehydrogenase; 2D-PAGE, two-dimen-sional polyacrylamide gel electrophoresis.

Supporting Information Available: Table S1: The fulllist of 280 proteins identified via the iTRAQ approach. TableS2: The number of proteins used in protein identification andquantitation. This material is available free of charge via theInternet at http://pubs.acs.org.

Acknowledgment. This research was supported bygrants from PROMET (project no. FP6-LSH-5-2004-018858);P-MARK (contract no. LSHC-CT-2004-503011), under thesixth EU Framework programme; NCRI (MRC 58152) to F.C.Hamdy; and an Engineering and Physical Sciences Research



Grant to P. C. Wright (EPSRC GR/S84347/01). Authors AdamGlen and Chee S. Gan contributed equally to this work.

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