Mobile phase A was 0.1% formic acid in water, mobile phase B, 0.1% formic acid in acetonitrile and the flow rate was 0.25 ml/ min. A linear gradient was used from 25% B to 35% B over 0 to 5 min, then a further gradient from 35% B to 99% B over 6 min followed by 99% B for 4 min. The column was re-equilibrated for a total of 3 min. The injection volume was between 5–20 ml. All MS experiments were conducted on a 4000 QTRAP hybrid triple- quadrupole linear ion trap mass spectrometer using Analyst version 1.4.1 software (Applied Biosystems, http://www.applied- biosystems.com/) equipped with a TurboIon source used in positive ion electrospray mode. The probe capillary voltage was optimized at 4200 V, desolvation temperature set to 400uC, curtain gas, nebulizing and turbo spray gas were set to 40, 10 and 20, respectively (arbitrary values). Declustering potential was ramped between 50–120 V. Nitrogen was used for collisionally induced dissociation (CID). The peak-width was set on Q1 and Q3 at 1.0 Th (measured at half height) for all MS and MS/MS experiments. Spectra were obtained over the range m/z 800–2000 with scan times of 1–2 sec. Operating in LIT mode Q0 trapping was activated and dynamic fill time used, the scan rate was set to 250 Th/s for enhanced product ion (EPI) scans, excitation time was 150 msec, excitation energy 25 V and entry barrier 4 V. For EPI spectrum acquisition the precursor ions of interest for conjugates of SF with insulin (m/z 1183.9 MH55+), EGF (m/z 1088.8, MH56+) and TGFb (m/z 1981.9, MH513+) were selected, the collision energy was ramped between 30–120 V and spectra were obtained over the range m/z 100–1500 with a scan time of 1.9 sec. MS3 settings were identical to MS2 except that the collision energy was 50–80 V and declustering potential was 50– 80 V. LC-MS/MS analysis of TGFb1 incubated with SF following electrophoresis and tryptic digestion 1 mg aliquots of the TGFb1 protein, supplied with bovine serum albumin as carrier, were incubated with either DMSO or 1.2 mmoles of SF for 30 minutes at 37uC and run onto denaturing 4–12% Bis-Tris NuPAGE gels (Invitrogen, http://www.invitro- gen.com). Bands were excised and digested with trypsin (Promega, http://www.promega.com/) after reduction with dithiothreitol (DTT) and alklyation with iodoacetamide (Sigma-Aldrich, http:// www.sigmaaldrich.com/). Extracted peptides were lyophilized and re-dissolved in 1% acetonitrile, 0.1% formic acid for analysis by mass spectrometry. LC-MS/MS analysis was performed using a LTQ mass spectrometer (Thermo Electron Corporation, http:// www.thermo.com/) and a nanoflow-HPLC system (Surveyor, Thermo Electron). Peptides were applied to a precolumn (C18 pepmap100, LC Packings, http://www.lcpackings.com/) connect- ed to a self-packed C18 8-cm analytical column (BioBasic resin ThermoElectron; Picotip 75 mm id, 15 mm tip, New Objective, http://www.newobjective.com/). Peptides were eluted by a gradient of 2 to 30% acetonitrile in 0.1% formic acid over 40 min at a flow rate of approximately 250 nL min21. Data- dependent acquisition of MS/MS consisted of selection of the five most abundant ions in each cycle: MS mass-to-charge ratio (m/z) 300 to 2000, minimum signal 1000, collision energy 25, 5 repeat hits, 300 sec exclusion. In all cases the mass spectrometer was operated in positive ion mode with a nano-spray source and a capillary temperature of 200uC, no sheath gas was employed; the source voltage and focusing voltages were optimized for the transmission of angiotensin. Raw data were processed using BioWorks 3.3 (Thermo Electron Corporation). Searches were performed with Mascot (Matrix Science, http://www. matrixscience.com/) against SPtrEMBL (4719335 sequences) restricted by taxonomy to Homo sapiens (68982 sequences), oxidized Broccoli & Signalling Pathways methionine and carbamidomethyl cysteine residues were allowed as variable modifications as was putative SF. The error tolerance of the parent ion was 61.2 Da and the fragment mass tolerance was 60.6 Da, one missed cleavage was permitted. Error tolerant searches in Mascot against TGFb were routinely performed and extracted ion chromatograms and manual inspection of spectra were prepared using Qual Browser and BioWorks 3.3 (Thermo Electron Corporation). Luciferase reporter gene assay NIH 3T3 cells stably transfected with a CAGA12-luc plasmid, which responds to Smad activation [23], were cultured in DMEM supplemented with 10% fetal calf serum (FCS), 1% penicillin, 1% streptomycin, 1% L-glutamine and 0.4 mg/ml geneticin. Cells were seeded into complete growth medium in a six-well tissue culture dish for 24 h, after which the medium was replaced with low serum medium (0.5% FCS) containing one of three treatments: (1) TGFb1 (to achieve a final concentration of 2 ng ml21) in PBS buffer, (2) TGFb1+10 mM DTT in PBS buffer (3) TGFb1+2 mM SF in PBS buffer. To simulate SF pharmacokinetics, all test samples were incubated at 37uC for 30 minutes prior to dialysis, performed in PBS buffer for 4 hours using Slide-A-Lyzer Dialysis Cassettes MWCO 3.5K (PIERCE, http://www.piercenet.com/). Dialysis reduced SF concentration to 34 nM. As additional controls, cells were treated with PBS without TGFb1 and PBS with SF (34 nM). The luciferase activity was determined 16 h following treatment using the Luciferase Reporter Gene assay (Roche Applied Science, http://www.roche. com/) in a Perkin Elmer Wallac Victor 2 1420 multilabel counter plate reader (http://las.perkinelmer.com/). Briefly, cells were washed twice with PBS and lysed in cell lysis buffer supplied with the assay. Chemiluminescence was immediately quantified following the addition of luciferase assay substrate. Luciferase values were normalized to protein concentration quantified using the BCA assay (Sigma-Aldrich, http://www.sigmaaldrich.com/). The experiment was repeated four times, with three replicates of each treatment per experiment. Statistical analysis was performed using 1-way ANOVA with the statistical software, R [24]. Results Comparison of gene expression of benign and malignant TURP tissue samples We compared global gene expression profiles in surgically resected benign and malignant prostate TURP tissue using RNA extracted from heterogeneous tissue (such as we intended to use in the intervention study). Unsupervised clustering distinguished unambiguously the benign and malignant samples (data not shown). Pathway analyses for genes that were significantly different between the two groups were undertaken with the use of GenMapp software, and identified pathways that are frequently reported to be perturbed during carcinogenesis (Tables 2a and 3a). To validate further our methods of data analysis and to determine whether microarray data from gross heterogeneous tissue are comparable to data generated from LCD epithelial cells, we analyzed independent data sets of LCD epithelial cells (GEO Accession: GDS1439) from benign, localized and metastatic prostate cancer. We used our benign and malignant samples as a training set for linear discriminant analyses (LDA) and the independent data as test sets, and found that the LDA model correctly distinguished the benign, localized and metastatic LCD epithelial cell samples (Figure 2). Thus, this preliminary study provides validation for our approach to the statistical analyses of array data. PLoS ONE | www.plosone.org 5 July 2008 | Volume 3 | Issue 7 | e2568