night at 37uC. The iTRAQ protocol was as follows: cells were lysed in a lysis solution containing 7 M urea, 2 M thiourea and 100 mM n- octyl-b-D-glucopyranoside, then mixed by intermit- tent vortexing over 20 min at room temperature, followed by centrifugation at 12000 RPM for 15 min. Protein concentrations in the supernatant were estimated using Bradford assay (BioRad Laboratories) and subjected to acetone precipitation before digestion. A 100 mg aliquot from each sample was used for the precipitation. Digestion and labeling using isobaric reagents (iTRAQH Reagents Multiplex Kit, Applied Biosystems, Foster City, CA) were performed following the protocol described previously [28]. Eighty micrograms each of these biological, duplicate control samples and R1881-treated samples were labeled with different isobaric tags (114: control_1, 116: control_2, 115: R1881_1, 117: R1881_2). All four samples were combined and subjected to fractionation by SCX and RP-LC MALDI plating. Mass-spectrometry Online 2D LC-MS/MS (MudPIT) of non-labeled tryptic peptides using LTQ. Paradigm MG4 HPLC system (Michrom Bioresources, Inc., Auburn, CA) was used for the separation of the peptides through a strong cation exchange trap and a reverse phase C18 microtrap (Michrom Bioresources, Inc.). Progressively increasing concentrations of ammonium formate (1.75 mM to 500 mM) were used for stepwise elution, and the trapped peptide salts at the microtrap were washed with buffer A (5% ACN, 0.0025% HFBA, 95% H20). The peptides were eluted using a 60 minute gradient starting with 5% ACN to 70% ACN with a two buffer system (Buffer A: as above; Buffer B: 95% ACN, 0.0025% HFBA, 5% H2O). The C-18 microtrap was coupled to an analytical C-18 column with a pulled tip (75 mm i.d., 10 cm length; New Objective Inc, Woburn, MA). Peptides were sprayed directly into the LTQ Mass Spectrometer (Thermo Fisher Corporation, Waltham, MA). Equal amounts of proteins (7.5, 15, and 50 mg each) from either control or R1881-treated LNCaP cells were trypsinized as described above and purified using a desalting peptide macrotrap (Michrom Bioresources, Inc.) prior to online SCX and C18 separation. LC-MS/MS was performed for each sample in data dependent mode using Xcalibur software (Thermo Fisher Corporation). Each MS full scan (m/z = 400–2000) was collected with an average of 10 micro scans, and the three most abundant ions were subjected to fragmentation (MS/MS). Collision energy was set to 30% and with dynamic exclusion duration of 3 minutes. Offline 2D LC-MS/MS of iTRAQ labeled peptides. SCX fractionation. Four iTRAQ-labeled samples were combined and fractionated on 2 sulfoethyl aspartamide SCX spin columns (SEM HIL-SCX, PolyLC, The Nest Group, Inc. Southboro, MA) equilibrated with 10 mM KH2PO4, (pH 4.5), 20 % ACN and run in parallel. Peptides were eluted with 50 ml each of 14 salt steps (35 to 500 mM KCl) and the paired eluates were combined and dried. RP LC MALDI: A Zorbax 300SB-C18 (3.5 um, 15060.1 mm) column was used to fractionate each of the above listed fractions. A 90 min gradient method with a flow rate of 0.4 ml/min was employed (1100 series nanoflow LC system, Agilent Technologies, Santa Clara, CA). Column effluent was mixed (micro Tee, Agilent) with matrix (2.5 mg/ml a-CHCA and 10 mM NH4H2PO4 in methanol: isopropanol: ACN: H2O: acetic acid (14:30:22:33:0.6) delivered with an infusion pump (PHD200, Harvard Apparatus, Holliston, MA) at 0.9 ml/min and directly spotted (microFC, Agilent) at 0.42 min intervals onto a stainless steel MALDI target plate from 12 to 92 minutes (192 wells/plate, Applied Biosystems, Foster City, CA). MALDI- TOF MS/MS analysis: Data were acquired using 4800 Proteomics Analyzer -TOF/TOFTM Proteomics of Prostate Cancer (Applied Biosystems) linked to 4000 Series Explorer software (v. 3.0). Spectra from m/z 800–3500 were acquired using 750 laser shots. Spectra with signal to noise (S/N) of 120 were selected for MS/MS by applying 750–6000 laser shots. Atmospheric gas was used as the collision gas with a pressure of ,6610-7 Torr and collision energy of 1 kV. A seven point Gaussian smoothing and S/N of 15 were applied using cluster area S/N optimization for peak detection. Instrument default calibration was updated using monoisotopic masses for angiotensin I (m/z 1296.685), Glu1- fibrinopeptide B (m/z 1570.677), ACTH (18–39) (m/z 2465.199), des-Arg1-bradykinin (m/z 904.468), ACTH (1–17) (m/z 2093.087) and ACTH (7–38) (m/z 3657.923) (ABI). RNA Isolation and Microarray Analysis Total RNA isolation was performed using TRIZOL reagent per the manufacturer’s instructions (Invitrogen). Briefly, cells were washed with PBS (Phosphate buffer saline) and scraped in TRIZOL reagent. Approximately 250 ml of chloroform was added to 1 ml of sample and mixed by inversion. The sample was centrifuged at 13,000 rpm for 15 min at 4uC. Approximately 500 ml of isopropyl alcohol was added to supernatant and centrifuged at 13,000 rpm for 15 min at 4uC. The pellet was washed with 70% ethanol, and centrifuged at 13,000 for 10 minutes at 4uC. After ethanol removal, RNA was dried and dissolved in RNAse- and DNAse-free water. The RNA was subsequently purified using RNeasy mini kit (QIAGEN Inc., Valencia, CA). The amount and integrity of purified RNA was checked by NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE) and Agilent Bioanalyzer (Agilent Technologies). Total RNA from the individual samples was analyzed on Affymetrix U133 Plus 2.0 arrays (Affymetrix, Inc., Santa Clara, CA). Complementary DNA synthesis, cRNA synthesis, hybridization, washing, and scanning were done following the manufacturer’s protocols (Affymetrix, Inc.). The experimental details and raw data have been deposited in the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm. nih.gov/geo) and are accessible through GEO Series accession number GSE17044. Mass-spectrometry data analysis MudPIT ESI- Ion Trap MS/MS. Raw files were converted into mzXML files and peptides were assigned to MS/MS spectra using SEQUEST [18] search against the Human IPI database version 3.24. The following search parameters were selected: 3 da precursor mass tolerance, average mass, semi-tryptic search with two or fewer missed cleavages, and oxidized methionine as a variable modification. In total, 120290 search results were obtained in the R1881 treated data set (32178, 47810, 39793, and 509 assignments to spectra of 1+, 2+, 3+, and 4+ charged peptide ions, respectively) and 113925 in the control data set (31643, 44,772, 36699, and 811 assignments to spectra of 1+, 2+, 3+, and 4+ charged peptide ions, respectively). Peptide assignments were validated using PeptideProphet [19], and the protein inference performed using ProteinProphet [20], as these tools are freely available as a part of the Trans-Proteomic Pipeline (www.proteomecenter.org). The list of protein identifications was filtered using a 0.9 probability threshold, which corresponds to less than 1% estimated false discovery rate (FDR). MS/MS data sets acquired on control and R1881 treated LNCaP cells were analyzed separately, resulting in 670 and 660 protein groups in each set (with indistinguishable protein accession numbers collapsed into a single group), respectively. iTRAQ MALDI- TOF MS/MS analysis. MS/MS spectra were extracted from the raw data in Mascot Generic File format PLoS ONE | www.plosone.org 9 September 2009 | Volume 4 | Issue 9 | e7075