Serum samples from 5 patients representing each of the 4 patient groups were pooled in equal volumes to give a total volume of 200 ml for each group (40 ml of each sample). The pooled serum samples were shipped on dry ice to Genway (Digilabs Biovision, Germany), for immuno-depletion using the Seppro Ig-Y14 system. The flow-through fraction (depleted of albumin, immunoglobulin IgG, fibrinogen, transferrin, IgA, IgM, haptoglobin, alpha2- macroglobin, alpha1-acid glycoprotein, alpha1-antitrypsin, Apo A-I HDL, Apo A-II HDL, complement C3 and LDL (ApoB)), was used for subsequent iTRAQ analysis. iTRAQ sample labelling and SCX fractionation Prior to iTRAQ analysis, samples were concentrated and buffer exchanged using 5 kDa molecular weight cut-off spin concentra- tors (Millipore). The samples were buffer exchanged three times against 500 ml of 1 M triethylammoniumbicarbonate (TEAB) buffer and concentrated to a volume of approximately 80 ml. Samples were labelled with the iTRAQ reagents according to the manufacturers instructions, and as previously described [14,15]. Each sample was labelled with one of the four iTRAQ reagents (iTRAQ reporter ions of 114.1, 115.1, 116.1, 117.1 mass/charge ratio). The tag labelling order was BPH- 117; localised non- progressing cancer-116; progressing cancer-115; metastatic dis- ease-114). Labelled samples were pooled and fractionated by strong cation exchange (SCX), using a BioLC HPLC column (Dionex, Surrey, UK), and analyzed by LC-MS/MS as previously described [14,15]. Tandem mass spectrometry analysis Mass spectrometry (MS) was performed using a QStar XL Hybrid ESI Quadrupole time-of-flight tandem mass spectrometer, ESI-qQ-TOF-MS/MS (Applied Biosystems, Framingham, MA; MDS-Sciex, Concord, Ontario, Canada), coupled with an online capillary liquid chromatography system (Famos, Switchos and Ultimate from Dionex/LC Packings, Amsterdam, The Nether- lands). The dried samples were resuspended in 60 l of 3% acetonitrile and 0.1% formic acid ready for the MS, and 10–15 l (depending on the peptide concentration as seen in the peak intensity of the SCX chromatogram) were injected to the nano- LC-ESI-MS/MS system for each analysis. Initial separation took place on a PepMap C18 RP capillary column (LC Packings) with a constant flow rate of 0.3 l/min. LC buffers A and B were made up as 3% acetonitrile, 0.1% formic acid and 97% acetonitrile, 0.1% formic acid, respectively. The gradient was started as 97% buffer A and 3% buffer B for 3 minutes, followed by 3 to 25% buffer B for 120 minutes, 90% buffer B for 7 minutes and finally 97% buffer A for 7 minutes. Data acquisition in the mass spectrometer was set to the positive ion mode, with a selected mass range of 350–1800 m/z. Tandem mass spectrometry was performed on peptides with +2, +3, +4 charge states across a scan range of 65– 2000 m/z. Protein identification and relative quantification Protein identification and relative quantification was carried out as previously described [23,24]. Identification of peptide precursor and fragments was performed by database searching against the Swiss-Prot and Trembl Homo sapiens protein database (41070, 71449 ORFs respectively, downloaded from UniProt, May 2010). Parameters for searching were set up as follows: MS tolerance was 0.4 and MS/MS tolerance were set at: peptide tolerance 0.4 Da, charge +2, +3 and +4, min peptide length, z-score, max p-value and AC score were 6, 6, 1026 and 6 respectively. Phenyx default ‘turbo’ scoring was enabled with mass tolerance restriction of 0.1 Da for MS and MS/MS and the minimum percentage of the Serum Biomarkers for Prostate Cancer Metastasis peptide sequence coverage by b+ (b), b2+ (b++), y+ (y) or y2+ (y++) fragment series was set to the default value of 20%. Target database search space was restricted to tryptic peptides with a maximal of 1 miscleavage. Modifications were set as: 4-plex iTRAQ mass shifts (+144 Da, K and N-term), methylthiol (+46 Da) and oxidation of methionine (+16 Da). False discovery rates (FDR) were estimated using a concatenated target-decoy database as described by Elias and Gygi [25]. Protein changes were qualified using a t- test algorithm developed in house [23]. Immunohistochemistry for eEF1A1 Immunohistochemistry was performed essentially as previously described [18]. Sections of bone and prostatic tissues were cut (4 m) and mounted on superfrost slides (VWR International, Germany). Slides were incubated with mouse monoclonal anti- eEF1A1 antibody (Santa-Cruz Biotechnology, CA, USA Cat. No sc21758), at 0.4 mg/ml in 2% horse serum overnight at 4uC. Sections were washed twice in PBS-Tween 20 (PBST), and incubated for 30 min with anti-mouse IgG ImmPRESS HRP (Vector Laboratories, Cat. No MP-7402). After further washing in PBST, localisation of antibody/antigen complex was visualized using the ImmPACT DAB system (Vector Laboratories, Cat. No SK-4105). Control sections were incubated with anti-mouse IgG isotype control (Vector laboratories I-2000), diluted to 0.4 mg/ml in 2% normal horse serum. eEF1A1 immunostaining was assessed for both intensity and cellular localization by an experienced histopathologist (Dr. Simon Cross), who was blinded to the study. Each case was assigned a staining intensity, ranging from 0–3, where 0 = absent; 1 = weak; 2 = moderate and 3 = Intense staining as previously described [18]. Western blotting Western blotting was performed essentially as previously described [26]. Anti-EF-Tu goat polyclonal IgG primary antibody was used at a concentration of 1:1000 (Santa Cruz, Cat. No. sc12990). Secondary antibody was HRP-conjugated rabbit anti-goat (Abcam), and was used at a concentration of 1:1400 (Abcam). Dual color precision plus molecular weight markers were used for size estimation (Bio-Rad, Hertfordshire, UK). Reverse-Transcription PCR and Sequencing Total RNA was extracted from prostate cancer cell lines using TRI reagent (Sigma-Aldrich, UK), according to the manufacturer’s instructions. The RNA was quantified spectrophotometrically and 2 mg was reverse transcribed into cDNA using the SuperScript III Reverse Transcriptase kit with 250 ng of random primers, according to the manufacturer’s instructions (Invitrogen, UK). PCR primers specific to the eEF1A1 isoform were designed manually, using the Ensembl cDNA sequence: ENST00000316292 (http://www. ensembl.org/index.html). The eEF1A1-forward primer sequence was (59-39): TCCTTCAAGTATGCCTGGGTCT (eEF1A1-F1), corresponding to nucleotide positions 157–178. The eEF1A1-reverse primer sequence was: TGGCACAAATGCTACTGTGTCG (eEF1A2-R1), corresponding to nucleotide positions 555–576, to give an expected PCR product size of 420 bp. Similarly PCR primers specific for the eEF1A2 isoform were designed using the Ensembl cDNA sequence: ENST00000298049. The eEF1A2-forward primer sequence was: AGGAGGCTGCTCAGTTCACCT (eEF1A2-F3), corresponding to nucleotide positions 1004–1024; and the eEF1A2- reverse primer sequence was: CCGCTCTTCTTCTCCACGTTC (eEF1A2-R3), corresponding to nucleotide positions 1317–1336, with an expected PCR product size of 334 bp. Primers were synthesized using the commercial facility at Eurofins MWG Operon (http:// www.eurofinsdna.com/products-services/oligonucleotides0.html). PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e30885