indolent from aggressive forms of the disease [6,7]. Additionally, PSA is associated with a high degree of false-positive and false- negative test results, as levels may be elevated in non-cancer conditions of the prostate, including benign prostatic hyperplasia (BPH). Thus, additional biomarkers are urgently needed which could improve the diagnostic specificity of PSA and predict the likelihood of disease progression. Blood and its products, such as plasma and serum are ideal fluids for the identification of cancer biomarkers since they contain proteins both secreted and shed from cancer cells, combined with the ease of sampling. However, the variable composition and large dynamic range of proteins present in plasma (estimated to be 1010 orders of magnitude or more), pose formidable challenges in identifying clinically relevant biomarkers amongst the background of abundant proteins such as albumin, immunoglobulin and transferrin [8]. Of the 22 or so most abundant proteins in plasma, these constitute more than 99% of the mass of the total plasma proteins, while the remaining 1% are thought to be composed of the medium/low abundance proteins and include the biomarker pool [9]. The large orders of magnitude in protein concentration have hampered previous mass spectrometry based efforts aimed at identifying clinically relevant biomarkers, mainly due to a suppres- sion of ionization of the low abundance proteins by the higher abundance proteins [10]. However, prior removal of some of the most highly abundant proteins has been shown to improve the detection of relatively lower abundant proteins [11,12]. Although there are many different protein fractionation methodologies based on differences in molecular weight, shape, charge, pI, hydrophobic- ity and affinity through specific biomolecular interactions, it has been reported that high abundance protein separation using the antibody based IgY-12 immunodepletion system is highly reproducible [13]. Amongst the proteomic technologies used for biomarker identification, ‘isobaric Tags for Relative and Absolute Quantita- tion’ (iTRAQ) has the advantages of being relatively high throughput, and can simultaneously provide information on peptide quantitation and identification, as previously reported by us and others [14–16]. Briefly, in a typical workflow samples are reduced, alkylated and proteolytically digested to generate peptides. The peptides are labeled with a set of iTRAQ reagents (in a 4 or 8-plex format), pooled and fractionated by strong cation exchange (SCX). The fractions are then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), with the resultant mass spectra providing sequence information (from the peptide fragments), and relative quantification (from the reporter group ions). In an effort to identify novel proteins associated with the metastatic progression of human prostate cancer, we have performed a 4-plex iTRAQ analysis using pooled serum samples collected prospectively from 4 well defined groups of patients who were actively monitored for at least 5 years, and selected to represent the spectrum of prostatic disease. Following data analysis, a number of candidates were found to be significantly differentially expressed in cancer samples compared with benign samples. One of the candidates identified as being significantly up-regulated in cancer groups was eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), and was further investigated by immunohistochemistry using prostate tissue samples from localized and metastatic cases. The biomarker leads identified in our ‘discovery’ phase study, including eEF1A1 are discussed in relation to their significance to prostate cancer progression. Materials and Methods Patients and serum collection Peripheral blood was collected prospectively from patients attending the Urology clinic at the Royal Hallamshire Hospital at Serum Biomarkers for Prostate Cancer Metastasis the time of their initial visit, following written informed consent. Blood sample collection was approved by the Ethics Committee of the University of Sheffield. Serum was collected by allowing the blood to coagulate for 30 min, centrifuged at 1,2006 g for 10 min at 4uC and then stored at 280uC in 100 ml aliquots. All blood samples were collected prior to the administration of any treatment. Twenty serum samples were carefully selected to represent the various stages and grades of prostate disease and pooled (n = 5 patients/group), to form 4 patient groups. All patients were actively monitored for at least 5 years from the time of their initial blood sampling. The 4 patient groups were: Group 1: histological diagnosis of benign prostatic hyperplasia ‘BPH’, with no evidence of cancer by at least 2 independent sets of prostatic biopsies, and a PSA level below 10 ng/ml (mean age of 61 yrs). Group 2: histological diagnosis of prostate cancer with a PSA level below 10 ng/ml, and no evidence of a rising PSA following 5 yrs active monitoring - ‘non-progressing’ group, (mean age of 67 yrs); Group 3: histological diagnosis of clinically localised cancer with an initial PSA level below 13 ng/ml, followed by 3 consecutive rises in PSA levels during 5 yrs of active monitoring - ‘progressing’ group, (mean age of 69 yrs); Group 4: patients with a PSA.19 ng/ml and evidence of bone metastasis from a positive radionucleotide bone scan - ‘metastatic’ group, (mean age of 73 yrs). The differences in the median ages of the patients were found not to be statistically significant between the 4 groups (p = 0.146, Kruskal-Wallis test). The disease characteristics of the 20 patients comprising the 4 groups are shown in Table S1. Patient tissue material Tissue microarrays (TMAs), comprised of 56 cases of prostatic adenocarcinoma ranging in single Gleason grades (i.e. grade 2, n = 5; grade 3, n = 32; grade 4, n = 9; grade 5, n = 10), and 40 cases of adjacent non-malignant tissue, and were constructed as previously described [17,18]. An additional 23 cases of bone biopsies from patients both with and without prostate cancer skeletal metastasis were obtained by 8 mm trephine biopsy performed under general anesthesia. Informed patient consent and Ethics Committee approval was obtained prior to the study (South Sheffield Research Ethics Committee, SSREC/02/155 and 00/172). Cell lines Human prostate cancer cell lines LNCaP, PC-3, DU145 and VCaP were purchased from the American Type Culture Collection (ATCC), (http://www.lgcstandards-atcc.org/). DuCaP cells were obtained via the PRIMA project consortium (http:// www.primaproject.org/participants.php). The LNCaP-Pro5, LNCaP-LN3, PC-3M and PC-3M-LN4 cells were a kind gift from Dr. Curtis Pettaway (University of Texas, M.D. Anderson Cancer Centre), [19]. The LNCaP-C4-2 and LNCaP-C4-2B cell lines were obtained from Prof. George Thalmann [20]. The TE- 85 osteosarcoma cells were a kind gift from Prof. J.A. Gallagher, University of Liverpool. All prostate cancer cell lines were cultured as previously described and confirmed to be free from Mycoplas- ma [15]. IgY-14 affinity depletion of serum samples Pooled serum samples were depleted of the 14 most common plasma proteins using the Seppro IgY-14 depletion system [21]. Previous studies have shown that serum pooling followed by depletion of the most highly abundant proteins is an effective strategy to reduce the dynamic range of proteins, and thus enhance the identification of serum biomarkers, as demonstrated using the quantitative proteomic method of iTRAQ(R) [22]. PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e30885