Figure 3. Representative images showing the immunoexpres- sion of eEF1A1 in prostatic tissue, bone metastatic lesions and control bone. (A), eEF1A1 immunoexpression can be seen in the malignant cells of the primary cancer (staining intensity = 2), and in the matched bone metastatic lesion from Patient 1 (staining intensity = 3). (B), eEF1A1 immunoexpression in the malignant cells of the primary cancer (staining intensity = 1), and in the matched bone metastatic lesion from Patient 2 (staining intensity = 3). (C), Intense eEF1A1 immunoexpression can be seen in the osteoblasts lining bone (staining intensity = 3, arrowheads), which are in close proximity to tumour cells, whereas osteoblasts lining normal bone show weak expression (staining intensity = 1, arrow heads). doi:10.1371/journal.pone.0030885.g003 Confirmation of candidates identified by iTRAQ To confirm the differential expression of candidate proteins identified by iTRAQ, we subsequently performed a 1D-gel electrophoresis using pooled serum from the 4 patient groups. Following staining with Coomassie blue, a faint band was visually seen to be present at a slightly higher intensity in the samples from patients with metastatic disease relative to the other 3 groups of patients (data not shown). This band was excised from the gel, digested with trypsin and the resultant peptides analysed by LC- MS/MS. The mass spectrometry data identified 7 peptides matching to CRP with a sequence coverage of 27.6%, and confirmed the iTRAQ data. The mass spectrum of a represen- tative iTRAQ labelled peptide from CRP protein following MS/ MS is shown in Figure S2. eEF1A1 immuno-expression in prostatic tissue During our iTRAQ analysis, we identified eEF1A1 to be increased in expression in all of the cancer groups relative to BPH, with relatively higher levels seen in the progressing (+1.8 fold), and metastatic groups (+1.4 fold), (Table S2, and Figure 2). eEF1A1 was of particular interest to us for a number of reasons. Our Serum Biomarkers for Prostate Cancer Metastasis previously published iTRAQ study had shown its levels to be increased in higher metastatic variant prostate cancer cells [14]. Furthermore, a previous study had shown that down-regulation of eEF1A1 by RNA interference (RNAi), in Du145 cells reduced cell proliferation, and inhibited cell migration and invasion [30]. Thus, immunohistochemical (IHC) staining for eEF1A1 was performed using clinical tissue material from patients with BPH, organ confined cancer, and bone from patients both with and without metastatic prostate cancer. Representative immunostain- ing images are shown in Figures 3A–C. Although eEF1A1 immunoexpression was seen in cancer cells, there was no significant difference in the staining intensity between organ confined disease and metastatic disease (p = 0.1720, Mann Whitney U). Interestingly, intense immunoexpression of eEF1A1 was seen in osteoblasts and in particular those osteoblasts in the vicinity of metastatic prostate tumour cells (Figure 3C). Assessment of the immunostaining intensity in bone metastatic lesions showed that there was a statistically significant higher median immuno- staining intensity in the osteoblasts adjacent to metastatic prostate cancer cells (n = 6 cases, median = 2.0), compared to osteoblasts in normal control bone samples (n = 15 cases, median = 0), (p = 0.0353, Mann Whitney U). Prostate cancer cell lines express both the eEF1A1 and eEF1A2 isoforms eEF1A occurs as two isoforms i.e. eEF1A1 and eEF1A2 with the proteins sharing 92% sequence identity (http://omim.org/entry/ 602959). In order to investigate the expression of both isoforms we performed Western blotting using 11 human prostate cancer cell lines and an osteosarcoma cell line (TE-85), with an antibody directed against the N-terminus of both isoforms. As expected a single immunoreactive band was detected at ,50 kDa in all cell lines tested (Figure 4A), and in the osteosarcoma cell line (data not shown). To investigate further the relative expression of the two isoforms, we performed semi-quantitative reverse transcription PCR using mRNA extracted from LNCaP, PC-3, VCaP and DuCaP prostate cancer cell lines with isoform specific primers. The expression of both the eEF1A1 and eEF1A2 isoforms was detected in all 3 cell lines tested with a relative equal intensity i.e. an expression ratio of 1.0 (Quantity One software, Bio-Rad), (Figure 4B) . The specificity of the PCR primers was confirmed by sequencing the PCR products (Figure 4C). Discussion In an effort to identify leads for potentially useful serum biomarkers for prostate cancer diagnosis and progression, we profiled pooled serum samples from 4 carefully selected groups of patients representing the various stages of prostate cancer development and progression using a 4-plex iTRAQ approach. Following the GO annotations of the 75 proteins identified and quantified ($2 peptides), the majority of these were found to class to diverse biological pathways such as protein metabolism and modification; blood clotting; proteolysis; immunity and defence; complement mediated immunity; blood circulation and gas exchange. Regarding the differentially expressed proteins, some of these have previously been reported as candidate prostate cancer biomarkers such as CRP, alpha-2-macroglobulin, cerulo- plasmin, zinc-alpha-2-glycoprotein, beta-2-microglobulin and fibronectin, which provides confidence to our dataset and provides an independent confirmation of these candidates [31–34]. Of the proteins previously associated with prostate cancer; CRP is an acute phase reactant (APR) protein produced by the liver in response to inflammation. Elevated levels of CRP have been PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e30885