metastatic prostate cancer [40]. Serum B2M levels have been shown to be elevated in patients with metastatic, androgen- independent prostate cancer [31]. Additionally, B2M has functionally been implicated in prostate cancer as its over- expression in cancer cells induced rapid tumour growth in bone, while disrupting B2M signaling by specific small interfering RNA produced a regression of previously established prostate tumours [41]. Thus, B2M may potentially serve as a biomarker for prostate cancer progression and a novel drug target for the treatment of bone metastasis which requires further study. Other candidates up-regulated in one or more of the cancer groups compared with the BPH group were fibronectin 1, afamin, alpha-2-HS-glycoprotein chain B, ceruloplasmin and beta-2- glycoprotein 1 (Figure 2). Interestingly, fibronectin was very recently shown to be amongst the five-protein signature panel with potential for Gleason score prediction [34]. Furthermore, in a recent study fibronectin has been shown to be involved in initiating lung cancer metastasis [42], thus making fibronectin an attractive candidate biomarker and therapeutic target. Another promising candidate identified in our study was Afamin, which was found to be up-regulated in the non- progressing group compared with BPH group, but was down- regulated in the progressing and metastatic disease (Figure 2). Afamin is a member of the albumin gene family expressed by the liver and kidneys, and has been shown to be a specific binding protein for vitamin E. Interestingly, our data is consistent with a previous study which showed a decrease in afamin levels in ovarian cancer where it has been proposed as a candidate biomarker [43]. Thus, the potential of afamin as a candidate prostate cancer biomarker requires further study. eEF1A1, is a member of the elongation factor proteins that normally functions to mediate the selection and binding of the aminoacyl-tRNA to the ribosome during protein synthesis, and ensures translational accuracy. eEF1A1 was of particular interest to us as we had previously shown its levels to be increased in higher metastatic variant prostate cancer cells [14], and another study had reported that down-regulation of eEF1A1 by RNA interference (RNAi) in Du145 cells led to an inhibition of cell proliferation, invasion and migration [30]. Interestingly a truncated form of eEF1A1 known as prostate tumour inducing gene 1 (PTI-1) has been shown to be expressed in prostate carcinoma patient-derived blood samples and proposed to be a sensitive biomarker for prostate cancer [44]. Additionally, PTI-1 was shown to be expressed in prostate cancer tissues but not in BPH or normal prostate tissues [45]. Our finding of increased eEF1A1 expression in osteoblasts in the vicinity of metastatic prostate cells, is in line with previous reports indicating a cross-talk between prostate cancer cells and osteoblasts during bone metastasis [46]. Prostate cancer bone metastasis are typically osteoblastic (i.e. involve excessive bone formation), and it is well known that tumour cells can stimulate osteoblasts to proliferate and differentiate. Thus, eEF1A1 over-expression seen in osteo- blasts may occur as a response to the presence of tumour cells. The increased expression of eEF1A1 in the serum of patients with cancer compared with BPH is an important finding as is suggests that the search for novel cancer biomarkers should encompass not only those factors secreted directly by the cancer cells but also ‘surrogate markers’ produced indirectly as a reaction to the presence of tumour cells. During our analysis we identified and quantified 122 unique proteins (of which 75 were qualified by $2 unique peptides). This is in line with a previous iTRAQ study analysing serum samples with similar MS instrumentation [47]. Although depletion strategies can improve sensitivity for detection of less abundant proteins, this Serum Biomarkers for Prostate Cancer Metastasis approach itself is associated with limitations as the higher abundant proteins are known to act as a ‘sponge’ by complexing with lower molecular weight proteins. For instance, a targeted analysis of alpha-2-macroglobulin binding partners indicated that this highly abundant plasma glycoprotein can bind various cytokines, growth factors and heat shock proteins [48]. Consistent with this, we were unable to detect PSA during our iTRAQ analysis despite it being known to be present at relatively high concentrations in the cancer and metastatic patient groups. PSA is known to complex with alpha- 1-antitrypsin and alpha-2-macroglobulin, both of which were targets for removal by the IgY-14 affinity column [49]. Thus, it is important that future biomarker studies analyze both the bound and flow-through fractions in order to increase the repertoire of disease candidates identified. Although our study identified the differential expression of a number of proteins, it is becoming apparent that a single biomarker is unlikely to provide the required sensitivity and specificity, due to the heterogeneity and dynamic nature of prostate cancer [50]. Thus, it has been proposed that accurate disease diagnosis and prognosis are likely to depend on the measurement of a panel of biomarkers, perhaps utilising emerging multiplex technologies which could fast forward these panels into the clinic. In support of this, our data has shown that levels of certain combinations of proteins could be seen to fluctuate as the disease developed and progressed. Certain proteins were seen to be differentially expressed either individually, as pairs or as panels (Figure 2). Interestingly, a previous study has reported that by using a combination of proteins (complement component 4a (C4a) and protein C inhibitor), a statistically significant value for predicting prostate cancer recurrence was demonstrated in men who underwent prostatectomy [51]. Thus, our study adds weight to other published studies demonstrating the importance of evaluating pairs or panels of carefully selected proteins to increase the diagnostic and prognostic accuracy of cancer. To our knowledge, our study appears to be the first iTRAQ based approach aimed at identifying leads for potentially useful biomark- ers of progression and metastasis in prostate cancer, using patient serum. If the findings are validated in a larger cohort of patients, then the detection of eEF1A1 in the serum of patients with prostate cancer at the time of their initial diagnosis, may be able to predict the likelihood of disease progression and those patients be offered radical treatment options at an early stage of their disease. With a number of biomarker studies already conducted over the years and others in the pipeline, what is becoming evident is that in order for a biomarker (or panel of biomarkers) to reach routine clinical use it must pass through five phases of development [52,53]. Thus, our study represents one of the initial steps (‘exploratory phase’) along this process. Many of the candidates identified in our and other studies await rigorous clinical validation using large cohorts of patient samples, together with robust long- term clinical and pathological information in subsequent phase 2– 5 studies, as proposed by Pepe et al., [52,53]. In addition to the leads identified, what has emerged is that due to the sizable inflammatory response to the presence of a tumour, future biomarker identification strategies could benefit by the pre- fractionation of APR proteins from the sera of cancer patients in addition to the removal of common highly abundant serum proteins, so that the cancer proteome can be mined even more deeply. The panel of proteins identified, including eEF1A1 warrant further investigation and validation. Supporting Information Figure S1 Metacore pathway analysis of proteins dif- ferentially expressed between metastatic and progress- PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e30885