and progression i.e. as the cancer developed from non-malignant epithelium and progressed to locally advanced and metastatic disease (Figure 2). The lists of differences are based on comparisons between the non-progressing versus BPH group; progressing versus non-progressing group; and metastatic versus progressing cancer groups. From Figure 2, it is apparent that individual proteins, ‘pairs’ of proteins and ‘panels’ of proteins (defined as $3 proteins), were seen to be differentially expressed at certain stages of disease development and progression. For instance, individual proteins such as alpha-2-macroglobulin, lumican and serum amyloid P-component were seen to be differentially expressed between the non-progressing versus the BPH group. Other proteins such as beta-2-glycoprotein 1 and somatomedin-B (blue font), were both seen to be relatively decreased as a ‘pair’ in progressing versus non-progressing samples, while both Apolipoprotein A-1V and Complement component 4B were seen to be relatively increased in expression in metastatic samples versus progressing samples (orange font). Additionally, two ‘panels’, were seen to be altered as the disease developed and progressed. The first panel comprised of 3 proteins: afamin, alpha-2-HS-glycoprotein chain B and fibronectin 1 (shown in red font), and was seen to be relatively increased in expression in the non-progressing versus the BPH group, but decreased as the cancer progressed and remained relatively low as Serum Biomarkers for Prostate Cancer Metastasis the cancer metastasized. The second panel comprised of 7 proteins: alpha-1-antichymotrypsin; cDNA FLJ55673, highly similar to complement factor B; cDNA FLJ54228, highly similar to leucine- rich alpha-2-glycoprotein; cDNA FLJ58564, highly similar to plasma protease C1 inhibitor; Ceruloplasmin, Complement C5 and Complement component C9b (green font), and were seen to be relatively decreased in expression in the non-progressing group compared with the BPH group, and relatively increased in expression as the cancer progressed i.e. were relatively increased in the progressing group and remained elevated in the metastatic group. Interestingly, eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), (a.k.a EF-Tu), was seen to show significant increased expression in non-progressing cancer relative to BPH, and its expression was further increased with disease progression, and was maintained during metastasis (Table S2 and Figure 2). eEF1A1 was the top hit following the blastp search of the VETGVLKPGMVVTFAPVNVTTEVK peptide identified in the serum samples. Comparison of the full length amino acid sequence of eEF1A1 with its isoform eEF1A2, indicated that the peptide sequence was unique to eEF1A1. The corresponding peptide in eEF1A2 differs by a single amino acid where valine is substituted by isoleucine. Since these amino acids have a 14 Da difference in molecular mass we could confidently assign the identified peptide to correspond to the eEF1A1 isoform. Figure 2. Proteins showing significant differential expression (up-regulated and down-regulated) according to disease progression. The list of differentially expressed proteins shown are based on comparisons between non-progressing versus BPH; progressing versus non-progressing and metastatic versus progressing groups. Note the differential expression of proteins either individually (black font), as pairs (blue, orange and purple fonts), or as a panel ($3 proteins, green and red fonts). (*) = identified as a single high confidence peptide. doi:10.1371/journal.pone.0030885.g002 PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e30885