Proteomics of Prostate Cancer Figure 2. Total and androgen-regulated proteins identified from two mass-spectrometric platforms. A) Venn diagram showing the number of total and the sub-sets of androgen-regulated proteins identified from iTRAQ MALDI TOF- MS/MS analysis. A minimum threshold of 1.25 SD from the global mean in both androgen-treated samples and not less than 1.5 SD in one of the androgen treated samples was set. B) Venn diagram showing the numbers of total and androgen-regulated LNCaP proteins identified in MudPIT (ESI ion trap- MS/MS) analysis. Proteins shown in brackets were not considered for differential analysis because of their lower spectral counts. C) Venn diagram showing the total number of proteins identified from two mass-spectrometric platforms and their overlap. D) Venn diagram showing the total number of androgen up-regulated proteins identified from two mass-spectrometric platforms and their overlap. E. Venn diagram showing the total number of androgen down-regulated proteins identified from two mass-spectrometric platforms and their overlap. doi:10.1371/journal.pone.0007075.g002 Antigen[22], iTRAQ ratio 1.44; MudPIT ratio 11:0). A direct quantitative comparison of proteins identified on both the MS platforms showed a high positive correlation (Rˆ2 = 0.92) for three proteins (ACSL3, FASN, and AARS; see Supplementary figure S1). These findings independently validated our normalization procedure and the thresholds that were used to build the combined list of androgen-regulated proteins. Further, immuno- blot analysis was employed to confirm the androgen-regulation of a subset of up-regulated proteins (Figure 3A). Notably, in addition to validating our mass spectrometry results, the immunoblot analysis revealed significant compression in the fold change of protein expression delineated by iTRAQ experiment (Figure 3A). A similar orthogonal verification performed for the androgen up- regulated protein fatty acid synthase by immunofluorescence- microscopy is shown in Figure 3B. Analysis of androgen up-regulated biological processes and pathways by MCM analysis To gain insight into the functional consequence of androgen- action, A large-scale association analysis was performed comparing our androgen up-regulated protein data set of 126 proteins with each of the 13364 concepts in the MCM [23]. The procedure involved in selection of significant molecular concepts is described in ‘Materials and Methods’ section. The enrichment network resulted in the MCM analysis with the combined set of androgen up-regulated proteins is presented in Figure 4. Importantly, the analysis showed enrichment PLoS ONE | www.plosone.org 4 September 2009 | Volume 4 | Issue 9 | e7075