Proteomics of Prostate Cancer Figure 4. Molecular concepts of androgen up-regulated proteins from two mass spectrometry platforms. Network view of molecular concepts or sets of biologically related genes enriched in androgen up-regulated protein set obtained through Molecular Concepts Mapping (MCM) analysis. Each node represents a biological concept; the node size is proportional to the number of genes in each concept. Each edge represents a statistically significant enrichment. P-value of each concept and the MCM number of the concept are given in brackets. The most enriched concept is indicated by a thick edge. Red colored edges indicate enrichment of known prostate cancer-specific and androgen-regulated concepts. Interconnected aminoacyl tRNA synthetase concepts are indicated in blue edges at the bottom of the network. doi:10.1371/journal.pone.0007075.g004 proteomic platforms: quantitative iTRAQ MALDI-TOF MS and semi-quantitative MudPIT Ion Trap MS. Each of the platforms independently identified in excess of 850 high confidence proteins, yielding a total of 1306. The gain in cumulative protein counts is consistent with earlier reports describing the advantage of interrogating global proteomes using complementary mass spec- trometry platforms [21,27]. We were able to identify 126 proteins that were up-regulated upon androgen treatment. Included were known targets of androgen action: KLK3, FKBP5, FASN, AGR2 (anterior gradient homolog 2), SORD (sorbitol dehydrogenase), NDRG1, ACSL3 and FOLH1 (folate hydrolase 1). Additionally, nested in the compendia of differentially regulated proteins, were novel targets of androgen action that included PCID1 (PCI domain containing 1), RAB10 (ras-related GTP-binding protein RAB10), and PEA15 (phosphoprotein enriched in astrocytes 15). An additional advantage of using the ion trap- based profiling strategy in tandem with TOF-TOF based quantitative profiling was that data from the former helped offset the loss of resolution resulting from iTRAQ ratio compression seen in the latter. This permitted the use of lower threshold level from iTRAQ-based quantitation for perturbation analysis. The ratio compression has been widely reported by others, e.g. [28], and is well illustrated by the direct comparison of iTRAQ ratios for androgen-regulated proteins with the corresponding expression as assessed by immunoblot analysis (Figure 3). As an example, protein FASN was identified with 17 peptides; among them the most differen- tially expressed peptide had an iTRAQ ratio of 2.79. Notably, averaging all iTRAQ ratios for 17 peptides resulted in a further compressed protein iTRAQ ratio of 1.85, while densitometry- based analysis of its expression by immunoblot revealed fold PLoS ONE | www.plosone.org 6 September 2009 | Volume 4 | Issue 9 | e7075