Figure 3. Androgen-induced expression of LNCaP proteins. A) Expression fold change of androgen-regulated proteins in mass- spectrometric quantitation compared to immunoblot assessment. Shown are the immunoblot bands and their intensities for androgen up-regulated proteins and their calculated intensity ratios as well as iTRAQ ratios and MudPIT spectral counts for the corresponding proteins. Beta-tubulin is used as a sample loading control. B) Immunofluorescence staining of AR (Red) and FASN (Green) following vehicle (ethanol) and 1 nM R1881 treatments for 48 h on LNCaP cells. Treatment was given after 48 h of androgen deprivation. doi:10.1371/journal.pone.0007075.g003 of known prostate cancer-specific and androgen-regulated concepts, thus providing a validation of the proteomic data generated in this work (Figure 4, red colored edges). Nested within these previously known prostate-specific concepts was one that described a set of co- enriched genes down-regulated in prostate cancer patients after post- neoadjuvant (anti-androgen) therapy [17]. Additional concepts of significance included those for aminoacyl-tRNA biosynthesis and ER to Golgi transport (both GO biological process), aminoacyl-transfer RNA synthetase Class II (InterPro), and RSRFC4 (MEF2A) and NKX3A (both Transfac). We were intrigued by those concepts that referred to aminoacyl tRNA synthetases (KEGG pathway: http:// www.genome.jp/kegg/ [24,23]), as they potentially reflect an elevation in amino acid utilization and thus an increase in downstream protein biosynthesis upon androgen treatment (Figure 4, blue colored edges). This observation is in agreement with our earlier gene expression-based prediction of increased protein biosynthesis as one of the hallmarks during prostate cancer development [17]. Elevated aaRS activity was also among the significant concepts enriched by matched transcriptome data for androgen treated LNCaP cells (data not shown). Thus, such observations led us to select the aminoacyl tRNA synthetase concept for further analyses. Proteomics of Prostate Cancer Androgen regulation of aminoacyl tRNA synthetases There were 7 proteins in the aaRS category that exceeded the set threshold in the combined list of proteins defined using both MS platforms: aaRS for alanine (AARS), phenylalanine (FAR- SLA), glycine (GARS), histidine (HARS), asparagine (NARS), threonine (TARS) and tryptophan (WARS). Of these only AARS and HARS were nominated by the MudPIT platform (Table S3) while AARS and the rest were nominated by the iTRAQ study (Table S1). Although, the total numbers of aaRS identified in iTRAQ and MudPIT experiments were 14 and 13, respectively, several of these proteins were identified with less than four peptides in MudPIT experiment making it less reliable for their spectral count based quantitation. This also indicates the low abundant nature of this critical class of proteins. Therefore, we focused to further analyze the iTRAQ identified aaRSs and examined if there is compression in expression ratio despite their elevated expression. Of the 14 aaRSs identified in the iTRAQ study, six had iTRAQ ratios 1.2 and above, and three of them had ratios 1.1 and above in both androgen-treated (115 and 117) replicate samples. Over-expression of majority these (nine of 14) aaRSs is depicted in a heat map (Figure 5A). A subset of these iTRAQ identified aaRSs (GARS, KARS, and WARS) were validated using androgen-treated LNCaP cells by immunoblot analysis (Figure 5C). Further, we examined their transcript levels using matched gene expression data for androgen-treated LNCaP cells (see Materials and Methods section). Nine of the fourteen proteins identified by our protein profiling were elevated at the transcript level across independent gene expression experiments (Figure 5B). This revealed potential regulation of these proteins by transcrip- tional activation by androgen. These data also support the observation of compression in fold change of protein expression in iTRAQ ratios. Androgens mediate their positive effect on transcription by binding to androgen receptor, which in turn binds to androgen response elements on the promoters of target genes [25]. Thus, we proceeded to examine androgen binding to the promoters of aminoacyl tRNA synthetases using chromatin immunoprecipita- tion (ChIP, see Materials and Methods). Figure 5E shows representative example of increased occupancy of AR on the promoter of GARS and KARS confirming androgen regulation of its expression at the transcript level. We next extended our cell line observation to prostate cancer tissue specimens. First, we used the ONCOMINE database (www. oncomine.org) to perform a meta-analysis for aminoacyl tRNA synthetase transcripts across multiple prostate cancer studies [26]. A subset of aaRSs was found to be up-regulated in localized prostate cancer samples in more than one study (Figure 5F). This observation was confirmed using immunoblot analysis of prostate- derived samples that included benign adjacent, localized prostate cancer, and metastatic disease. Importantly, four of the five localized PCa and four of the five metastatic PCa showed elevated expression of KARS and GARS when compared to three of the four benign samples tested (Figure 5D). These lend credence to a possible role of androgen-regulated aminoacyl tRNA synthetases action during prostate cancer progression. Discussion To delineate the biological processes and pathways that are dysregulated by androgen in prostate cancer, we adopted a strategy of global MS-based proteomic profiling coupled to enrichment-based pathway mapping in the androgen-responsive prostate cancer cell line LNCaP. We profiled the proteome of LNCaP cells with and without androgen treatment using two PLoS ONE | www.plosone.org 5 September 2009 | Volume 4 | Issue 9 | e7075