change of eight and the immunofluorescence analysis also showed a highly elevated expression. This conclusion is supported by the spectral counting of the MudPIT data, wherein FASN was represented by 417 spectra in the androgen-treated sample compared to only 45 spectra in the control sample. Such ratio compression, although seen in other studies, was more prominent in our iTRAQ MALDI- TOF/TOF data compared to other quantitative MS platforms such as ICAT [9]. This difference may be explained by a wider mass tolerance window for selection of peptide ions for MS/MS fragmentation in the ABI 4800 TOF/ TOF mass analyzer used here [29]. We believe that this compression in ratio could partly explain the poor overlap between the sets of androgen-regulated proteins nominated from each of the two MS platforms. Additionally, factors such as differences in peptide processing (labeled versus unlabeled), peptide fractionation (on-line and offline), MS data acquisition (ion trap versus TOF/TOF mass analyzer), data normalization (ratios of spectral counts versus ratios of iTRAQ label peak intensities), and the properties of peptides selected for MS/MS sequencing could play pivotal roles in the lack of global concordance. These factors, in addition to the biological variations such as the multiple splice forms for a single accession could also have contributed to those three proteins that showed discordant trends between the two platforms observed. Indeed, it has been established that different proteomic platforms show substantial differences in the physico-chemical properties of the identified peptides [30]. Thus, the strategy of using two different mass spectrometers, which differ both in their ionization source and principle of peptide detection, allowed us to build an additive list of proteins and gain deeper insight into the androgen-regulated proteome. With higher level enrichment analyses, our study showed that subsets of proteins, nominated by each of the two platforms, map to similar concepts including those that are androgen-derived and prostate cancer-derived. With a stringent threshold setting for the spectral count based semi- quantitation, the MudPIT experiment had nominated only two aaRSs candidates as androgen regulated. However, due to the nomination of higher number of aaRSs from iTRAQ experiment, molecular concept network with combined data set showed a set of interconnected aaRS concepts with significant scores (Figure 4). Even more intriguing, however, was that each of the two independent lists of proteins, when analyzed separately using MCM analysis, enriched for concepts that were known to be associated with prostate cancer progression but were not identified in the analysis of the combined dataset (Figure 4, Supplementary Figure S2). An example of this was the enrichment of ETS (ETV1 and ERG) over-expression concepts by the androgen-regulated proteins nominated by MudPIT analysis. This was interesting in the context of earlier reports that describe the existence of recurrent gene fusions involving ETS transcription factors to be pathonomonic to prostate cancer development [31,32]. These gene fusions have been shown to bring ETS transcription factors under androgen control, and ETV1 (chromosome 7p21) is known to be over-expressed by androgen through its rearrangement to 14q13.3–14q21.1 region of chromosome 14 in LNCaP cell line studied here [31]. As the number of ETS regulated proteins identified in MudPIT was sufficient to map the ETS over- expression concept, lack of similar proportion of this class of proteins in iTRAQ platform possibly resulted in the disappearance of the same concept in the combined analysis. Thus, use of multiple mass spectrometric platforms helps to elucidate multiple roles of androgen action. In addition to previously known androgen-regulated biological processes associated with prostate cancer progression, such as Proteomics of Prostate Cancer NDRG1 [33] that helped to validate our proteomic study, our list of androgen-regulated proteins was enriched for aminoacyl tRNA synthetases. Several aaRSs that are known to play a pivotal role in protein biosynthesis were included in this concept. This finding was interesting in the context of earlier gene expression-based predictions from our laboratory: an increase in protein synthesis during prostate cancer progression driven by actions of ETS transcription factors [17]. These observations lead us to the validation of this finding at multiple levels; starting at the level of regulatory controls with a combination of matched gene expression and chromatin immunoprecipitation assays. Finally, we used Western for orthogonal verification of the MS findings on the LNCaP cells, and showed that indeed the class of aaRSs are altered both at the transcript and protein level in prostate cancer. Materials and Methods Materials Synthetic androgen R1881 (methyltrienolone) was purchased from Perkin Elmer (Boston, MA). A 10 mM R1881 stock was prepared in 100% ethanol and stored in 220uC. RPMI 1640 medium and fetal bovine serum was purchased from Invitrogen (Carlsbad, CA). Sequencing grade modified trypsin was from Promega (Madison, WI). Sample preparation reagents formic acid, trifluoroacetic acid, and HPLC solvents were from Fisher Scientific (Waltham, MA). Mass spectrometriy grade water was from Burdick and Jackson (Muskegon, MI, USA). Antibodies used were: KLK3 (kallikrein 3, rabbit polyclonal, DakoCytomation Carpinteria, CA ), NDRG1 (N-myc downstream regulated 1, goat polyclonal, Santa Cruz Biotechnology Inc., Santa Cruz, CA.), FASN (fatty acid synthase, mouse monoclonal, BD Transduction Labs, Franklin Lakes, NJ), FKBP5 (FK506 binding protein 5, mouse monoclonal, BD Transduction Labs), AR (mouse mono- clonal-AR441, Lab Vision, Fremont, CA), GARS (glycyl-tRNA synthetase, rabbit polyclonal, Abcam, Cambridge, MA), WARS (tryptophanyl-tRNA synthetase, rabbit polyclonal, Abcam), KARS (lysyl-tRNA synthetase, rabbit polyclonal, Bethyl Laboratories, Montgomery, TX), b-tubulin (rabbit polyclonal, Santa Cruz), b- actin (rabbit polyclonal, Cell Signaling Technology, Inc., Danvers, MA). LNCaP cells were purchased from American Type Culture Collection (University Boulevard, Manassas, VA). Prostate spec- imens comprising adjacent normal, localized prostate cancer, and metastasis tissue utilized for immunoblot experiments were from the University of Michigan Rapid Autopsy Program which is part of University of Michigan Prostate Cancer Specialized Program of Research Excellence (SPORE) tissue core. Cell culture and sample preparation LNCaP (ATCC number: CRL-1740TM) cells were grown to 70% confluence in RPMI 1640 medium containing 10% FBS under 5% CO2 and 90% humidity. Phenol-red free RPMI 1640 supplemented with charcoal-stripped FBS was used to deplete endogenous androgen for two days. R1881, solubilized in ethanol, was added at a final concentration of 1 nM for 48 h, and ethanol was used as vehicle control in parallel experiments. Cells were washed with tris-buffered saline (TBS) and harvested using a cell scraper. MudPIT: cells were lysed in RapiGestTM SF cell lysis buffer as per the manufacturer’s procedure (Waters Corporation, Milford, MA). Protein concentrations in the supernatant were estimated using Bradford assay (BioRad Laboratories, Hercules, CA). Proteins were reduced using 2 mM tris(2-carboxyethyl) phosphine hydrochloride at 60uC and alkylated by 5 mM Iodoacetamide at 37uC. Sequencing grade trypsin (Promega, Madison, WI), at the ratio of 1:50, was added for digestion over- PLoS ONE | www.plosone.org 8 September 2009 | Volume 4 | Issue 9 | e7075