reagent followed by 2D liquid chromatography (LC) MALDI- TOF MS/MS analysis on an ABI 4800 MALDI tandem time-of flight (TOF/TOF) instrument [11] and the other a label-free approach based on spectral counting [12,13,14,15] using Multi- dimensional Protein Identification Technology (MudPIT) [16] with Electrospray Ionization (ESI)- ion trap MS/MS on a linear ion trap (LTQ) instrument. Second, we elucidated the proteomic alterations in the context of functional pathways using Molecular Concepts Mapping (MCM, also called Oncomine Concepts Mapping (OCM)) [17], to gain deeper insight into the biology of androgen action in prostate cancer. Notably, such combinatorial analyses revealed a striking increase in the level of aminoacyl tRNA synthetases (aaRS) that could underlie the increase in protein biosynthesis predicted in prostate cancer by various gene expression studies. Results We employed an integrative strategy (Figure 1) to understand androgen action in prostate cancer. In brief, we performed mass spectrometry-based proteome profiling of androgen-deprived and androgen-stimulated LNCaP cells. Trypsin-digested proteins were identified and quantified using two mass spectrometric platforms: iTRAQ MALDI-TOF MS/MS and MudPIT ESI- ion trap MS/ MS. The data from each of these platforms were normalized independently and combined to generate a list of androgen regulated proteins. The androgen regulated proteome was interrogated for biological associations using Molecular Concepts Mapping (MCM). From the many molecular concepts, including the androgen-regulated and prostate cancer concepts that were enriched by our androgen up-regulated data set, the concept describing elevated aminoacyl tRNA synthetases (aaRSs) was selected for further examination. A subset of these proteins was validated by immunoblot analysis. Using transcriptomic profiling and chromatin immunoprecipitation, we showed that androgen drives the expression of aaRSs at the transcript level. Finally, we showed the existence of the elevated aaRS niche during prostate cancer progression using clinical specimens. Identification and relative quantitation of LNCaP proteins by iTRAQ MALDI-TOF MS/MS Using iTRAQ MALDI-TOF MS/MS platform, we identified 904 proteins with high confidence scores as determined with SEQUEST [18], PeptideProphet [19] and ProteinProphet [20] computational tools. Of these, 879 were quantified post-data normalization (Figure 2A), with the overall protein identification FDR of less than 1 % (see Materials and Methods for detail). Procedurally, the androgen proteome was assessed by iTRAQ using two independent biological replicates. A double duplex iTRAQ experiment was carried out wherein androgen-treated samples in each replicate were labeled with the 115 and 117 isobaric tags while their corresponding control samples were labeled using the 114 and 116 isobaric tags. In order to derive a threshold to define protein expression changes, we normalized the relative protein ratio for each of the androgen-treated samples and one of the controls (labeled with isobaric tag 116) to the protein expression in the control sample labeled with the 114 isobaric tag. The distribution of all protein ratios for the androgen-treated sample (tag 115) versus control (tag 116) is shown in Supplemen- tary Figure S1A. More than 80% of proteins had ratios within the range of 0.8–1.2. The median-centered normalization resulted in a mean close to unity for all samples, and the standard deviation of the control replicate (tag 116, SD = 0.16) was used as a scale to set the threshold ratio for differentially expressed proteins. Accord- Proteomics of Prostate Cancer ingly, changes with a minimum threshold of 1.25 SD (ratio of 1.2 for up-regulated and 0.83 for down-regulated) were considered as significant (see Materials and Methods for details). The lists of all 70 androgen up-regulated and 39 down-regulated proteins identified from this analysis are given in Supplementary Table S1 and Supplementary Table S2, respectively. Semi-quantitative comparison of proteins using MudPIT analysis Using MudPIT ESI- ion trap MS/MS proteomic profiling approach 857 proteins were identified with the minimum protein probability score of 0.9 (estimated FDR of less than 1%); 67% (574) of them were identified in common between control and androgen-treated samples, while 126 proteins and 157 proteins were identified only in control and androgen-treated samples, respectively (Figure 2B). In addition to determining the presence or absence of proteins, we performed a statistical analysis to compare the relative spectral counts for proteins in each treatment. The total number of spectra from either control or androgen-treated data was independently normalized, and the normalized cumulative spectral counts of all the peptides for each protein were used as a measure of protein abundance. Based on the statistical analysis of data, proteins were designated as differentially expressed if they were identified only in either the control (down-regulated) or androgen treated sample (up-regulat- ed) with four or more peptides. For proteins identified in both samples, normalized spectral count ratios of two standard deviations above or below the mean of all proteins in that group (which corresponds to a four-fold change) was used as cut-off (see Materials and Methods; see Supplementary Figure S1B for distribution of the normalized spectral count ratios for proteins identified in control and androgen-treated samples). Based on these criteria, 65 and 57 proteins were designated as androgen up- regulated and down-regulated, respectively (Supplementary Table S3, Supplementary Table S4). Combined list of differentially expressed proteins identified from the two MS platforms With the understanding that the list of peptides profiled by mass spectrometry is influenced by the ionization source and the mass analyzer [21] (in this case MALDI- TOF vs. ESI-ion trap), we combined the protein lists derived from the two MS platforms to derive a composite list. This was facilitated by the fact that the two platforms identified similar numbers of high confidence proteins (857 and 879 proteins from MudPIT and iTRAQ, respectively, with comparable FDR of less than 1 %). As a first step in the process, non-redundant proteins were culled from each experi- ment by converting their IPI accession numbers to gene IDs. Three proteins (lectin, mannose-binding 2, LMAN2; CLPX, caseinolytic peptidase X homolog; and 40S ribosomal protein S9, RPS9) that showed discordant trends in their expression between the two platforms were removed. These procedures yielded a total of 844 and 875 proteins, respectively, for MudPIT and iTRAQ data sets (Figure 2B, 2A). Together 1306 proteins were observed in the combined dataset (Figure 1 and Figure 2C). Of these, 126 unique proteins were elevated upon androgen treatment. Notably, 9 of these androgen-up regulated proteins overlapped between the two proteomic platforms (Figure 2D): KLK3, ACSL3, FASN, FKBP5, AARS (alanyl-tRNA synthetase), FDFT1 (farnesyl- diphosphate farnesyltransferase 1), UAP1 (UDP-N-acteylglucosa- mine pyrophosphorylase 1), ENDOD1 (endonuclease domain containing 1), and NDRG1. The combined down-regulated list contained 95 proteins, and one (HNRPL, heterogeneous nuclear PLoS ONE | www.plosone.org 2 September 2009 | Volume 4 | Issue 9 | e7075