Sage‐N's Sorcerer 2 provides an integrated data analysis system for comprehensive protein identification and characterization. It runs on a proprietary version of SEQUESTR, the most widely used search engine for identifying proteins in complex mixtures. The protocol presented here describes the basic steps performed to process mass spectrometric data with Sorcerer 2 and how to analyze results using TPP and Scaffold. The unit also provides an overview of the SEQUESTR algorithm, along with Sorcerer‐SEQUESTR enhancements, and a discussion of data filtering methods, important considerations in data interpretation, and additional resources that can be of assistance to users running Sorcerer and interpreting SEQUESTR results. Curr. Protoc. Bioinform. 28:13.3.1‐13.3.21. © 2009 by John Wiley & Sons, Inc.

Androgen-independent (AI)2 prostate cancer (CaP) typically develops from the selective outgrowth of tumor cells to castrate levels of testosterone in response to androgen-deprivation therapy (1). AI CaP cells have evolved different strategies for overriding the androgen-dependent (AD) growth and survival characteristics of early stage, organ-confined CaP or early stage metastatic CaP (2). Aberrant AR activation is the primary mechanism for the growth and survival of AI CaP in response to castrate levels of androgen (2). With the exception of prostatic small cell neuroendocrine carcinoma (3), the most parsimonious model of AI CaP arises from the inappropriate activation of AR-dependent cell growth and survival pathways. Unfortunately, this model is incomplete because examples of AR-independent CaP do exist. Most notably, the well established human CaP cell lines DU145 and PC3, which were derived from brain and bone metastases, respectively (4, 5), are supposedly devoid of AR mRNA and protein (6, 7) and thus represent bona fide models of AR-independent CaP. Interestingly, this classification was recently called into question as detectable levels of AR mRNA and protein were observed in both DU145 and PC3 cells (8). More importantly, several studies have demonstrated low AR activity in PC3 cells, suggesting these cells also utilize AR-dependent mechanisms for growth and survival similar to other established AD CaP cell lines (e.g. LNCaP, 22Rv1, and C4-2) (8 -11).AR displays variable immunoreactivity in individual tumor cells of AI CaP, with cells showing a strong, weak, or undetectable AR signal (12). These results illustrate the heterogeneity of AR expression in AI CaP and suggest that both AR-dependent and AR-independent mechanisms of growth and survival are present in AI CaP. Practically speaking, determining whether AI CaP utilizes AR-dependent or AR-independent mechanisms for growth and survival is a controversial and arduous endeavor that has profound clinical ramifications on how to treat advanced stage, AI CaP. Here we have investigated whether DU145 and PC3 cells are representative models of AR-independent CaP by testing if they respond to androgens or require AR expression for cell growth in vitro. We show that DU145 and PC3 cells are androgen-responsive and require AR for optimal growth in vitro, thus demonstrating each cell line is a bona fide model of androgen-responsive, AR-dependent CaP. We show AR gene transcripts in DU145 and PC3 cells harbor nucleotide transitions suggesting the AR pre-mRNA is the target of multiple RNA editing enzymes. We propose that RNA editing enzymes are modulators of AR activity through the introduction of loss-of-function or gain-of-function mutations into AR gene transcripts in advanced stage AI CaP.

Purpose To compare vitreous biopsy methods using analysis platforms employed in proteomics biomarker discovery. Methods Vitreous biopsies from 10 eyes were collected sequentially using a 23-gauge needle and a 23-gauge vitreous cutter instrument. Paired specimens were evaluated by UV absorbance spectroscopy, SDS-PAGE, and mass-spectrometry (LC-MS/MS). Results The total protein concentration obtained with a needle and vitrectomy instrument biopsy averaged 1.10 mg/ml (SEM = 0.35) and 1.13 mg/ml (SEM = 0.25), respectively. In eight eyes with low or medium viscidity, there was a very high correlation (R2 = 0.934) between the biopsy methods. When data from two eyes with high viscidity vitreous were included, the correlation was reduced (R2 = 0.704). The molecular weight protein SDS-PAGE profiles of paired needle and vitreous cutter samples were similar, except for a minority of pairs with single band intensity variance. Using LC-MS/MS, equivalent peptides were identified with similar frequencies (R2 ≥ 0.90) in paired samples. Conclusion Proteins and peptides collected from vitreous needle biopsies are nearly equivalent to those obtained from a vitreous cutter instrument. This study suggests both techniques may be used for most proteomic and biomarker discovery studies of vitreoretinal diseases, although a minority of proteins and peptides may differ in concentration.

We have developed a novel androgen receptor (AR) expression system in the 293 human embryonic kidney cell line that recapitulates AR biochemical activity as a steroid hormone receptor in prostate cancer cells. We used this system to identify putative AR-binding proteins in the cytosolic and nuclear compartments of mammalian cells using a large scale co-immunoprecipitation strategy coupled to quantitative mass spectrometry. For example, the heat shock 70 and 90 chaperones, which are known regulators of steroid hormone receptor, were identified as AR-binding proteins. AR purification enriched for proteins involved in RNA processing, protein transport, and cytoskeletal organization, suggesting a functional link between AR and these protein modules in mammalian cells. For example, AR purification in the nuclear compartment led to the specific enrichment of ␣-actinin-4, clathrin heavy chain, and serine-threonine protein kinase C ␦. Short interfering RNA knockdown studies and co-transcriptional reporter assays revealed that clathrin heavy chain possessed co-activator activity during AR-mediated transcription, whereas ␣-actinin-4 and protein kinase C ␦ displayed both co-activator and co-repressor activity during AR-mediated transcription that was dependent upon their relative expression levels. Lastly immunohistochemical staining of prostate tissue showed that ␣-actinin-4 levels decreased in the nucleus of high grade cancerous prostate samples, suggesting its possible deregulation in advanced prostate cancers as previously observed in late stage metastatic breast cancers. Taken together, these findings suggest AR binds to specific protein modules in mammalian cells and that these

Increasing evidence suggests that the disruption of androgen-mediated cellular processes, such as cell proliferation and cell differentiation, contributes to the development of early-stage androgen-dependent prostate cancers. Large-scale mRNA profiling experiments have paved the way in identifying androgen-regulated gene networks that control the proliferation, survival, and differentiation of prostate cancer cells. Despite these extensive research efforts, it remains to be determined whether all androgen-mediated mRNA changes faithfully translate into changes in protein abundance that influence prostate tumorigenesis. Here, we report on a mass spectrometry-based quantitative proteomics analysis that identified known androgen signaling pathways and also novel, androgen-sensitive microsome-associated proteins and protein networks that had not been discovered by gene network studies in human LNCaP prostate cancer cells. Androgen-sensitive microsome-associated proteins encoded components of the insulin growth factor-1 (IGF-1), phosphoinositide 3-kinase (PI3K)/AKT, and extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling pathways. Further bioinformatic analyses showed most of the androgen-sensitive microsome-associated protein networks play roles in cell proliferation and differentiation. Functional validation experiments showed that the androgen-sensitive microsome-associated proteins Janus kinase 2 (JAK2) and I-kappa B kinase complex-associated protein (IKAP) modulated the expression of prostate epithelial and neuronal markers, attenuated proliferation through an androgen receptor-dependent mechanism, and co-regulated androgen receptor-mediated transcription in LNCaP cells. Further biochemical analyses showed that the increased proliferation in JAK2 knockdown cells was mediated by activation of the mammalian target of rapamycin (mTOR), as determined by increased phosphorylation of several downstream targets (p70 S6 kinase, translational repressor 4E-BP1, and 40S ribosomal S6 protein). We conclude that the expression of microsome-associated proteins that were previously implicated in the tumorigenesis of prostate epithelial cells is strongly influenced by androgens. These findings provide a molecular framework for exploring the mechanisms underlying prostate tumorigenesis and how these protein networks might be attenuated or potentiated in disrupting the growth and survival of human prostate cancers.