Quantitative proteomic analysis of proteins released by neoplastic prostate epithelium.

Prostate cancer is unusual among neoplasms in that it may be diagnosed at a curable stage through detection of a protein in serum, the serine protease prostate-specific antigen (PSA). PSA is secreted by both normal and neoplastic prostate epithelial cells in response to androgenic hormones and has found widespread use in cancer screening. Because PSA screening is controversial due to sensitivity and specificity issues, efforts continue to focus on the identification and characterization of additional markers that may be used for diagnostic and therapeutic purposes. In this study, we report the application of quantitative proteomic techniques that incorporate isotope coded affinity tag reagents and tandem mass spectrometry to comprehensively identify secreted and cell surface proteins from neoplastic prostate epithelium. LNCaP cells, a prostate tumor-derived cell line that secretes PSA in response to androgen exposure, were grown in a low protein-defined media under androgen-stimulated (A+) and -starved (A-) conditions. Proteomic analysis of the media identified in excess of 600 proteins, 524 of which could be quantified. Nine percent of the proteins had A+/A- ratios > 2.0, including PSA, and 2.5% had ratios < 0.5. A subset of these androgen-regulated proteins appeared to be expressed in abundance. Of these, selected mass spectrometry observations were confirmed by Western analysis. The findings suggest that androgen-mediated release of proteins may occur through the activation of proteolytic enzymes rather than exclusively through transcriptional or translational control mechanisms. On the basis of their known functional roles, several of the abundant androgen-regulated proteins may participate in the progression of neoplastic epithelial cell growth and should be considered as potential serum markers of neoplastic prostate diseases.

Identification of phosphorylation sites using microimmobilized metal affinity chromatography.

One of the most important roles that mass spectrometry (MS) has played in the late twentieth and early twenty-first centuries has been to assist in the growth of knowledge of dynamic phosphorylation events. Not only has MS allowed researches to pinpoint the site of phosphorylation, but it has also enabled them to identify the kinase/phosphatase pairs responsible for regulation of a specific modification as well as to follow the functional consequences of the observed phosphorylation events on the biology of the system. For phosphorylation analysis, the important contribution of MS has been critical but not definitive. There are numerous methods that have been applied with success, yet none are generally applicable to all analyses. So, for the time being, researchers in the field must select from a panel of methods to find (de)phosphorylation events. In the work described in this chapter, a collection of integrated methods are presented. A detailed account is provided for phosphorylation capture via on- and off-line immobilized metal affinity chromatography (IMAC). This is followed by a suite of useful strategies for discovery of phosphorylation positioning through sequence determination by phosphate-specific diagnostic ion scans, including precursor and product ion scans, neutral loss scans, and in-source dissociation and post-source decay.

Proteomic analysis of the intestinal epithelial cell response to enteropathogenic Escherichia coli.

We present the first large scale proteomic analysis of a human cellular response to a pathogen. Enteropathogenic Escherichia coli (EPEC) is an enteric human pathogen responsible for much childhood morbidity and mortality worldwide. EPEC uses a type III secretion system (TTSS) to inject bacterial proteins into the cytosol of intestinal epithelial cells, resulting in diarrhea. We analyzed the host response to TTSS-delivered EPEC effector proteins by infecting polarized intestinal epithelial monolayers with either wild-type or TTSS-deficient EPEC. Host proteins were isolated and subjected to quantitative profiling using isotope-coded affinity tagging (ICAT) combined with electrospray ionization tandem mass spectrometry. We identified over 2000 unique proteins from infected Caco-2 monolayers, of which approximately 13% are expressed differentially in the presence of TTSS-delivered EPEC effector proteins. We validated these data in silico and through immunoblotting and immunofluorescence microscopy. The identified changes extend cytoskeletal observations made in less relevant cell types and generate testable hypotheses with regard to host proteins potentially involved in EPEC-induced diarrhea. These data provide a framework for future biochemical analyses of host-pathogen interactions.