Identifying prostate cancer biomarkers by profiling glycoproteins in human prostate tissue (591.6)

Abstract

Prostate‐specific antigen (PSA), the current clinical biomarker for prostate cancer, suffers from high false positive and high false negative rates that leave many men without a conclusive diagnosis. Virtually all clinically approved cancer biomarkers are cell‐surface or secreted glycoproteins. We seek to identify more specific clinical biomarkers of prostate cancer by developing an efficient method for specifically labeling and enriching glycoproteins, followed by MS‐based analysis of normal and cancerous human prostate tissue. We present a novel biomarker identification strategy where human prostate tissue derived from radical prostatectomies, at varying states of prostatic disease is chemically tagged. To identify cell‐surface or secreted glycoproteins from prostate tissue, we employ metabolic labeling, a technique in which unnatural azide or alkyne‐functionalized monosaccharides are incorporated, into cell‐surface glycoproteins via the cells’ own metabolic machinery. Tissue slices incubated with these labeled sugars are subsequently reacted with a biotin‐functionalized probe for the capture of sialylated glycoproteins. Analysis of labeled tissue by Western blot confirmed azide or alkyne‐dependent labeling. Proteins were then digested with trypsin and the resultant peptides were analyzed on a linear ion trap mass spectrometer with collision‐induced dissociation (CID). Greater than 60% of the proteins identified by LC‐MS/MS were cell‐surface or secreted proteins, indicating that this strategy achieves enrichment of low abundance proteins. Subsequently, we performed a comparative proteomics experiment between diseased and healthy tissue and identified proteins unique to each tissue type, which are undergoing further validation as potential biomarkers. This work extends the application of existing metabolic labeling techniques, while developing new methodologies for cell surface labeling to discover biomarkers associated with prostate cancer.