The rs17632542 SNP results in lower serum PSA levels which may further mitigate against its clinical utility as a prostate cancer biomarker. Post-DRE urine is a minimally invasive fluid that is currently utilized in prostate cancer diagnosis. We have used targeted MS to detect and quantitate the variant protein in urine. Experimental Design Fifty-three urines collected after digital rectal exam (post-DRE urine) from rs17632542 genotyped individuals were processed and analyzed by LC-MS in a double-blinded randomized study. The ability to distinguish between homozygous wild-type, heterozygous, or homozygous variant was examined prior to unblinding. Results Stable-isotope labeled peptides were used in the detection and quantitation of peptides of interest in each sample. Three peptides were monitored by LC-MS using a PRM method. Analysis of the raw data using Skyline allowed for peak detection and area extraction. Using these data, groupings were predicted using hierarchical clustering in R. Accuracy of the predictions showed 100% concordance across the 53 samples, including individuals homozygous and heterozygous for the SNP. Conclusions and clinical relevance The study demonstrates that MS based peptide variant quantitation in urine could be useful in determining patient genotype with high accuracy to complement currently used PSA screening.

Blinded and randomized post-DRE urines were processed through an adapted MStern protocol first published by Berger et al.[30] The protocol utilizes a 96-well plate with porous polyvinylidene fluoride membranes (Sigma Millipore MSIPS4510). Briefly, three 250µL pre-spun aliquots of 53 post-DRE urine samples were reduced using 5mM dithiothreitol (DTT) for 30 minutes at 56°C. After cooling, samples were alkylated with 25mM iodoacetamide (IAM) to prevent disulfide bond reformation. Samples were bound to the membranes using a vacuum manifold. Digestion was carried out on the membrane at 37°C for four hours using 50µL per well of a solution containing 1µg Trypsin/Lys-C, 100mM ammonium bicarbonate, 5% acetonitrile, and 1mM CaCl2. Peptides were collected by passing 50µL of 50% acetonitrile through each well twice by centrifugation for 2 minutes at 2500xg. Samples were dried using a SpeedVac. Finally, peptides were purified using solid-phase extraction (SPE) C18 tips (Pierce 87784) according to manufacture instructions.