Targeted Mass Spectrometry of a Clinically Relevant PSA Variant from Post‐DRE Urines for Quantitation and Genotype Determination

Abstract

Purpose: The rs17632542 single nucleotide polymorphism (SNP) results in lower serum prostate specific antigen (PSA) levels which may further mitigate against its clinical utility as a prostate cancer biomarker. Post-digital rectal exam (post-DRE) urine is a minimally invasive fluid that is currently utilized in prostate cancer diagnosis. To detect and quantitate the variant protein in urine. Experimental design: Fifty-three post-DRE urines from rs17632542 genotyped individuals processed and analyzed by liquid chromatography/mass spectrometry (LC-MS) in a double-blinded randomized study. The ability to distinguish between homozygous wild-type, heterozygous, or homozygous variant is examined before unblinding. Results: Stable-isotope labeled peptides are used in the detection and quantitation of three peptides of interest in each sample using parallel reaction monitoring (PRM). Using these data, groupings are predicted using hierarchical clustering in R. Accuracy of the predictions show 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 expression. This assay provides a tool to evaluate the utility of PSA variant (rs17632542) in parallel with current and forthcoming urine biomarker panels.

Direct N-Glycosylation Profiling of Urine and Prostatic Fluid Glycoproteins and Extracellular Vesicles

“…The rapidity and efficiency of the slide-based MALDI assay will facilitate evaluation of larger cohorts. Possibly pairing the N-glycan data with the quantitative MRM proteomic assay developed for EPSu proteins could address both the changes in N-glycan levels and protein concentrations in the same sample ( Kim et al, 2016 ; Otto et al, 2020 ). It also does not preclude direct targeting of specific glycoproteins present for N-glycan content analysis, as is done so often for PSA.…”

“…Our group has previously characterized the proteomic composition of EPSu and prostatic secretions, identifying hundreds of different prostate-derived glycoproteins ( Drake et al, 2009 ; Drake et al, 2010 ; Kim et al, 2012 ; Principe et al, 2012 ). Development and evaluation of extensive targeted proteomic assays to these proteins in EPSu are in progress for use in prostate cancer diagnosis ( Kim et al, 2016 ; Otto et al, 2020 ). The prostatic fluids, EPSu and urine are also rich in extracellular vesicles (EV), which are a source for many ongoing non-coding RNA and related oligonucleotide-targeted diagnostic assays for prostate cancer and multiple diseases ( Van Gils et al, 2007 ; Laxman et al, 2008 ; Linxweiler and Junker, 2020 ).…”

Targeted protein quantitation in human body fluids by mass spectrometry

“…With the optimization of the digestion of urinary EV proteins (C. L. , only 1-2 μg of proteins are required to perform multiplexed protein quantification, which enables the statistical analysis of a biomarker panel through LC-MRM-MS (C. L. by using limited amounts of urinary proteins from EVs (C. L. Sequeiros et al, 2017) or postdigital rectal exam urine samples (Kim et al, 2016;Otto et al, 2020). Crucial clinically relevant PSA variants could be detected through LC/MRM-MS, and this can be an attractive alternative to ELISA for the reliable measurement of mature and precursor forms or the genotype-specific protein expression of PSA, a well-known prostate cancer biomarker (Y.-T. Chen et al, 2015;Otto et al, 2020). Studies exploring biomarker verification through targeted proteomics in the urine samples of patients with malignant or benign diseases other than those of the urological system indicated the applicability of targeted proteomics in clinical practice (Chutipongtanate & Greis, 2018;Duangkumpha et al, 2019;Rauniyar et al, 2018;Wu et al, 2020).…”

“…Because only 0.1–150 μg of proteins are extractable from urinary EVs, protein quantification through western blot analysis or other antibody‐based immunoassays is difficult. With the optimization of the digestion of urinary EV proteins (C. L. Chen et al, 2012), only 1–2 μg of proteins are required to perform multiplexed protein quantification, which enables the statistical analysis of a biomarker panel through LC‐MRM‐MS (C. L. Chen et al, 2012) by using limited amounts of urinary proteins from EVs (C. L. Chen et al, 2012; Sequeiros et al, 2017) or postdigital rectal exam urine samples (Kim et al, 2016; Otto et al, 2020). Crucial clinically relevant PSA variants could be detected through LC/MRM‐MS, and this can be an attractive alternative to ELISA for the reliable measurement of mature and precursor forms or the genotype‐specific protein expression of PSA, a well‐known prostate cancer biomarker (Y.‐T.…”

Prostate Cancer Biomarker Development: National Cancer Institute's Early Detection Research Network Prostate Cancer Collaborative Group Review

“…One such SNP encodes a PSA variant (rs17632542) that results in a single amino acid change in the PSA protein associated with reduced relative secretion into blood and association with lower serum PSA levels (59,60). In a recent collaborative group study, investigators developed a PRM-MS assay that could accurately detect and quantify PSA wild-type and variant proteins in patient urine (61). Although genotyping is readily available, it is not routinely ordered and does not address the relationship between heterozygosity and protein expression.…”