Abstract
Expressed prostatic secretions (EPS), also called post digital rectal exam urines, are proximal fluids of the prostate that are widely used for diagnostic and prognostic assays for prostate cancer. These fluids contain an abundant number of glycoproteins and extracellular vesicles secreted by the prostate gland, and the ability to detect changes in their N-glycans composition as a reflection of disease state represents potential new biomarker candidates. Methods to characterize these N-glycan constituents directly from clinical samples in a timely manner and with minimal sample processing requirements are not currently available. In this report, an approach is described to directly profile the N-glycan constituents of EPS urine samples, prostatic fluids and urine using imaging mass spectrometry for detection. An amine reactive slide is used to immobilize glycoproteins from a few microliters of spotted samples, followed by peptide N-glycosidase digestion. Over 100 N-glycan compositions can be detected with this method, and it works with urine, urine EPS, prostatic fluids, and urine EPS-derived extracellular vesicles. A comparison of the N-glycans detected from the fluids with tissue N-glycans from prostate cancer tissues was done, indicating a subset of N-glycans present in fluids derived from the gland lumens. The developed N-glycan profiling is amenable to analysis of larger clinical cohorts and adaptable to other biofluids.
Highlights
In the search and characterization of disease biomarkers for use in liquid biopsy applications, proximal fluids like blood and urine are commonly used
Based on an adaptation of a recently published workflow for rapid characterization of serum and plasma N-glycans (Blaschke et al, 2020), we report a more efficient slide-based approach combined with MALDI imaging mass spectrometry (IMS) workflows to detect total N-glycan profiles of urine, expressed-prostatic secretion in urine (EPSu) and prostatic fluid samples
To complement previous proteomic studies of proximal prostatic fluids obtained in the urology clinic as related to prostate cancers (Drake et al, 2009; Drake et al, 2010; Kim et al, 2012; Principe et al, 2012) a series of different EPSu, EPS fluids (EPSd) and EPS-derived extracellular vesicles (EPSev) samples were used to develop a MALDI-based N-glycan profiling method
Summary
In the search and characterization of disease biomarkers for use in liquid biopsy applications, proximal fluids like blood and urine are commonly used. For prostate cancer and other genitourinary diseases, proximal fluids are represented by seminal plasma and expressed-prostatic secretion in urine (EPSu) The prostate gland secretes many proteins and other biomolecules in a prostatic fluid that combines with seminal fluid and sperm from the seminal vesicles during ejaculation. 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 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). EV obtained from urine and EPSu continues to be a highly active area for diagnostic assay development (Wang et al, 2020; Erdbrügger et al, 2021)
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