Elsevier

Journal of Proteomics

Volume 72, Issue 6, 20 August 2009, Pages 907-917
Journal of Proteomics

Clinical collection and protein properties of expressed prostatic secretions as a source for biomarkers of prostatic disease

https://doi.org/10.1016/j.jprot.2009.01.007Get rights and content

Abstract

The prostate gland secretes many proteins in a prostatic fluid that combines with seminal vesicle derived fluids to promote sperm activation and function. Proximal fluids of the prostate that can be collected clinically are seminal plasma and expressed-prostatic secretion (EPS) fluids. EPS represents the fluid being secreted by the prostate following a digital rectal prostate massage, which in turn can be collected in voided urine post-exam. This collection is not disruptive to a standard urological exam, and it can be repeatedly collected from men across all prostatic disease states. A direct EPS fluid can also be collected under anesthesia prior to prostatectomy. While multiple genetic assays for prostate cancer detection are being developed for the shed epithelial cell fraction of EPS urines, the remaining fluid that contains many prostate-derived proteins has been minimally characterized. Approaches to optimization and standardization of EPS collection consistent with current urological exam and surgical practices are described, and initial proteomic and glycomic evaluations of the of EPS fluid are summarized for prostate specific antigen and prostatic acid phosphatase. Continued characterization of the prostate specific protein components of EPS urine combined with optimization of clinical collection procedures should facilitate discovery of new biomarkers for prostate cancer.

Section snippets

Overview

Large scale clinical detection of prostate specific antigen (PSA) levels in serum as a biomarker of prostate cancer has been ongoing since the 1990s [1], [2]. The assay continues to evolve to reflect new knowledge about disease specific isoforms, free PSA versus bound complexes with serum proteins, PSA velocity and PSA density considerations [3], [4], [5], [6]. While the overall benefits and risks of population PSA screening for prostate cancer continues to be assessed [7], [8], the known

The clinical need for new prostate cancer biomarkers

Prostate cancer (PCa) continues to have a high incident rate with over 186,000 new cases estimated in 2008 in the U.S., and second only to lung cancer in cancer death rate (29 per 100,000 men) [13]. Early detection screening for prostate cancers has relied on PSA detection in serum [8]. Although far from ideal, PSA screening is one of the widest utilized oncological screening methods in current practice, and has facilitated treatment by prostatectomy to remove cancers at their earliest, more

Expressed prostatic secretions (EPS) in urine as a new source of biomarkers

The prostate gland secretes many protein molecules in a prostatic fluid that combines with seminal fluid and sperm from the seminal vesicles during ejaculation. In seminal fluids, many of these prostatic proteins perform functions that promote the ability of sperm to become activated and escape the vaginal immune micro-environment. PSA performs the critical function of liquefaction in this process by degrading semenogelin, while the exact role of PAP is not known but is suspected to be involved

Clinical collection of expressed prostate secretions (EPS) in urine

We have concentrated on collecting EPS urine samples from men reporting to the clinic for a prostate biopsy procedure, as this group of men reflects a population where new biomarker assays would be most useful for distinguishing benign prostatic disease from cancers. The EPS samples are initially classified using the currently accepted risk stratification system [6], [59], [60], and include the results of a biopsy with a minimum of 12 cores. Within this group, individuals with no evidence of

Concentration of PSA and PAP in EPS

From a diagnostic perspective, two of the most abundant and assayable proteins that should be present in the EPS fluids are PSA and PAP. An ELISA assay format was adapted from one previously reported for use with nipple aspirate fluids [62], which involves coating each plate well with unprocessed EPS sample and then assaying for individual proteins with an appropriate primary/secondary antibody combination. Using this assay, for most EPS urines we can detect 10–40 μg/ml PSA and 3–10 μg/ml PAP.

Proteomic characterization of PAP and PSA in EPS urines

Based on the determined concentrations of PSA and PAP in the EPS fluids, these proteins should be readily detectable using mass spectrometry based methods. To facilitate their analysis, we applied a thiophilic adsorption chromatography (TAC) approach previously reported for purification of PSA from seminal plasma [63]. As described in Fig. 3, pooled samples from prostate cancer patients for seminal plasma, EPS urine and direct EPS were applied to TAC resins. PAP does not bind to this resin when

MALDI-TOF profiling of total glycans from urine EPS and direct EPS fractions

As an initial assessment for global glycan profiling, pooled EPS proteins (0.3 mg) from BPH and cancer subjects and 0.3 mg of direct EPS protein pools from low and intermediate risk prostate cancer subjects have been used. Each protein solution was digested with trypsin for 18 h, followed by overnight digestion with PNGase F to release N-linked glycans. Total glycans were permethylated [64] and spotted for preliminary MALDI-TOF profiling. Based on the identified normalized mass ions, we have

Time and temperature stability of EPS urine proteins

Like for any clinical fluid, it will be necessary to standardize collection procedures for EPS urines and determine time and temperature parameters that facilitate sample stability. We have determined that for most samples, removal of the cell pellet from the supernatant can be done routinely within 20–30 min of collection. We have not yet assessed the effect of the sediment components on stability of proteins beyond 30 min exposure. A pilot study was done on an EPS urine and direct EPS sample

Summary and future directions

EPS urine is routinely collected for diagnosing prostatitis and increasingly for development of genetic and metabolic markers of prostate cancer. It is also a rich source of prostate derived proteins, particularly secreted glycoproteins, and by extension, their glycan constituents. Our initial proteomic studies have focused on characterizing the most prevalent constituents in these EPS fluids that we believe will be the initial targets for biomarker assay development. The relative high

Acknowledgement

This work was funded in part by NIH/NCI grant 2U01 CA98028 (OJS).

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