Electrophoresis 2014, 35, 1017–1024 1017 Tijl Vermassen 1 Charles Van Praet 2 Dieter Vanderschaeghe 3 Thomas Maenhout 4 Nicolaas Lumen 2 Nico Callewaert 3 Piet Hoebeke 2 Simon Van Belle 1 Sylvie Rottey 1 Joris Delanghe 4 1 Department of Medical Oncology, Ghent University Hospital, Ghent, Belgium 2 Department of Urology, Ghent University Hospital, Ghent, Belgium 3 Unit for Medical Biotechnology, Department for Molecular Biomedical Research, VIB – Ghent University, Ghent, Belgium 4 Department of Clinical Chemistry, Ghent University Hospital, Ghent, Belgium Received July 22, 2013 Revised October 17, 2013 Accepted October 27, 2013 Research Article Capillary electrophoresis of urinary prostate glycoproteins assists in the diagnosis of prostate cancer Prostate marker assays are widely used for detection of prostate cancer (PCa) but are as- sociated with considerable sensitivity and specificity problems. Therefore, we investigated prostatic protein glycosylation profiles as a potential biomarker. We determined the uri- nary asparagine-linked glycan (N-glycan) profile of prostatic proteins of healthy volunteers (n = 25), patients with benign prostate hyperplasia (BPH; n = 62) and newly diagnosed PCa patients (n = 42) using DNA-sequencer-assisted fluorophore-assisted carbohydrate electrophoresis. Through squeezing of the prostate, a sufficient amount of prostatic pro- teins was obtained for direct structural analyses of N-glycan structures. N-glycans of PCa compared to BPH were characterized by a significant decrease in triantennary structures (p = 0.047) and overall fucosylation (p = 0.026). Prostate-specific antigen (PSA) and the urinary glycoprofile marker showed comparable overall receiver operating characteristic curve analysis as well as in the diagnostic gray zone with serum PSA values between 4 and 10 g/L. However, when combining PSA and the urinary glycoprofile marker, the latter gave an additive diagnostic value to serum PSA (p 0.001). In conclusion, N-glycosylation profiling demonstrated differences between BPH and PCa. These changes could lead to the discovery of a new biomarker for PCa. Keywords: Asparagine-linked glycosylation / Benign prostate hyperplasia / Diagnostic marker / Prostate cancer / Prostate-specific antigen DOI 10.1002/elps.201300332 1 Introduction Prostate cancer (PCa) is one of the most common malignan- cies in men. Worldwide PCa is the second most common cancer diagnosed among males next to lung cancer. With 258 400 men dying from PCa in 2008, it is the sixth most common cause of death in males [1]. In prostate disease, such as PCa, the basement mem- brane can be disrupted and prostate-specific antigen (PSA) can access the peripheral circulation [2]. PSA, a glycopro- tein that consists of one N-glycosylation site at asparagine-45 residue, is produced primarily by epithelial cells lining the acini and prostatic ducts of the prostatic gland and is largely secreted as into the seminal plasma fluid (0.5–3 g/L) [3]. Correspondence: Professor Joris R. Delanghe, Department of Clinical Chemistry, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium E-mail: Joris.Delanghe@UGent.be Fax: +32-9-332-36-59 Abbreviations: AUC, area under the curve; BPH, benign prostate hyperplasia; fPSA, noncomplexed or free PSA; %fPSA, ratio free PSA on total PSA; HV, healthy volunteer; N- glycan, asparagine-linked glycan; PCa, prostate cancer; PSA, prostate-specific antigen; ROC, receiver operating character- istic; tPSA, total PSA However, in serum, PSA reference values are much lower (1–4 g/L) [4]. In urine, PSA is only detected in its free form with concentrations largely exceeding (till hundredfold) the values observed in serum or plasma [5]. PSA assays are widely used for early detection of PCa [6]. However, those analyses are associated with considerable sen- sitivity and specificity problems, especially in the diagnostic gray zone with serum PSA concentration of 4–10 g/L [2, 7]. PSA is a prostate-specific but not cancer-specific marker and an increased PSA concentration can be associated with other prostatic diseases, such as benign prostate hyperplasia (BPH) and prostatitis, and does not discriminate between high- and low-risk PCa. Even below the threshold of 4 g/L aggressive PCa can be present [7]. In order to increase the diagnostic accuracy new tech- niques, such as ratio of noncomplexed or free PSA (fPSA) on total PSA (tPSA) (%fPSA) [8] and multiple immunoassays for different molecular forms of PSA (inactive PSA, intact PSA, and BPH-associated PSA) have been proposed without a sub- stantial improvement in the ability to distinguish between benign and malignant disease [9, 10]. New genetic markers and biomarkers have recently emerged but only a few of them (e.g. PCA-3) have shown clinical value [11–15]. Colour Online: See the article online to view Fig. 2 in colour. C  2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com