Development of MS-based Prostate-Specific Antigen Test with In-Depth Glycosylation Analysis Guinevere S.M. Lageveen-Kammeijer (Presenter) Center for Proteomics and Metabolomics, LUMC Presenter Bio: Guinevere S. M. Lageveen-Kammeijer received a BSc degree in Biotechnology - Forensic Sciences from University of Applied Science van Hall Larenstein, Leeuwarden, NL followed by a MSc in Analytical Chemistry at the VU University, Amsterdam, NL. Since June 2013, she is a PhD candidate at the Center for Proteomics and Metabolomics at Leiden University Medical Center, NL under the supervision of Prof. Dr. Manfred Wuhrer. Her main research project involves exploring the glycosylation of prostate-specific antigen for a better defined biomarker for prostate cancer. In addition, she studies the possibilities for in-depth analysis of glycans and intact glycoproteins with CE-ESI-MS for biomarker discovery and the characterization of biopharmaceuticals. Furthermore, she is a member of the organization committee of the Netherlands Area Biotech (NLab) Discussion group supported by the CASSS.

Authors: Guinevere S.M. Kammeijer(1), Jan Nouta(1), Anna Kałuża(1,2), Wei Wang(1), Noortje de Haan(1); L. Renee Ruhaak(3), Kasper M. Siliakus(3); Mervin M. Pieterse(3), Theo M. de Reijke(4), Yuri E.M. van der Burgt(1,3), Christa M. Cobbaert(3); Manfred Wuhrer (1)
(1) Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands, (2) Department of Chemistry and Immunochemistry, Wrocław Medical University, Wrocław, Poland, (3) Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, The Netherlands, (4) Urology Department, Academic Medical Center, Amsterdam, The Netherlands

The diagnostic test for prostate cancer (PCa) based on glycoprotein prostate-specific antigen (PSA) is widely known for its lack in specificity. Recently, we established a PSA glycomics assay that allows identifying and relatively quantifying the glycosylation profile of PSA in a non-invasive manner after affinity capturing from patients’ urine. The applicability of this assay is further explored for other biofluids such as seminal plasma and serum. Potentially, alterations in the glycosylation pattern will provide better diagnostic and prognostic tools for fertility conditions (seminal plasma) and PCa (serum and urine) by studying different cohort studies.

Introduction

Prostate cancer (PCa) is one of the most frequent cancers in men.(1) As an early screening method the concentration of the glycoprotein prostate-specific antigen (PSA) is measured in serum. Even though this clinical test is applied worldwide, it exhibits a rather low sensitivity, specificity and moreover a poor predictive value.(2) As a result, high PSA-levels often lead to a high number of unnecessary biopsies. Literature suggests that knowledge on specific alterations in the glycosylation profile of PSA may provide a more specific yet non-invasive PCa diagnosis.(3) However, conventional PSA glycomics assays lack the in-depth isoform differentiation (e.g. lectin-based assays) that is needed to provide a better understanding of the alterations of molecular features of PSA glycosylation (such as antenna modification and core fucosylation).(4-6) Recently, we have developed an in-depth PSA glycosylation assay (PGA) based on capillary electrophoresis of tryptic glycopeptides that enables the differentiation of α2,6- and α2,3-sialylated isomers,(7,8) the latter one being suggested to be a hallmark of aggressive types of cancer.(9,10) Besides PCa, it has been suggested that alterations in the glycosylation profile could also provide a better insight into the fertility of the men which has recently been studied in total seminal plasma.(11,12)

Methods

We intend to further expand the established, non-invasive, PSA glycomics assay(8) in order to allow the quantitation and in-depth glycosylation analysis of PSA to other biofluids next to urine: PSA from seminal plasma and serum will be investigated for their diagnostic and prognostic potential for fertility conditions (seminal plasma) and PCa (serum). In addition, the assay is currently being expanded to cover the glycosylation analysis of prostatic acid phosphatase (PAP). Similar to PSA, PAP concentration in serum has previously been used as an early detection marker of PCa, but gradually became replaced by PSA. Notably, the diagnostic potential of PAP glycosylation has hitherto remained largely unexplored.

Results

Preliminary results already revealed that for capturing PSA from other biofluids than urine, the same procedure (PGA) can be applied after some slight modifications (e.g. volume of the biofluid and antibody:volume ratio). For the high-throughput analysis with MALDI-TOF-MS, a derivatization method was developed that allows the differentiation of α2,3- and α2,6-sialylated glycopeptides and the same derivatized sample was used for the quantification of glycopeptide candidates with LC-MS. In addition, analysis of a PAP standard revealed that two of the three N-linked glycosylation sites mainly carried complex N-glycans while the other N-linked glycosylation site only contained high mannose types. On glycosylation site N₉₄ 31 different complex N-glycans were identified and on site N₂₂₀ 24 different complex N-glycans, most of them contained one up to three sialic acids. In addition, the analysis with CE-ESI-MS revealed the presence of differently linked sialic acids (α2,3- and α2,6-sialylated glycopeptides). On glycosylation site N₃₃₃ only four different N-glycans were identified varying from five mannoses up to eight mannoses on PAP. Overall PAP showed signficantly lower glycosylation heterogeneity per glycosylation site and a total of 59 different N-glycopeptides as compared to the single N-linked glycosylation site on PSA that exhibited 75 different N-glycopeptides.

Conclusions & Discussion

Overall, by including PAP as a second target antigen next to PSA and by expanding the PGA to other biofluids, we aim to define protein-specific glycosylation profiles that provide information on both PCa progression and possibly also fertility status. In addition, we will define biofluid specific alterations -as well as protein-specific perturbations of glycosylation, providing insights into disease-associated changes in glycosylation as well as protein release and secretion mechanisms. Future research will focus on applying the mass spectrometry glycomics assays to large clinical cohorts for establishing diagnostic markers.

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