Protein biomarkers for epithelial ovarian cancer are critical for the early detection of the cancer to improve patient prognosis and for the clinical management of the disease to monitor treatment response and to detect recurrences. Unfortunately, the discovery of protein biomarkers is hampered by the limited availability of reliable and sensitive assays needed for the reproducible quantification of proteins in complex biological matrices such as blood plasma. In recent years, targeted mass spectrometry, exemplified by Selected Reaction Monitoring (SRM) has emerged as a method, capable of overcoming this limitation. Here, we present a comprehensive SRM-based strategy for developing plasma-based protein biomarkers for epithelial ovarian cancer and illustrate how the SRM platform, when combined with rigorous experimental design and statistical analysis, can result in detection of predictive analytes. Our biomarker development strategy first involved a discovery-driven proteomic effort to derive potential N-glycoprotein biomarker candidates for plasma-based detection of human ovarian cancer from a genetically engineered mouse model of endometrioid ovarian cancer, which accurately recapitulates the human disease. Next, 65 candidate markers selected from proteins of different abundance in the discovery dataset were reproducibly quantified with SRM assays across a large cohort of over 200 plasma samples from ovarian cancer patients and healthy controls. Finally, these measurements were used to derive a 5-protein signature for distinguishing individuals with epithelial ovarian cancer from healthy controls. The sensitivity of the candidate biomarker signature in combination with CA125 ELISA-based measurements currently used in clinic, exceeded that of CA125 ELISA-based measurements alone. The SRM-based strategy in this study is broadly applicable. It can be used in any study that requires accurate and reproducible quantification of selected proteins in a high-throughput and multiplexed fashion.

Formerly N-linked glycosylated peptides from each plasma sample were isolated using the N-linked glycopeptide capture procedure as described by Zhang et al (2003). SRM assays were retrieved from the N-glycoprotein SRM atlas (http://www.srmatlas.org/) (43), reanalyzed to select the best transitions for endogenous detection in plasma, split to multiple SRM methods, or used to optimize a single SRM method. Tier 2 SRM assays were used to quantify the N-glycosites employing internal standard peptides labeled with heavy isotopes at the C-terminal lysine or arginine, +8 or +10 Da, respectively, to identity peptides based on analogy of chromatographic and fragmentation properties to the reference and quantify peptides. For each peptide, the best 3-4 transitions for the internal standard as well as the endogenous peptide were monitored in a scheduled fashion. SRM analyses for pilot batch 1 was performed on a 4000QTRAP and 5500QTRAP (AB-Sciex) equipped with a nanoelectrospray ion source. All samples were analyzed on both instruments, biomarker candidates that were not detected on the 4000QTRAP were targeted on the 5500QTRAP. Samples from batch 2 were analyzed on TSQ Vantage (Thermo Fischer Scientific) equipped with a nanoelectrospray ion source.

The current study included blood plasma from patients in the biobank, 124 patients with epithelial ovarian cancer (EOC) and 110 healthy controls. Patients with borderline tumors were excluded from this study, due to the uncertain malignant potential of these benign tumors. The healthy controls were voluntary partners to cancer patients at Skåne University Hospital. Patients with suspected adnexal tumor that underwent surgery at the gynecological department, Skåne University Hospital between 2004 and 2013 were included in the Skåne University Hospital ovarian tumor biobank. The blood samples of healthy controls were collected between January 2004 and June 2008. Controls were age and gender matched to EOC cases and had no previous cancer disease. All individuals gave written informed consent for participation. Ethical permission for the biobank was obtained from the Lund University Ethics Committee. All blood samples were drawn into EDTA-tubes and centrifuged at 2000 x g for 10 minutes. The plasma samples were stored at -80 Celsius degrees within approximately two hours from sampling.