Article pubs.acs.org/jpr
Novel Glycobiomarker for Ovarian Cancer That Detects Clear Cell Carcinoma Maki Sogabe,†,# Hirofumi Nozaki,†,# Nana Tanaka,† Tomomi Kubota,† Hiroyuki Kaji,† Atsushi Kuno,† Akira Togayachi,† Masanori Gotoh,† Hayao Nakanishi,‡ Toru Nakanishi,§ Mikio Mikami,∥ Nao Suzuki,⊥ Kazushige Kiguchi,⊥ Yuzuru Ikehara,† and Hisashi Narimatsu*,† †
Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Central-2, 1-1-1 Umezono, Tsukuba 305-8568, Japan ‡ Division of Oncological Pathology, Aichi Cancer Center Research Institute and §Department of Gynecologic Oncology, Aichi Cancer Center Hospital, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan ∥ Department of Obstetrics and Gynecology, School of Medicine, Tokai University, 143 Shimokasuya, Isehara 259-1193, Japan ⊥ Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki 216-8511, Japan S Supporting Information *
ABSTRACT: Epithelial ovarian cancer (EOC) is often asymptomatic and thus diagnosed at advanced stages with a poor prognosis. Falsenegative results for the conventional marker CA125 frequently occur in cases of clear cell carcinoma (CCC), a type of EOC; therefore, it is necessary to develop biomarkers with greater sensitivity. We previously reported a strategy to discover glycobiomarker candidates by combined lectin microarray and IGOT−LC/MS analysis. We have now optimized this strategy for discovering EOC biomarkers. Glycopeptides possessing cancerous glycans were enriched from the ascites fluids and culture supernatants of cancer cell lines with a fucose-binding lectin, AAL. IGOT−LC/MS analysis of CCC samples yielded 144 candidate glycoproteins. We selected WFA by lectin microarray as the optimal lectin to distinguish EOC from gastric and colon cancer. The candidates were narrowed by Western analysis of the WFA-bound fraction of ascites fluids. One of the final candidates, WFA-reactive ceruloplasmin, produced higher signals in the ascites fluids of EOC patients, including CCC, in comparison with the benign samples, while CA125 levels were comparable in the sandwich ELISA. Thus, our glycoproteomic strategy featuring efficient enrichment of glycans with disease-related alterations is applicable to various diseases. KEYWORDS: glycoprotein, glycoproteomics, glycan alteration, epithelial ovarian cancer, clear cell carcinoma, WFA, ceruloplasmin, lectin microarray
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INTRODUCTION Glycosylation is a major post-translational modification of secreted and cell surface proteins; it is often altered by changes in the cellular environment that are coincident with the progress of various diseases, including cancer.1−4 A promising approach to discovering novel reliable biomarkers is to focus on such qualitative, not quantitative, changes of disease-specific glycan structures. Changes in the glycosylation pattern can be represented as altered lectin reactivity such as LCA (Lens culinaris agglutinin)-reactive alpha-fetoprotein (AFP).5 We developed a highly sensitive lectin microarray system optimized for comparative analysis in the discovery and development of disease-specific biomarkers.6 To develop a cholangiocarcinoma marker, we first identified the probe lectins that specifically distinguish diseased from nondiseased specimens by using the lectin microarray system with pathological tissue samples and body fluids from patients.7 After selection of the probe lectins, we enriched glycopeptides with the respective © 2014 American Chemical Society
lectin-bound glycoepitopes. These glycopeptides were analyzed to identify the core proteins through lectin−IGOT (isotopecoded glycosylation site-specific tagging)−LC/MS technology, which identifies protein molecules and their N-glycosylated sites.8 It is essential to identify glycoprotein disease markers with high specificity and sensitivity. Each glycoprotein undergoes various glycan modifications, and disease progression is reflected by various glycoproteins and their glycan motifs. We established an analytical flow for comprehensive and reliable biomarker development with glycoproteomic study of clinical specimens.9 We previously reported the discovery of new candidate biomarkers for liver fibrosis and hepatocarcinoma using this strategy.10,11 Received: November 13, 2013 Published: February 5, 2014 1624
dx.doi.org/10.1021/pr401109n | J. Proteome Res. 2014, 13, 1624−1635
Journal of Proteome Research
Article
free (i.e., protein-free) and antibiotic-free media for 48 h; culture supernatants were harvested for analysis.
It is difficult to discover novel candidate cancer markers directly from serum samples, except in the case of hepatocarcinoma. Because most serum proteins are secreted by the liver, serum proteins secreted by other organs are relatively minor and difficult to detect.12,13 Therefore, we modified the strategy to utilize ascites fluids instead of serum and added lectin column enrichment prior to analysis for discovery of novel candidate glycoproteins as ovarian cancer markers. Ovarian cancer is asymptomatic and insensible; most cases are diagnosed when the disease is advanced, and the 5 year survival rate is pyro-Glu (N-term Gln), oxidation (Met), pyro-carbamidomethyl (N-term Cys), IGOT (Asn > Asp +18O = +2.988261 Da, custom-made) (Asn); peptide mass tolerance: 200 ppm; fragment mass tolerance: 0.5 Da; max missed cleavage: 2. The false discovery rates of the Mascot search were
