Abstract B34: Tandem Mass Tag 10-plex isobaric labeling of Pap test proteins: A novel method for the identification of ovarian cancer protein biomarkers by mass spectrometry.
Authors: Skubitz Amy P.N. , Afiuni Somi , Boylan Kristin L.M. , Geller Melissa , Argenta Peter , Hoffman Samantha , Griffin Timothy 
Published: May 2016

Abstract
Early detection is the key to increased survival for women with ovarian cancer, yet a screening tool has yet to be developed that is adequately sensitive and specific enough for use in the general population. A need also exists for a reliable diagnostic method for women who present with an abdominal mass. In contrast, screening for cervical cancer by Pap tests has been routinely performed for over 50 years. In the liquid-based Pap test, cells are collected from the cervical os and placed into an alcohol-based fixative and then examined for abnormal cells. Since ovarian cancer cells have been observed in Pap tests, we reasoned that ovarian cancer peptide biomarkers may also be present. Our central hypothesis is that proteins shed by ovarian cancer cells can be detected in the Pap test fixative by Mass Spectrometry (MS)-based proteomics. The Pap test fixative is ideal for biomarker discovery since it is derived from a site near the ovarian cancer (i.e. proteins may be secreted or shed from the tumor and flow through the fallopian tube into the uterus and out the cervical opening). Recently, the fimbria of the fallopian tube have been suggested to be the true precursor to ovarian cancer, strengthening our hypothesis that ovarian cancer proteins will be found in the lower genital tract, perhaps even at early stages. To demonstrate the feasibility of this approach, we previously examined the proteins present in residual Pap test fixative samples from women with normal cervical cytology by MS, and described 152 proteins in the “Normal Pap Proteome.” (Boylan et al., Clinical Proteomics, 2014).
The objective of this study is to discover ovarian cancer biomarkers in Pap test fixative samples by MS-based proteomic techniques. We used isobaric tags to quantify the proteins in liquid Pap test samples from women with ovarian cancer compared to women with benign gynecological disease and healthy women. Proteins were concentrated from “Mock” Pap tests, and samples were trypsin digested utilizing the STrap technique to increase the likelihood that proteins would be recovered. We then performed Tandem Mass Tag™ (TMT) 10-plex labeling of Pap test fixative samples from post-menopausal women: 3 with ovarian cancer, 3 with benign gynecological conditions, 3 healthy, and a Reference Control consisting of pooled samples. The 10 labeled samples were combined and run on 2D LC-MS/MS, followed by bioinformatics integration to discover proteins in Pap test fixatives that are unique and/or differentially expressed in the samples. We identified &gt;3800 proteins total, and 22 proteins that were expressed at least 2-fold higher in all three ovarian cancer samples compared to the controls. We conclude that quantification of proteins from Pap test samples will prove a rich source of biomarkers for ovarian cancer detection.
Citation Format: Amy P.N. Skubitz, Somi Afiuni, Kristin L.M. Boylan, Melissa Geller, Peter Argenta, Samantha Hoffman, Timothy Griffin. Tandem Mass Tag 10-plex isobaric labeling of Pap test proteins: A novel method for the identification of ovarian cancer protein biomarkers by mass spectrometry. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B34.