Poster Session - Abstract # 26

Affinity-free Enrichment and Mass-spectrometry Characterization of CA125 (MUC16) from Ovarian Cancer Patient Ascites

Naviya Schuster-Little¹, Roberta Fritz-Klaus², Mark Etzel³, Niharika Patankar², Saahil Javeri², Manish S. Patankar² and Rebecca J. Whelan¹

¹Department of Chemistry, University of Kansas, Lawrence, KS, USA; ²Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, WI; ³Department of Food Science, University of Wisconsin-Madison, Madison, WI

Ovarian cancer 5-year survival rates exceed 90% when diagnosed at the localized stage; however, owing to a lack of clear signs and symptoms, over half of ovarian cancer patients are diagnosed with distant-stage disease, for which survival rates drop to 29%.  This decrease in survival rates can be attributed to the lack of a biomarker or diagnostic test approved for population-wide preventative screening.  The biomarker of greatest utility in the clinical management of ovarian cancer is CA125, the peptide epitope of the glycoprotein MUC16.  CA125 is currently detected using an immunoassay, and counts are tracked to monitor a patient’s response to treatment or recurrence of disease.  We aim to develop an antibody-free assay that uses mass spectrometry to detect and quantitate MUC16 isolated from the biofluids of individual patients.  Here, we report an optimized bottom-up proteomics workflow that involves suspension trapping (S-Traps™) and uses deoxycholic acid (DCA) as a passivating agent to reduce protein loss through adsorption.  Proteins are denatured, reduced, and alkylated in the presence of sodium dodecyl sulfate and DCA.  Following alkylation, sample is spun onto an S-Trap™, and proteins are digested using trypsin.  Peptides are eluted from the trap, desalted, and analyzed using nano LC-MS/MS on a Q-Exactive mass spectrometer.  Inclusion of DCA results in improved coverage of MUC16, from 3 to 12%. We applied the bottom-up proteomics workflow to ascites that contains tumor-associated proteins including MUC16. MUC16 is enriched from ascites using an immunoaffinity-free method that consists of filtration, ion exchange and size-exclusion chromatography.  Using this method, we detect MUC16 peptides in ascites samples from three patients with high grade serous ovarian cancer.  The peptides map to the tandem repeat and C-terminus of MUC16 and peptide abundance correlates with clinical CA125 counts.  These results will help us develop a mass spectrometry-based assay for detecting the CA125 cancer biomarker, which may result in earlier detection of ovarian cancer.