Elisa Coupled Anion Exchange Methodology For Separation Of Klk6 Glycoprotein Subpopulations In Biological Fluids

Ovarian cancer is the leading cause of death among all gynecological disorders. Elevated levels of Kallikrein 6 (KLK6), a secreted trypsin-like N-glycosyalted protease, have been shown to have unfavourable prognostic value in ovarian cancer patients. It has been revealed to be aberrantly up regulated at both transcriptional and translational levels in ovarian cancer. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in the cancer of the ovary. KLK6 isolated from ascites fluid of ovarian cancer patients has been shown to have enrichment of terminal alpha 2-6 linked sialic acid at its single N-glycosylation site when compared to protein isolated from cerebrospinal fluid (CSF), which is believed to be the major source of KLK6 present in circulation. However, due to the relatively low levels of KLK6 in serum (ng/ml range) neither mass spectrometry nor lectin based characterization of this protein found in serum has been achieved. Here, we report a novel HPLC anion exchange method, coupled to a KLK6 specific ELISA, capable of differentiating KLK6 glycoform subgroups in biological fluids, including serum, at physiologically relevant levels. In brief, biological fluids were injected onto a MonoQ bead matrix (GE Healthcare), bound proteins were eluted over a salt gradient and the resulting fractions were analyzed for KLK6 content resulting in a chromatographic elution profile containing four distinct peaks. Utilizing this assay, the KLK6 elution profile and distribution across peaks of a small set (n=8) of ovarian cancer patient matched serum and ascites fluid samples was found to be different than the profile of serum and CSF of normal individuals (n=10). Utilizing tandem mass spectrometry (MS/MS), recombinant KLK6 (rKLK6) purified from an immortalized human cell line was characterized and found to have a highly heterogeneous KLK6 population, encompassing the majority of glycoforms previously shown to be present in the protein from CSF and ovarian cancer ascites. This protein was subjected to the developed assay and was found to contain all of the four diagnostic KLK6 peaks present in the previously assayed biological fluids. Due to the available high quantity of protein from this source (mg levels) we were able to analyze the rKLK6 glycoform composition of each peak utilizing lectin affinity and MS/MS based glycopeptide quantification by single reaction monitoring. The combined results showed a significant increase in terminal alpha 2-6 linked sialic acid on the N-glycans found on KLK6 from ovarian cancer serum and ascites, as opposed to CSF and serum of normal individuals. Therefore, further development and application of this methodology might lead to improvement of KLK6 as a serum ovarian cancer biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2223. doi:10.1158/1538-7445.AM2011-2223