title: Separation of kallikrein 6 glycoprotein subpopulations in biological fluids by anion-exchange chromatography coupled to ELISA and identification by mass spectrometry.
abstract: Kallikrein 6 (KLK6) has been shown to be aberrantly glycosylated in ovarian cancer. Here, we report a novel HPLC anion exchange method, coupled to a KLK6-specific ELISA, capable of differentiating KLK6 glycoform subgroups in biological fluids. Biological fluids were fractionated using anion exchange and resulting fractions were analyzed for KLK6 content by ELISA producing a four-peak elution profile. Using this assay, the KLK6 elution profile and distribution across peaks of a set (n = 7) of ovarian cancer patient matched serum and ascites fluid samples was found to be different than the profile of serum and cerebrospinal fluid (CSF) of normal individuals (n = 7). Glycosylation patterns of recombinant KLK6 (rKLK6) were characterized using tandem mass spectrometry (MS/MS), and found to consist of a highly heterogeneous KLK6 population. This protein was found to contain all of the four diagnostic KLK6 peaks present in the previously assayed biological fluids. The rKLK6 glycoform composition of each peak was assessed by lectin affinity and MS/MS based glycopeptide quantification by product ion monitoring. The combined results showed an increase in terminal alpha 2-6 linked sialic acid in the N-glycans found on KLK6 from ovarian cancer serum and ascites, as opposed to CSF and serum of normal individuals.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Uros
authors.initials: U
authors.lastname: Kuzmanov
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Smith
authors.forename: Ihor
authors.initials: I
authors.lastname: Batruch
authors.forename: Antoninus
authors.initials: A
authors.lastname: Soosaipillai
authors.forename: Anastasia
authors.initials: A
authors.lastname: Diamandis
authors.forename: Eleftherios P
authors.initials: EP
authors.lastname: Diamandis
mesh_terms: D002852:Chromatography, Ion Exchange; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007610:Kallikreins / Q000032:analysis / Q000097:blood* / Q000134:cerebrospinal fluid*; D010051:Ovarian Neoplasms / Q000097:blood* / Q000134:cerebrospinal fluid*; D011994:Recombinant Proteins / Q000032:analysis; D053719:Tandem Mass Spectrometry
chemical_list: D006020:Glycopeptides; D011994:Recombinant Proteins; C103551:KLK6 protein, human; D007610:Kallikreins