title: Endo-β-N-acetylglucosaminidase H de-N-glycosylation in a domestic microwave oven: application to biomarker discovery.
abstract: Sample preparation is the rate-limiting step in glycan analysis workflows. Among all of the steps, enzymatic digestions, which are usually performed overnight, are the most time-consuming. In the current study, we report an economical and fast preparation of N-glycans from serum, including microwave-assisted enzymatic digestion in the absence of denaturing chemicals and solvents during the release. To this end, we used a household microwave oven to accelerate both pronase and endo-β-N-acetylglucosaminidase H (Endo H) digestions. Purification was then performed using self-made SP20SS and carbon tips. We were able to prepare samples in 55 min instead of 21 h. Finally, the method was applied in the context of oncological biomarker discovery exemplarily to ovarian and colon cancer. We observed a significant downregulation of sialylated hybrid structures in ovarian cancer samples using capillary electrophoresis-laser-induced fluorescence (CE-LIF). Furthermore, sepsis, a systemic inflammatory response syndrome, was also included in the study to understand whether the changes observed in ovarian cancer patients were due to the cancer itself or to the inflammation that usually accompanies its development. Because sialylated hybrid structures were upregulated in sepsis samples, the downregulation of these structures in ovarian cancer is specific to the cancer itself and, therefore, could be used as a biomarker.
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry, and Pathobiochemistry, Charité Medical University, 13353 Berlin, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Frisch
authors.forename: Christian
authors.initials: C
authors.lastname: Schwedler
authors.forename: Matthias
authors.initials: M
authors.lastname: Kaup
authors.forename: Elena
authors.initials: E
authors.lastname: Iona Braicu
authors.forename: Jörn
authors.initials: J
authors.lastname: Gröne
authors.forename: Johannes C
authors.initials: JC
authors.lastname: Lauscher
authors.forename: Jalid
authors.initials: J
authors.lastname: Sehouli
authors.forename: Matthias
authors.initials: M
authors.lastname: Zimmermann
authors.forename: Rudolf
authors.initials: R
authors.lastname: Tauber
authors.forename: Markus
authors.initials: M
authors.lastname: Berger
authors.forename: Véronique
authors.initials: V
authors.lastname: Blanchard
mesh_terms: D053000:Analytic Sample Preparation Methods / Q000191:economics / Q000379:methods*; D014408:Biomarkers, Tumor / Q000097:blood / Q000378:metabolism*; D001774:Blood Chemical Analysis; D003110:Colonic Neoplasms / Q000097:blood; D005260:Female; D006031:Glycosylation; D006798:Housing; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism*; D008872:Microwaves; D009584:Nitrogen / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000097:blood; D011134:Polysaccharides / Q000097:blood / Q000378:metabolism; D018805:Sepsis / Q000097:blood; D013997:Time Factors
chemical_list: D014408:Biomarkers, Tumor; D011134:Polysaccharides; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009584:Nitrogen