Purification and method optimisation for identification of carbohydrate moieties on human chorionic gonadotropin glycoforms using MALDI IT–TOF MS

The expression of human chorionic gonadotropin (hCG) is com-monly associated with a variety of cancers including testicular,bladder and gestational trophoblast diseases. All trophoblastictumours secrete very high amounts of hCG however hyperglyco-sylated form of hCG was found to be highly elevated in chorio-carcinoma patients. Current analytical methods, measuringbranched N-linked glycans in hCG, involve use of antibodieswhich may cause uncertainty due to issues of antibody specificity.In this project, highly sensitive and robust method to identify N-linked glycans, based on their specific chemical composition andmass tolerance, was developed. It was applied to variety of hCGforms, including hCG from pregnancy, choriocarcinoma andrecombinant hCG. hCG and its varied forms, with freebhCG,were isolated using IgG-coupled magnetic beads. N-linked gly-cans were then isolated from hCG by denaturation, reduction,alkylation (100°C, 10mM DTT, 20mM IAA) followed by degly-cosylation (PNGase, 37°C, 20h) and purification using porousgraphite carbon tips (Hypercarb). MALDI IT-TOF MS was usedto separate resulting glycans, which were then characterised usingProtein Scape software. Glycan peaks were reported, m/z 900–5,000 (Da), and carbohydrate library with precise glycan searchparameters (GlycoQuest, Bruker) was designed. A series of gly-cans were successfully identified from selected hCG sources, andsignificant differences in repertoire of glycans composition weredetected. Alteration in cellular glycosylation of proteins con-tribute to cancer metastasis, therefore strategies to understandtumour glycome and glycoproteome provide diagnostic potential,and targets for therapeutic intervention. Rapid and efficientdetection method of specific hCG glycoforms, from crudepatients urine samples, could help to speed and improve diagno-sis of hCG associated cancers including choriocarcinoma andnon-trophoblastic malignancies