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

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Gondek, M., Burczynska, B. and Hills, F. 2019. Purification and method optimisation for identification of carbohydrate moieties on human chorionic gonadotropin glycoforms using MALDI IT–TOF MS. 4th FEBS Congress. Krakow, Poland 06 - 11 Jul 2019 Wiley. https://doi.org/10.1002/2211-5463.12674
TitlePurification and method optimisation for identification of carbohydrate moieties on human chorionic gonadotropin glycoforms using MALDI IT–TOF MS
AuthorsGondek, M., Burczynska, B. and Hills, F.
Abstract

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.

Research GroupBiomarkers for Cancer group
Conference4th FEBS Congress
ISSN2211-5463
PublisherWiley
Publication process dates
Deposited31 Jul 2019
Accepted06 Jul 2019
Completed11 Jul 2019
Output statusPublished
Additional information

Abstract published as:
M. Gondek, B. Burczynska, F. Hills (2019). Purification and method optimisation foridentification of carbohydrate moieties onhuman chorionic gonadotropin glycoformsusing MALDI IT–TOF MS. FEBS Open Bio, 9 (Suppl 1): ShT-37-2. doi:10.1002/2211-5463.12674

Digital Object Identifier (DOI)https://doi.org/10.1002/2211-5463.12674
LanguageEnglish
Book titleFEBS Open Bio, 9 (Suppl 1)
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Carter, A., Hills, F., O'Gorman, D., Robets, C., Soorana, S., Watson, C. and Westwood, M. 2004. The insulin-like growth factor system in mammalian pregnancy: a workshop report. Placenta. 25 (Supple), pp. S53-S56. https://doi.org/10.1016/j.placenta.2004.01.019
Imprinting of IGF2 P0 transcript and novel alternatively spliced INS-IGF2 isoforms show differences between mouse and human
Monk, D., Sanches, R., Arnaud, P., Apostolidou, S., Hills, F., Abu-Amero, S., Murrell, A., Friess, H., Reik, W. and Stanier, P. 2006. Imprinting of IGF2 P0 transcript and novel alternatively spliced INS-IGF2 isoforms show differences between mouse and human. Human Molecular Genetics. 15 (8), pp. 1259-1269. https://doi.org/10.1093/hmg/ddl041
STOX1 is not imprinted and is not likely to be involved in preeclampsia.
Hills, F., Iglesias-Platas, I., Jebbink, J. and Monk, D. 2007. STOX1 is not imprinted and is not likely to be involved in preeclampsia. Nature Genetics. 39 (3), pp. 279-280. https://doi.org/10.1038/ng0307-279
Limited evolutionary conservation of imprinting in the human placenta
Hills, F., Apostolidou, S., Arnaud, P. and Monk, D. 2006. Limited evolutionary conservation of imprinting in the human placenta. Proceedings of the National Academy of Sciences. 103 (17), pp. 6623-6628. https://doi.org/10.1073/pnas.0511031103
Regulation of human villous trophoblast by insulin-like growth factors and insulin-like growth factor-binding protein-1
Hills, F., Chard, T., Elder, M. and Sullivan, M. 2004. Regulation of human villous trophoblast by insulin-like growth factors and insulin-like growth factor-binding protein-1. Journal of Endocrinology. 183 (3), pp. 487-496. https://doi.org/10.1677/joe.1.05867
Polyclonal anti-tumor necrosis factor-alpha Fab used as an ancillary treatment for severe malaria
Looareesuwan, S., Sjorstrom, L., Krudsood, S., Wilairatana, P., Porter, R., Hills, F. and Warrell, D. 1999. Polyclonal anti-tumor necrosis factor-alpha Fab used as an ancillary treatment for severe malaria. The American Journal of Tropical Medicine and Hygiene. 61 (1), pp. 26-33. https://doi.org/10.4269/ajtmh.1999.61.26
Mechanism of action of the intrauterine contraceptive device: evidence for a specific biochemical deficiency in the endometrium
Seleem, S., Hills, F., Salem, H., El-Nashar, E. and Chard, T. 1996. Mechanism of action of the intrauterine contraceptive device: evidence for a specific biochemical deficiency in the endometrium. Human Reproduction. 11 (6), pp. 1220-1222.