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Title

An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level

Authors
Camperi, Julien
Combes, Audrey
Guibourdenche, Jean
Pichon, Valerie
Fournier, Thierry
Delaunay, Nathalie
Published in Journal of Pharmaceutical and Biomedical Analysis. 2018, vol. 161, p. 35-44
Abstract For the first time, the human Chorionic Gonadotropin (hCG) hormone at the intact level was character-ized by reversed phase liquid chromatography (RPLC) coupled with high resolution mass spectrometry(HRMS). This heterodimeric protein is specific to human pregnancy, consists in an and a subunit, so-called hCG and hCG, respectively, and has 8 glycosylation sites leading to a high number of isoforms.First, the LC method was optimized to separate the largest number of isoforms and also to facilitate theMS ionization process and data treatment. The initial mobile phase composition, slope of the gradient,and column temperature were appropriately selected to maximize the number of separated isoforms.Moreover, the MS detection parameters were adjusted to i) promote the efficient transfer of the heaviestions, ii) avoid or limit the fragmentation of the ions and iii) improve the sensitivity. The repeatability ofthe final method in terms of retention times and peak areas was assessed. The method was next used tocharacterize two hCG-based drugs: Ovitrelle®(a recombinant hCG, r-hCG) and Pregnyl®(hCG isolatedfrom urine of pregnant women, u-hCG). After the deconvolution step, the analytical method did not allowto observe the isoforms of the hCG. This may be due to its dramatic higher heterogeneity induced byits 6 glycosylation sites and a lack of ionization in the MS source. Nevertheless, the results revealed thepresence of more than 30 hCG isoforms, which differ by their number and their nature in the two drugs.Then, the molecular weights of the N-glycans already described in the literature for hCG were compiledin a database to identify the hCG glycoforms by mass matching. This strategy was successfully appliedfor the identification of five glycoforms for both r-hCG and u-hCG. This work demonstrates for the firsttime the potential of RPLC-HRMS for the identification of the intact hCG glycoforms.
Keywords GlycosylationHuman Chorionic GonadotropinIntact proteinLiquid chromatographyQTOF high-resolution mass spectrometry
Identifiers
PMID: 30144627
Full text
Structures
Research group Sciences analytiques
Citation
(ISO format)
CAMPERI, Julien et al. An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level. In: Journal of Pharmaceutical and Biomedical Analysis, 2018, vol. 161, p. 35-44. doi: 10.1016/j.jpba.2018.07.056 https://archive-ouverte.unige.ch/unige:113097

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Deposited on : 2019-01-21

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