POSTER: Characterization of the glycoforms of hCG by Chromatographic and Electrophoretic Separation Techniques.

Nathalie Delaunay, Julien Camperi, Bart De Cock, Audrey Combès, Debby Mangelings, Yvan Vander Heyden, Jean Guibourdenche, Valerie Pichon, Thierry Fournier

Research output: Unpublished contribution to conferencePoster

Abstract

The discovery of new maternal markers from placental origin to improve the early prognosis of implantation pathologies represents a major challenge. Indeed, a poor placentation is directly involved in many diseases of pregnancy, foetal growth restriction and prematurity. The human Chorionic Gonadotropin (hCG) hormone is essential for the maintenance of the pregnancy and for the development of the placenta. Some studies demonstrated that different sources produce hCG and affect its glycosylation sites [1], leading to different biological activities [2,3]. This is why it seems relevant to think that some glycosylated forms of hCG could be maternal biomarkers to diagnose so-called "at-risk" pregnancies.

The heterodimer hCG is composed of two subunits, hCGα and hCGβ, and 8 potential glycosylation sites, which can give a very large number of glycoforms. The objectives of this study is to characterize and to quantify the different glycoforms of hCG in different biological samples: placental cell cultures, blood samples from pregnant women and their urines after catabolization. A chemometric approach will be next used to identify the biomarkers of placental implantation and development. A miniaturized prognosis tool will be finally developed.

Thus, the analytic challenge is complex and it is necessary to implement several complementary analytical techniques with orthogonal separation mechanisms, to characterize and quantify the different glycoforms, some of which are in very low concentrations in complex and varied samples. First, a top-down approach was selected to analyze the intact hCG glycoforms by capillary electrophoresis (CE) and liquid chromatography (LC). Two hCG-based drugs were used as hCG standards: Ovitrelle (recombinant hCG, r-hCG) and Pregnyl (hCG from the urines of pregnant women, u-hCG). A method of analysis by capillary isoelectric focusing (CIEF) was developed, testing different ampholyte compositions to improve the resolution in the pH range of interest. It allowed the determination of the isoelectric points (pI) of the different isoforms of r-hCG, u-hCG, and hCGβ. As an example, more than 15 r-hCG isoforms were detected with pI values between 3.5 and 5.0. Capillary gel electrophoresis (CGE) was also used to separate according to mass the isoforms of r-hCG and u-hCG, but also their hCGα and hCGβ subunits after reduction with 2-mercaptoethanol. The results obtained by CGE and CIEF were in agreement with results in literature obtained with SDS-PAGE but here with a significant improvement of the resolution. A method in capillary zone electrophoresis (CZE) with a UV detection was next developed with a volatile electrolyte (ammonium acetate) for future hyphenation with mass spectrometry (MS) with a triple quadrupole analyzer, after electrospray ionization with a sheath liquid interface. In parallel, the analysis of intact hCG by reversed phase liquid chromatography coupled with high-resolution MS (time-of-flight) detection was also developed.
Original languageEnglish
Publication statusPublished - 2017
EventEighth International Symposium on the Separation and Characterization of Natural and Synthetic Macromolecules (SCM-8) - Amsterdam, Netherlands
Duration: 1 Feb 20173 Feb 2017

Conference

ConferenceEighth International Symposium on the Separation and Characterization of Natural and Synthetic Macromolecules (SCM-8)
CountryNetherlands
CityAmsterdam
Period1/02/173/02/17

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