Nathalie DELAUNAY, researcher from Laboratoire Sciences Analytiques, Bioanalytiques et Miniaturisation – CNRS, ESPCI Paris, PSL Université will give a lecture on “Development of different sample handling and separation methods hyphenated to mass spectrometry for the characterization of glycoproteins at the intact scale ” the 10th October 2022 at the Institute for analytical science (11:00 am – seminar room).
Abstract: The human chorionic gonadotropin (hCG) is the hormone specific to human pregnancy. It is essential for the development of placenta and foetus. It is based on two subunits, hCGα and hCGβ, having in total 4 N- and 4 O-glycosylation sites. Some studies demonstrated that there is a correlation between the hCG glycosylation state and the foetus implantation. This is why the characterization of the hCG glycoforms is needed, but the high number of glycosylation sites and thus glycoforms constitutes an analytical challenge.
To address this issue, capillary electrophoresis (CE) methods based on three different separation mechanisms were first investigated for the characterization of the glycosylation of hCG at the intact level using two hCG-based drugs. Then, two liquid chromatography (LC) methods in reversed phase (RP) and HILIC modes were developed for the analysis of the intact hCG glycoforms and allowed, by their complementarity, the detection of a high number of hCGα glycoforms. The transfer of the RPLC method to the nano scale and its hyphenation with high resolution mass spectrometry, as well as optimization of chromatographic conditions, led to an improvement of the separation of hCGα and hCGβ glycoforms, which allowed for the first time the detection of 33 hCGβ glycoforms at the intact level. In addition, a higher number of hCGα glycoforms was detected.
Regarding the development of a selective extraction step of hCG glycoforms from serum of pregnant women, two anti-hCG antibodies were selected to target hCG regions that are expected to be not modified by glycosylation and thus should have affinity for all its glycoforms. They were immobilized on Sepharose and these immunosorbents were evaluated to extract hCG glycoforms using our hCG “standards”. A second approach using lectin-based sorbents was evaluated to try to fractionate hCG glycoforms to simplify the analysis and data treatment.