Development of a human chriogonadotropin alpha, beta PSAQTM heterodimerci standard Feasibility study

Code: T11A03VB

Human chorionic gonadotropin (hCG) may be abused by male athletes in sports and 1s included in the banned substance list of the Wortd Anti-Doping Agency.The goal of this prej!;l.ct Is t9 develop an new analytical tool for the detection of hCG In eith!etes biological samples. In particular. we wm develop an isotopically-labeled version of hCG (PSAQ standard) to be used as qUi,,ritificaUon reference in mass spectrometry (MS)-based assays. ParUli:IJlarly, we w ii develop optimized protocols for expression, stable-Isotope-labelling and purification of hum􀃧n choriogona9otropin (hCG).

hCG is an heterodfmer protein composed of an a chain (common to thyrotropin, lutropin and follitropin), and a ti chain. which confers its specific biplogical activity_ The a and (i subunM are non-covalently linked and carry several dtsulfide bonds (6 disulfide bonds per subunit). The 2 chains also harbour carbohydrate moieties. 2 N-glycosylations and 4 0-g!ycosylatlo s on the fi subunit and 2 N-glycosylatlp11s on the a subunit. For optimal assay accuracy and reliablUty. the PSAQ standard will be developed as an a􀆃 heterodimer. The PSAQ heterodimer will be purified and controlled for Isotope incorporation. MS-based analyses will also be perfomed to check the sequence, structure and post-translational modifications of the standard

Main findings

This program was a feasibility study aiming at developing expression, isotope-labelling and purification protocols to produce isotopically-labeled human choriogonadotropin PSAQ standard. Choriogonadotropin protein is a heterodimer composed of an alpha chain, which is also common to thyrotropin, lutropin, follitropin, and a beta chain, which confers its specific biological activity. The alpha and beta subunits are non-covalently linked and carry numerous disulfide bonds. The 2 chains also harbour carbohydrate moieties: 2 N-glycosylations and 4 O-glycosylations on the beta subunit and 2 Nglycosylations on the alpha subunit. The first step of evaluating the expression of a choriogonadotropin in human cell free expression system was failed. In a second step of the program, each subunit (alpha and beta) was cloned with its signal peptide into our dedicated mammalian expression vector. The DNA sequence coding for the poly-His purification tag was added at the C-terminal end of sequence coding for subunit alpha protein. Then both expression vectors coding for the alpha and beta chains were co-transfected in HEK 293 cells. The choriogonadotrophin heterodimer was successfully expressed and purified. The purity was estimated to be greater than 90% at this stage. The standard was treated with PNGase F which removes N-linked glycosylations. Alpha and beta subunits sequences were verified by LC-MS/MS analyses after reduction/alkylation and in-gel trypsin digestion. Sequences coverage was higher than 50% with 3 specific peptides detected for each subunit. Stable isotope incorporation was determined using MS data generated by LC-MS/MS analyses. This experiment confirmed that the isotope incorporation of arginine and lysine was greater than 98% with limited Arginine-to-Proline conversion.