En vigueur

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

Investigateur principal
V. Brun
Pays
France
Institution
Promise Advanced Proteomics
Année approuvée
2011
Statut
Complété
Themes
Autres facteurs de croissance

Description du projet

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.