Mass Spectrometry, Quantification, Isotope-Dilution Internal PSAQ Standard
Human chorionic gonadotropin (hCG) may be abused by male athletes in sports and is included in the banned substance list of the World Anti-Doping Agency. An ti - soping laboratories mainly use immunoassays to quantify hCG but cross-reactivity with the different forms of hCG can constitute a poblem for quantitative hCG determination especially in urine samples. Choriogonadotropin protein is a heterodimer composed of an alpha chain, whjich 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 ans 4O- glycosylations on the beta subunit and 2 N-glycosylations on the alpha subunit. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers analytical specificity superior to that of immunoassays and can be alternative method for wuantification of hCG in athletes biological samples. for optimal assay accuracy and reliability, a stable isotope labeled internal standard should be used. In a precious project, we developed a protpcol to produce an isotopically-labeled version of hCG (PSAQ standard). The choriogonadotrophin heretodimer was successfully expressed in mammalian cells and purified. Stable isotope incorporation was determined to be greater than 98%. The goal of this project is to produce 200 to 500μg of hCG PSAQ standard according to the protocol previously developed to deliver different anti-doping laboratories.
Recombinant hCG protein was produced and labelled in HEK293 cells. The choriogonadotrophin heterodimer was successfully expressed and purified using Ni-IMAC resin. To obtain higher purity level, the sample was submitted to a second purification step using Ion Exchange Chromatography (IEX). The purity was estimated to be greater than 95%.
In this program, to increase the sequence coverage, a sample of hCG was treated with a combination of five enzymes to remove N-linked glycans and many common O-linked glycans. Following deglycosylation, hCG alpha and beta subunits sequences were verified by LC-MS/MS analysis after reduction/alkylation and in-gel trypsin digestion of the purified protein. The sequence coverage of CGB is greatly improved after treatment with the Deglycosylation Mix compared to the previous feasibility study where the protein was only digested with PNGase F. Stable isotope incorporation was estimated using MS data generated by LC-MS/MS analysis.
hCG PSAQ™ standard will be delivered in aliquots of 20 μg. We recommend to store at -80°C upon receipt.