Identification and Relative Quantification of hFSH Glycoforms in Women’s Sera via MS–PRM-Based Approach

Abstract

Follicle-stimulating hormone (FSH) is a glycohormone synthesized by adenohypophysis, and it stimulates ovulation in women and spermatogenesis in men by binding to its receptor (FSHR). FSHR is involved in several mechanisms to transduce intracellular signals in response to the FSH stimulus. Exogenous FSH is currently used in the clinic for ovarian hyperstimulation during in vitro fertilization in women, and for treatment of infertility caused by gonadotropin deficiency in men. The glycosylation of FSH strongly affects the binding affinity to its receptor, hence significantly influencing the biological activity of the hormone. Therefore, the accurate measurement and characterization of serum hFSH glycoforms will contribute to elucidating the complex mechanism of action by which different glycoforms elicit distinct biological activity. Nowadays ELISA is the official method with which to monitor serum hFSH, but the test is unable to distinguish between the different FSH glycovariants and is therefore unsuitable to study the biological activity of this hormone. This study presents a preliminary alternative strategy for identifying and quantifying serum hFSH glycoforms based on immunopurification assay and mass spectrometry (MS), and parallel reaction monitoring (PRM) analysis. In this study, we provide an MS–PRM data acquisition method for hFSH glycopeptides identification with high specificity and their quantification by extracting the chromatographic traces of selected fragments of glycopeptides. Once set up for all its features, the proposed method could be transferred to the clinic to improve fertility treatments and follow-ups in men and women.

Development of an enzyme-linked immunosorbent assay for determining FSH plasma concentrations in green sea turtle (Chelonia mydas), using recombinant gonadotropins

“…In recent years the use of analytical methods to determine concentrations of gonadotropins such as LC-MS/MS and LC-MRM/ MS were implemented due to the limited availability of high-quality antibodies, and ability to distinguish between different FSH glycovariants and showed comparable sensitivity to ELISA assays, with advantage of being faster and more selective (Melchiorre et al, 2021). The majority of assays LC-MS is currently at least complementary if not superior to immunoassay and that the benefits LC-MS offers will see its increased use over immunoassay, especially as the technology advances but we are not there yet (Cross and Hornshaw, 2016).…”