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

Chiara Melchiorre,Andrea Carpentieri,Alessandro Conforti,Cerina Chhuon,Vincent Jung,Francesca Di Rella,Ida Chiara Guerrera,Angela Amoresano,Joanna Lipecka

doi:10.3390/pharmaceutics13060798

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.

Highlights

IntroductionFollicle-stimulating hormone (FSH) belongs to the gonadotropin glycoproteins hormone family, and it has a central and essential role in human reproduction
Protein identification was performed by the Byonic search engine, and each recognized glycopeptide was validated by manually interpreting mass spectrometry (MS)/MS spectra
To the best of our knowledge, we provided for the first time a MS–PRMPRM-based strategy for the characterization of an hFSH glycan moiety from serum and its based strategy for the characterization of an hFSH glycan moiety from serum and its sitesite-specific relative quantification

Summary

Introduction

Follicle-stimulating hormone (FSH) belongs to the gonadotropin glycoproteins hormone family, and it has a central and essential role in human reproduction. FSH characterization is essential to elucidating reproductive physiology-regulating fertility, and to improving the diagnosis and treatment disorders of reproduction [1]. FSH consists of two subunits, a 92-amino acid α-subunit and a 111-amino acid β-subunit [2]. The FSH α-subunit is common to all the gonadotropins, and its high degree of structural conservation among different mammalian species implies an important role in the evolutionary process [3]. The β-subunit is unique to FSH [2] and binds to its G protein-coupled receptor (FSHR).

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Conclusion