Urinary follicle stimulating glycoform analysis by automated Western blot
AdvisorBousfield, George R.
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Follicle-stimulating hormone (FSH) is a critical hormone for fertility in women. In females, FSH enables ovarian follicle development, thereby producing mature oocytes at ovulation. Studies from our laboratory have shown that human FSH exists as two major glycoforms, fully glycosylated FSH24, which possesses two α-subunit and two β-subunit asparagine-linked oligosaccharides, and hypo-glycosylated FSH21 or FSH18 glycoforms, which both possess one FSHβ oligosaccharide and two α-subunit oligosaccharides. Pituitary FSH21 abundance exhibits reduced relative abundance with increasing age in women. As FSH21 exhibits greater FSH biologic activity than FSH24, age- and cycle-related changes in glycoform abundance may contribute to fertility regulation. The goal of this project is to evaluate FSH glycoform ratios in female urine samples. Human FSH and other urinary proteins were precipitated with ethanol and FSH captured by immuno-affinity chromatography using the anti-FSHβ monoclonal antibody. Automated Western blotting of FSH samples was employed to measure the relative abundance of both FSH glycoforms based on the density of the 21kDa- and 24kDa-FSHβ subunit bands. FSH was captured from 6 postmenopausal urine samples by anti-FSHβ monoclonal antibody. Monoclonal antibody can capture the majority of FSH in precipitated urinary protein samples and it is feasible to measure FSH glycoform abundance in urinary protein samples. Finally, the results from automated western blotting is compared with published conventional western blot data.
Poster project completed at the Wichita State University Department of Biological Sciences. Presented at the 17th Annual Capitol Graduate Research Summit, Topeka, KS, February 26, 2020.