Identification and semi-relative quantification of intact glycoforms of human chorionic gonadotropin alpha and beta subunits by nano liquid chromatography-Orbitrap mass spectrometry.

摘要: Abstract The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is heterodimer made two non-covalently linked subunits. α-subunit structure, hCGα, has 2 N-glycosylation sites, while the beta subunit, hCGβ, 2 N- and 4 O-glycosylation sites. This leads numerous glycoforms. A method based on analysis hCG glycoforms at intact level by nano-reversed phase liquid chromatography coupled high resolution mass spectrometry (nanoLC-HRMS) with an Orbitrap analyzer was previously developed using recombinant hCG-based drug, Ovitrelle®, as standard. allowed detection about 30 hCGα glycoforms, but didn't allow hCGβ thus here significantly modified (addition pre-concentration step sample increase volume from 70 nl 1 µl, optimization gradient slope nature content acidic additive in mobile phase). led improvement separation which for first time 33 level. In addition, higher number (42 total, i.e. 40% increase) detected. figures merit this new were next assessed. relative standard deviations (RSDs) retention ranged between 0.02 0.95% (n = 3), average value 0.36% alpha 0.01 1.08% (n = 3) 0.23% RSDs peak area measured extracted ion chromatogram each glycoform below 20% 9.8%, allowing semi-relative quantification. Therefore, potential rapid quality control aiming comparison present glycoprotein-based pharmaceutical preparations.