Site-Specific Analysis of N-Linked Glycosylation Heterogeneity from Royal Jelly Glycoproteins.

Abstract

Royal jelly (RJ) is secreted by young worker bees, and it plays key roles in the development and physiological function in honeybees and can improve human health. Although there have been analyses on the glycosylation modification of RJ proteins, none of these methods have been conducted on a site-specific analysis of glycosylation from these glycoproteins. Here, a combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity of RJ glycoproteins. First, global characterization of the N-glycome of RJ was performed using a direct infusion ion trap-sequential mass spectrometry (IT-MSn) method. Second, tryptic glycopeptides were enriched and separated by hydrophilic interaction liquid chromatography-ion trap-sequential mass spectrometry (HILIC-IT-MSn). A total of 50 N-glycopeptides and 30 N-glycans have been site-specific glycosylation profiled in major royal jelly protein 1 (MRJP1) and MRJP2 of RJ for the first time. Eighteen of the identified N-glycans have been structurally characterized by IT-MSn, including oligosaccharide composition, sequence, branching, and linkage. Two N-glycosylation sites (N177 and N394), 3 sites (N145, N178, and N92), and 1 site of N183 were identified in MRJP1, MRJP2, and MRJP3, respectively. There were 18, 17, and 2 N-glycans attached to MRJP1, MRJP2, and MRJP3, respectively. The diversity of N-glycans attached to each single glycosylation site of these glycoproteins confirmed that MRJP1 and MRJP2 heterogeneity was mostly associated with their glycoform populations. Understanding the properties of the site-specific glycosylation heterogeneity of the RJ glycoproteins can be potentially useful for producing a glycoprotein with desirable pharmacokinetic and biological activity.