Glycosylation Profiling of IL-23: Determination of N-Glycosylation Sites and Structure Characterization of the Oligosaccharides by Mass Spectrometry

摘要

As a new member of the growing family of interleukin (IL)-12-related cytokines, IL-23 is a key immunoregulatory cytokine that coordinates innate and adaptive immune responses (1). IL-23 promotes end-stage inflammation, which makes it and the proteins in its down-stream pathway potential targets for the treatment of immune-mediated diseases. As a heterodimeric cytokine that comprises of disulfide-linked p19 and p40 subunits, IL-23 has four potential N-glycosylation sites ~(125)NKTF, ~(135)NYSG, ~(222)NYTS, and ~(303)NASI. Structural characterization of the glycoforms of IL-23 has not yet been reported. In this presentation, we describe the use of mass spectrometry (MS) to establish the glycosylation profiles of recombinant IL-23, which includes defining glycosylation sites, the degree of occupancy of each glycosylation site, and the structures of the oligosaccharides attached to each site. Single chain IL-23 (p19-peptide linker-p40) was expressed in adenovirus or baculovirus. LC/ESI-MS and MALDI MS were applied to detect the molecular weight (MW) of the purified recombinant IL-23. MS strategies involved enzymatic digestion of the glycoprotein and analysis of the resulting peptide fragments for the determination of the site specific glycosylation pattern. Specifically, identification of glycosylated peptides generated from trypsin digestion of IL-23 was accomplished by LC/ESI-MS peptide mapping followed by precursor ion scans of oxonium ions of GIcNAc~(+) (m/z 204) and Hex-GIcNAc~(+) (m/z 366) on a triple quadruple MS instrument (ABI API4000). To determine the glycosylation sites and the structures of the oligosaccharides of each site, the glycopeptides were fraction collected from the RP-HPLC separation and de-glycosylated with PNGase F. Both the glycopeptides and their corresponding apopeptides were further studied by MALDI MS (ABI DE-STR) and LC/ESI-MS/MS (Micromass Q-Tof2).