Structural analysis of the CHO‐derived interleukin‐4 by liquid‐chromatography/electrospray ionization mass spectrometry

Abstract

AbstractElectrospray (ES) ionization mass spectrometric analysis of CHO‐derived recombinant interleukin‐4 (IL‐4) before and after deglycosylation with peptide: N‐glycosidase F is described. That proved useful not only in deriving the size of the attached carbohydrate components but also in identifying the major glycoforms as the mono‐and disialylated complex‐type N‐linked oligosaccharides. Additional signals arising from glycoforms containing carbohydrate components with more extended or higher branching were also detected. Further mapping of CHO IL‐4 was carried out by combining proteolytic digestion and chromatographic separation of the resulting peptide mixture with on‐line ES mass spectrometric detection. Comparative analysis of the V8 protease digests of CHO IL‐4 and its deglycosylated product revealed the Asn38 N‐glycosylation site. Glycopeptide‐containing fractions were identified by searching the resulting raw ES data for signal pairs whose m/z values differ by the mass of various carbohydrate units adjusted for the signal's charge state (e.g. 97 u difference for a triply charged ES signal of a glycopeptide containing NeuAc units). Furthermore, ES mass spectrometric analysis at elevated orifice potentials allowed the rapid location of the glycopeptides in the total ion current chromatogram by monitoring several carbohydrate‐specific fragment ions. This high orifice‐induced fragmentation is a highly sensitive method for generating sugar diagnostic ions in chromatographically separated components, even when glycopeptide and peptide fragments are co‐eluting.