ISSN:
1076-5174
Keywords:
Chemistry
;
Analytical Chemistry and Spectroscopy
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Chemistry and Pharmacology
,
Physics
Notes:
Electrospray (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.
Additional Material:
11 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jms.1190301217
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