Ion mobility mass spectrometry for extracting spectra of n-glycans directly from incubation mixtures following glycan release: Application to glycans from engineered glycoforms of intact, folded HIV gp120

Harvey, David J., Sobott, Frank, Crispin, Matthew, Wrobel, Antoni, Bonomelli, Camille, Vasiljevic, Snezana, Scanlan, Christopher N., Scarff, Charlotte A., Thalassinos, Konstantinos and Scrivens, James H. (2011) Ion mobility mass spectrometry for extracting spectra of n-glycans directly from incubation mixtures following glycan release: Application to glycans from engineered glycoforms of intact, folded HIV gp120. Journal of the American Society for Mass Spectrometry, 22 (3), 568-581. (doi:10.1007/s13361-010-0053-0).

Abstract

The analysis of glycosylation from native biological sources is often frustrated by the low abundances of available material. Here, ion mobility combined with electrospray ionization mass spectrometry have been used to extract the spectra of N-glycans released with PNGase F from a serial titration of recombinantly expressed envelope glycoprotein, gp120, from the human immunodeficiency virus (HIV). Analysis was also performed on gp120 expressed in the a- mannosidase inhibitor, and in a matched mammalian cell line deficient in GlcNAc transferase I. Without ion mobility separation, ESI spectra frequently contained no observable ions from the glycans whereas ions from other compounds such as detergents and residual buffer salts were abundant. After ion mobility separation on a Waters T-wave ion mobility mass spectrometer, the N-glycans fell into a unique region of the ion mobility/m/z plot allowing their profiles to be extracted with good signal:noise ratios. This method allowed N-glycan profiles to be extracted from crude incubation mixtures with no clean-up even in the presence of surfactants such as NP40. Furthermore, this technique allowed clear profiles to be obtained from sub-microgram amounts of glycoprotein. Glycan profiles were similar to those generated by MALDI-TOF MS although they were more susceptible to double charging and fragmentation. Structural analysis could be accomplished by MS/MS experiments in either positive or negative ion mode but negative ion mode gave the most informative spectra and provided a reliable approach to the analysis of glycans from small amounts of glycoprotein.

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Contributors

Author: Matthew Crispin

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