Mass spectrometric characterization of the glycosylation pattern of HIV-gp120 expressed in CHO cells

An analytical approach is reported for the characterization of the specific glycans found on highly glycosylated proteins based on a combination of sp ecific proteolysis and deglycosylation combined with two different mass spe ctrometric approaches, matrix-assisted laser desorption/ionization mass spe ctrometry, and nanoelectrospray mass spectrometry/tandem mass spectrometry using a hybrid quadrupole-time-of-flight tandem mass spectrometer. The high resolution and mass accuracy of the mass spectrometric data obtained on th e hybrid instrument combined with the high parent mass capabilities are sho wn to be extremely useful in the site-specific assignment of heterogeneous glycans. Using this methodology, 25 of 26 consensus glycosylation sites on HIV-1(SF2) gp120, expressed in Chinese hamster ovary cells, could be assign ed. Good correlations between the relative abundances of members of heterog eneous series in the matrix-assisted laser desorption/ionization mass spect ra and the nanoelectrospray mass spectra were observed, indicating that the mass spectrometric data reflected the actual abundances of the members of the series. These data were incorporated with molecular modeling based on t he solved structure of a mutant truncated, highly deglycosylated gp120 to p ropose a structural model for the completely glycosylated form.