Extended Data Fig. 5: N-glycan analysis by quantitative mass spectrometry.
Electrophoretic separation of gp120 Env subunit using SDS-PAGE under denaturing and reducing conditions, stained with Coomassie G-250 (a-c): a, Total BG505, 007IgG-bound BG505, and 007IgG control; b, Total BG505 with and without PNGase F treatment; c, 007IgG-bound BG505 with and without PNGase F treatment. The shift in mobility of PNGase F-treated samples in (b-c) indicates removal of N-glycans from gp120. Arrows mark gp120, deglycosylated gp120, IgG heavy and light chains, and PNGase F. Asterisks indicate bands that were excised and stored for LC-MS analyses. Red text and arrows indicate gp120 or deglycosylated gp120 bands. d, Comparison of glycoform abundance in total unliganded BG505 and 007IgG-bound BG505 at N156gp120/N160gp120, N234gp120, N295gp120, N301gp120, and N448gp120. Data are presented as a side-by-side bar graph, where different high-mannose glycoforms are differentiated, but hybrid and complex-type glycans are presented as single groups. Points represent the replicative measurements (three for total BG505 and two for bound BG505) and bar graphs represent the mean of the replicative measurements. For visualization purposes, data are also presented as a stacked bar with individual glycoforms separated by a vertical line, and the most prevalent glycoform(s) labeled.
