Overcoming Challenges in Structural Characterization of HIV-1 Envelope Glycoprotein by LC-MS/MS.

Characterization of HIV Env glycoprotein with 28 glycosylation sites is the essential step of structure-based vaccine design programs. A comprehensive LC-MS/MS peptide mapping analysis was applied to assess the primary sequence, glycosylation profiles, and glycosite occupancy of Env to ensure the adequate mimicking of the native immunogen. Another structural feature was reported, related to its cleaved subunits within the trimeric assembly. We bring attention to the importance of thorough inspection of the results generated by the informatics tools which are currently available for the biopharmaceutical characterization. The complexity of Env translates into a vast amount of data with occasional information gaps that could not possibly be filled by means of the automatic data analysis. A series of data validation steps was applied, followed by the illustrations on how the high-quality results may be misinterpreted. It was shown that the glycan sites can only be characterized to a certain limit, and that any claim of full structural characterization of this molecule beyond these limits should be treated with caution. Following the result verification, the percent glycan occupancy was reported for 25 N-glycan sites, including 3 critical antibody-recognition sites. The exact glycan profiles were provided for 20 individual sites, whereas only the glycosylation type could be deduced for 5 sites, dictated by their location within Env sequence. The distribution of the unprocessed high mannose-type glycans correlated with the expected "mannose patch." Experimental procedure optimization and a workflow for glycan characterization with a focus on stringent data testing are presented in the current study.