(a) Glycoengineering strategies direct N-glycan biosynthetic enzymes in the endoplasmic reticulum (ER) and early Golgi to generate homogeneous glycoforms. Small molecule inhibitors such as NB-DNJ, kifunensine, and swainsonine, as well as the HEK293 GnT-I knock-out cell line, are particularly useful for delineating glycan composition. (b) For example, kifunensine, which blocks processing to produce Man9GlcNAc2 N-glycans, was recently used to assign glycosylation occupancy of recombinant HIV-1 gp120 (adopted from PDB: 5ACO). (c) Global N-glycan occupancy of gp120 could be quantified simply by evaluating the number of Man9GlcNAc2 moieties (inset). Additionally, simplification of protein N-glycosylation revealed the presence and number of O-glycans. Figure adapted from Ref. [45].