Relating glycoprotein structural heterogeneity to function - insights from native mass spectrometry

Abstract

Glycosylation is the most complex and prevalent protein modification that influences attributes ranging from cellular localization and signaling to half-life and proteolysis. Glycoconjugates are fundamental for cellular function and alterations in their structure are often observed in pathological states. Most biotherapeutic proteins are glycosylated, which influences drug safety and efficacy. Therefore, the ability to characterize glycoproteins is important in all areas of biomolecular and medicinal research. Here we discuss recent advances in native mass spectrometry that have significantly improved our ability to characterize heterogeneous glycoproteins and to relate glycan structure to protein function.

Figure 1

Mass spectrometry strategies for glycoproteins focus on structural analysis of isolated glycans (glycomics), compositional and positional glycosylation from enzyme digested peptides (glycoproteomics) and global evaluation of intact glycoprotein assemblies by native analysis. Glycoprotein heterogeneity arrises from variable glycan structures (microheterogeneity) and different degrees of occupancy at a given site (macroheterogeneity). N-Glycans are covalently attached to asparagine residues containing an N-X-S/T sequon and are classified as oligomannose, complex or hybrid type. O-Glycans do not have specific amino acid sequence requirements and can be linked to serine or threonine residues. Both N-glycans and O-glycans are synthesized in a non-template driven manner producing a range of structures with different monosaccharide compositions, linkages and topologies.

Figure 2

Native mass spectrum of monoclonal IgGs (150 kDa) display a characteristic charge-state distribution of 21–27+ centering at 6000 m/z (trastuzumab (Herceptin) is shown). The pairing of the two conserved complex-type N-glycans, commonly referred to as GO, G1, and G2, present on each heavy chain (HC) in the Fc domain (Asn 297) are well-defined. Native MS can easily identify glycan structural features such as core fucosylation (red triangles) and terminal galactosylation (yellow circles), which are important attributes in biotherapeutic activity.

Figure 3

(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].