Characterization of Stability and Small-scale Structural Differences of Intact Glycoprotein Complexes Using Native Mass Spectrometry-Travelling-Wave Ion Mobility Spectrometry

Glycosylation is one of the most common types of post-translational modifications (PTMs) of proteins, which affects protein properties including conformation, stability and solubility, and plays essential roles in functions including molecular recognition, signaling and immune defense. Characterizing the higherorder structures and dynamics of glycoprotein complexes at the intact level is essential for studying the biological functions of glycoproteins. Particularly, accessing structural information such as glycosylation pattern, proteoform distribution, protein interaction, and dynamic conformational evolvement requires direct measurement of intact species. Native mass spectrometry (nMS) allows preservation of noncovalent interprotein interactions and direct analysis of topology, stoichiometry and dynamic assembly/disassembly at the intact complex level, highly complementing the conventional biophysical techniques. The combination of nMS with travellingwave ion mobility spectrometry (TWIMS) provides not only an additional orthogonal dimension of separation, but also geometric features of the analytes through collisional cross-section (CCS) determination, thereby facilitating the structural characterization. The macro- and micro-heterogeneity caused by glycosylation, as well as binding with small molecules, induce structural changes of glycoproteins and their complexes on a scale smaller than those resulting from unfolding or macromolecular association/dissociation. However, the benefits of nMSIMS for characterization of smallscale structural changes or differences of glycoprotein complexes remain to be further demonstrated, in aspects including proteoformspecific structural analysis, distinguishing different binding states of glycoprotein complexes in complex with small molecules, and solutionparameterinduced conformational or stability changes. In this work, aiming at characterizing the stability and smallscale structural differences of glycoprotein complexes, two formats of avidin (tetrameric biotin-binding glycoprotein) that were expressed from different species and exhibit differing carbohydrate contents and proteoform patterns were used as model systems, and the benefits of implementing ion mobility analysis in nMS for structural analysis of both intact complexes and subunits released in the gasphase were evaluated. It was demonstrated that IMS allows separation of subpopulations of proteoforms, which facilitates not only profiling of proteoform distribution for intact glycoproteins by MS, but also investigation of correlation between glycosylation and CCS. We also demonstrated the power of IMS in measuring the geometric features of the protein complexes in complex with small molecules, and characterizing the extent of conformational changes of glycoproteins under conditions including gasphase dissociation, supercharging, chargereduction, and chargemanipulated dissociation. These advantages enable the stability evaluation for glycoprotein complexes.