Structural Analysis of the Glycoprotein Complex Avidin by Tandem-Trapped Ion Mobility Spectrometry–Mass Spectrometry (Tandem-TIMS/MS)

Abstract

Glycoproteins play a central role in many biological processes including disease mechanisms. Nevertheless, because glycoproteins are heterogeneous entities, it remains unclear how glycosylation modulates the protein structure and function. Here, we assess the ability of tandem-trapped ion mobility spectrometry–mass spectrometry (tandem-TIMS/MS) to characterize the structure and sequence of the homotetrameric glycoprotein avidin. We show that (1) tandem-TIMS/MS retains native-like avidin tetramers with deeply buried solvent particles; (2) applying high activation voltages in the interface of tandem-TIMS results in collision-induced dissociation (CID) of avidin tetramers into compact monomers, dimers, and trimers with cross sections consistent with X-ray structures and reports from surface-induced dissociation (SID); (3) avidin oligomers are best described as heterogeneous ensembles with (essentially) random combinations of monomer glycoforms; (4) native top-down sequence analysis of the avidin tetramer is possible by CID in tandem-TIMS. Overall, our results demonstrate that tandem-TIMS/MS has the potential to correlate individual proteoforms to variations in protein structure.

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry

“…The native S-RBD without any deglycosylation could not be isotopically resolved due to multiple overlapping ion signals from all of the diverse glycoforms/proteoforms carrying different charges ( Figure S2C and S2D ), as observed in complex glycoproteins previously. 36 , 50 …”

“… 61 Moreover, the CCS distribution for the native S-RBD O-glycoform dimer (Region 2, 15+ charge state, TIMS CCS N2 3246 ± 7 Å 2 ) showed a slightly broader envelope relative to the monomer, which is consistent with the increased structural heterogeneity due to the additional glycoform combinations. 50 A plot of the specific CCS values determined for each of the detected monomer (Region 1) and dimer (Region 2) charge states illustrates the global conformational landscape of the native S-RBD O-glycoforms ( Figure 3 E, Table S1 ). The variations in the conformational heterogeneity of the S-RBD O-glycoforms were found to be mobility region specific and separable under TIMS-MS analysis, illustrating the potential of this approach for investigating the gas-phase structural variations of glycoproteins.…”

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

“…The native S-RBD could not be resolved due to overlapping ion signals from all diverse glycoforms/proteoforms carrying different charges, similarly as observed in complex glycoproteins previously. 40,53 The S protein is known to carry many complex-type N-glycans, with two glycosites (Asp331 and Asp343) previously reported and extensively characterized on the S-RBD. 21,22 By contrast, the O-glycans of the S-RBD are less studied and their glycoforms poorly understood, despite the potential insights they may provide to understand viral structure-function relationship.…”

“…[40][41][42] In contrast, by combining glycoproteomics with the top-down MS approach, which preserves the intact glycoprotein enabling high-resolution proteoform-resolved analysis, [43][44][45] we could achieve the simultaneous characterization of the molecular structures, the site specificity, and the relative abundance of various glycoforms. Furthermore, by integrating native MS, which has recently emerged as a powerful structural biology tool to study protein structure-function relationships, [46][47][48][49][50][51][52] with trapped ion mobility spectrometry (TIMS), [53][54][55] we can investigate the gas phase structural variants to achieve the direct quantification of individual glycoproteoforms.…”

Surface-Induced Dissociation of Protein Complexes Selected by Trapped Ion Mobility Spectrometry

“…Trapped ion mobility spectrometry (TIMS), initially optimized and implemented for small molecules, has been gaining traction for the analysis of native proteins and protein complexes. 16,17 TIMS differs from the previously mentioned techniques in that an electric field that opposes the ion motion is used to hold ions stationary against a bath gas which pushes ions along the TIMS analyzer (an ion funnel). Ions of different mobilities are trapped at different points (potentials) along the ion optical axis using the electric field gradient and are then eluted from the device over time, based on their mobility, as the TIMS potential gradient is reduced.…”