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. 2024 Aug 12;15(35):14431-14439.
doi: 10.1039/d4sc03672g. Online ahead of print.

Sialylation-induced stabilization of dynamic glycoprotein conformations unveiled by time-aligned parallel unfolding and glycan release mass spectrometry

Yifei Jia  1 , Yichang Liu  2 , Yamei Wang  1 , Jinyu Li  3 , Gongyu Li  1   4
Affiliations

Sialylation-induced stabilization of dynamic glycoprotein conformations unveiled by time-aligned parallel unfolding and glycan release mass spectrometry

Yifei Jia et al. Chem Sci. .

Abstract

Sialylation, a critical post-translational modification, regulates glycoprotein structure and function by tuning their molecular heterogeneity. However, characterizing its subtle and dynamic conformational effects at the intact glycoprotein level remains challenging. We introduce a glycoform-resolved unfolding approach based on a high-throughput ion mobility-mass spectrometry (IM-MS) platform. This method integrates high-throughput unfolding with parallel fragmentation, enabling simultaneous analysis of sialylation patterns, stoichiometries, and their impact on conformational stability. Applying this approach to fetuin, we identified distinct sialylation patterns and their differential influence on protein conformation, namely sialylation-induced stabilization during early unfolding and increased flexibility in later unfolding stages. IM-MS-guided molecular dynamics simulations revealed that increased sialylation enhances the initial conformational stability, likely through enhanced electrostatic interactions and hydrogen bonding. These findings highlight the complex interplay between sialylation and protein dynamics and establish glycoform-resolved unfolding IM-MS as a powerful tool for characterizing glycoprotein conformational landscapes.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. Glycosylation patterns of bFT and hFT. (a) Structural alignment of bFT (AlphaFoldDB: P12763) and hFT (AlphaFoldDB: P02765) and representative glycoforms of each glycosylation site. (b) Overall glycosylation pattern highlighting sialylation and fucosylation. (c) Overall sialylation pattern. SA, sialic acid.
Fig. 2
Fig. 2. Glycoform-resolved all ion unfolding. (a) Scheme for the TAP module for parallel protein unfolding and glycan release equipped with AIU on a Synapt XS instrument. (b) Characteristic oxonium ions and well-resolved hFT protein sialoforms. (c) Representative AIU fingerprints for different hFT sialoforms.
Fig. 3
Fig. 3. Quantitative analysis of fetuin conformational stability with glycoform-resolved unfolding. Feature CCS, AIU50 values, unfolding ratios based on CCS changing percentages, and RMSD values among different sialoforms were compared for bFT (a–d) and hFT (e–h), respectively. Statistical analysis was performed based on the unpaired t-test method, *p < 0.05, ***p < 0.001.
Fig. 4
Fig. 4. Stabilizing effects of sialylation on fetuin conformation. (a) Representative native MS spectra, (b) AIU and RMSD fingerprints, (c) unfolding feature CCS and AIU50 values, and (d) theoretical CCS calculated from gas-phase heating MD simulation trajectories of bFT with (bFT, red) or without sialylation (asialo, yellow). (e) Percentage of remnant secondary structures, (f) remaining proportion of the α-helix and β-sheet, and (g) representative structures of four unfolding features of bFT and its asialo-form during gas-phase heating MD simulation. Statistical analysis was performed based on the unpaired t-test method, ***p < 0.001.
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