Characterization of Conformational Ensembles of Protonated N-glycans in the Gas-Phase

Abstract

Ion mobility mass spectrometry (IM-MS) is a technique capable of investigating structural changes of biomolecules based on their collision cross section (CCS). Recent advances in IM-MS allow us to separate carbohydrate isomers with subtle conformational differences, but the relationship between CCS and atomic structure remains elusive. Here, we characterize conformational ensembles of gas-phase N-glycans under the electrospray ionization condition using molecular dynamics simulations with enhanced sampling. We show that the separation of CCSs between isomers reflects folding features of N-glycans, which are determined both by chemical compositions and protonation states. Providing a physicochemical basis of CCS for N-glycans helps not only to interpret IM-MS measurements but also to estimate CCSs of complex glycans.

Figure 1

(a) Symbolic representation of the ten PA-glycans studied. (b) Experimentally observed arrival time distributions of the ten PA-glycans. (c) Plot of calculated Collision Cross Sections (CCSs) in N₂ gas against experimental values obtained in N₂ gas. P3 and P6 indicate the different protonation states (P3: H⁺ at α1-3 (white) and P6: H⁺ at α1-6 branch (black)) (Dotted lines: regression lines with the correlation coefficient of 0.90 (P3, blue) and 0.89 (P6, red), respectively).

Figure 2

(a) RMSFs of heavy atoms calculated for the ten PA-glycans. The protonation states, P3 and P6, are marked using red and black lines, respectively. (b) Plot of calculated Collision Cross Sections (CCSs) along molecular mass. P3 and P6 indicate the different protonation states (P3: H⁺ at α1-3 (white), P6: H⁺ at α1-6 branch (black)).

Figure 3

(a) CCS distributions, cluster populations that were obtained from k-means clustering using MMTSB toolset (threshold: RMSD = 2.5 Å), and major conformers making up more than 10% of the population (orange bars). The CCS values of major conformers are also given. Blue, green, red, and gold colors for the core chitobiose, α1-3 arm, α1-6 arm, and fucose residue respectively. (b) Differences in CCS values (ΔCCS^calc) are listed with the corresponding values from experiment (ΔCCS^exp).

Figure 4

Comparison of major conformers: (a) three major conformers in G0F and (b) two major conformers in G1(3). Blue, green, red, and yellow colors for the core chitobiose, α1-3 arm, α1-6 arm, and fucose residue, respectively. The pyridylamino (PA) group is marked by a black circle.

Figure 5

(a) Molecular structures of compact globular and rod-like “backfolding” forms. (b) Schematic illustration of H-bond networks (solid line for >70% and dashed line for >50% probability). The geometric definition was used to identify H-bonds: R_XY < 3.5 Å and θ_HXY < 30°, where R_XY is the distance between heavy atoms X and Y, and θ_HXY is the angle between X–H bond and X–Y vectors. (c) Key inter-arm H-bonds in “backfolding” structure (H-bond with the first GlcNAc residue (I) and H-bonds with the second GlcNAc residue (II)).

Figure 6

Residue-residue H-bond maps calculated for three pairs of isomeric PA-glycans with selected protonation states. The geometric definition was used to identify H-bonds: R_XY < 3.5 Å and θ_HXY < 30°, where R_XY is the distance between heavy atoms X and Y, and θ_HXY is the angle between X–H bond and X–Y vectors. The regions of inter-branch interactions are highlighted with Greek numbers (I: core chitobiose-α1-6 branch, II: core chitobiose-α1-3 branch, III: α1-6 branch-α1-3 branch).