Conformationally Restricted Glycopeptide Backbone Inhibits Gas-Phase H/D Scrambling between Glycan and Peptide Moieties

Abstract

Protein glycosylation is a common post-translational modification on extracellular proteins. The conformational dynamics of several glycoproteins have been characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS). However, it is, in most cases, not possible to extract information about glycan conformation and dynamics due to the general difficulty of separating the deuterium content of the glycan from that of the peptide (in particular, for O-linked glycans). Here, we investigate whether the fragmentation of protonated glycopeptides by collision-induced dissociation (CID) can be used to determine the solution-specific deuterium content of the glycan. Central to this concept is that glycopeptides can undergo a facile loss of glycans upon CID, thereby allowing for the determination of their masses. However, an essential prerequisite is that hydrogen and deuterium (H/D) scrambling can be kept in check. Therefore, we have measured the degree of scrambling upon glycosidic bond cleavage in glycopeptides that differ in the conformational flexibility of their backbone and glycosylation pattern. Our results show that complete scrambling precedes the glycosidic bond cleavage in normal glycopeptides derived from a glycoprotein; i.e., all labile hydrogens have undergone positional randomization prior to loss of the glycan. In contrast, the glycosidic bond cleavage occurs without any scrambling in the glycopeptide antibiotic vancomycin, reflecting that the glycan cannot interact with the peptide moiety due to a conformationally restricted backbone as revealed by molecular dynamics simulations. Scrambling is also inhibited, albeit to a lesser degree, in the conformationally restricted glycopeptides ristocetin and its pseudoaglycone, demonstrating that scrambling depends on an intricate interplay between the flexibility and proximity of the glycan and the peptide backbone.

Figure 1

Structure of the complex-type N-linked glycan of glycopeptide 83–95 obtained from ART. Adapted from ref (14). NH groups in N-glycosidic bonds, including glycan acetamido groups, are indicated by red circles. These NH groups exchange slowly at pH 2.5, and they retain deuterium in addition to the backbone amide groups in HDX-MS experiments. In contrast, the hydroxyl groups exchange several orders of magnitude faster at pH 2.5. Therefore, the hydroxyl groups do not retain any deuterium in HDX-MS experiments, as it is completely lost due to rapid back-exchange with the solvents during liquid chromatography. The hydroxyl groups and NH groups are the only exchangeable hydrogens in the depicted glycan. The symbols in the schematic structure are rhombus: sialic acid; open circle: galactose; filled square: N-acetylglucosamine, GlcNAc; filled circle: mannose; triangle: fucose. Glycosidic cleavages by specific glycosidases to make the DS-ART, Endo-ART, Degala-ART, and Degly-ART are shown with dotted line.

Figure 2

Cleavage of the O-glycosidic bond in the Endo-ART glycopeptide proceeds with H/D scrambling between the peptide and glycan moieties. (A) Fragmentation scheme of the Endo-ART glycopeptide, ⁸³MDVNSTWRTVDRL⁹⁵, with the N-linked glycan GlcNAc-fucose. CID spectrum of the triply protonated Endo-ART glycopeptide in its (B) nondeuterated state and (C) deuterated state. CID spectrum is shown along with isotope distributions of the following fragment ions, y₂ at m/z 288.2, peptide at m/z 796.89, peptide + GlcNAc at m/z 898.44, m/z values refer to the monoisotopic peak from the nondeuterated peptide. The precursor ion average deuterium content is 7.5 Da.

Figure 3

Peptic glycopeptide 83–95 from Endo-ART undergoes complete H/D scrambling upon gas-phase fragmentation by CID. Deuterium content of fragment ions from CID of deuterated Endo-ART glycopeptide (⁸³MDVNSTWRTVDRL⁹⁵ with the N-linked glycan GlcNAc-Fuc), normalized to the precursor ion deuterium content (7.5 Da). The normalized deuterium content of fragment ions and neutral loss of fucose (black line and crosses) is very similar to the theoretical values in the case of 100% scrambling (red line). The deuterium content of the neutral fucose fragment was determined by the mass difference between the precursor ion and the peptide+GlcNAc fragment ion. In the case of 0% scrambling, the neutral fucose fragment would be devoid of deuterium (green symbol). Note that the 0% scrambling value is only displayed for the neutral fucose fragment as the solution labeling pattern of Endo-ART’s backbone amide groups is not known. Error bars represent mean ± standard deviation (n = 3).

Figure 4

Characterization of the hydrogen-bonding network between the Endo-ART ⁸³MDVNSTWRTVDRL⁹⁵ peptide and the GlcNAc-fucose glycan attached covalently to the asparagine, N, residue of the peptide. (A) Molecular rendering of the system featuring 10 possible hydrogen bonds, labeled as d1–10 (Movie S3 highlights these hydrogen bonds and the glycan). The atoms participating in hydrogen bond formation are indicated following the CHARMM force field nomenclature. Only heavy atoms (no hydrogens) are shown for the sake of clarity. (B) Molecular rendering of the investigated system showing all atoms, including hydrogens. (C) Time evolution of the hydrogen-bonding contacts computed for the 10 possible hydrogen bonds between the peptide and the sugar subsystems; see panel (A). Color indicates the probability of hydrogen bond formation. (D) Time evolution of the individual hydrogen bond length, as defined in panel (A). The numbers to the left of the plot indicate the average hydrogen bond lengths with the standard deviation of the average distances shown as the ± values. (E) Probability density functions of the hydrogen bond lengths, corresponding to panel (D). (F) Time evolution of the total number of hydrogen bonds between the peptide and the sugar subsystems; see panel (A), while panel (G) shows the corresponding probability density. The glycan residues are labeled as follows according to AMBER 9 notation: WYB: N-acetylglucosamine; 0fA: fucose.

Figure 5

Cleavage of the O-glycosidic bond in vancomycin proceeds without H/D scrambling between the peptide and glycan moieties. (A) Chemical structure of vancomycin. CID spectrum of doubly protonated (B) nondeuterated vancomycin and (C) deuterated vancomycin. Full CID spectrum is shown along with isotope distributions of the precursor ion at m/z 725.72, vancosamine fragment ion at m/z 114.10, and desvancosamine–vancomycin fragment ion at m/z 1305.33. The precursor ion average deuterium content is 4.5 Da. The residues in the heptapeptide scaffold are numbered. The average mass of the vancosamine fragment ion does not increase upon deuteration of vancomycin, providing direct evidence for the absence of scrambling involving the vancosamine moiety.

Figure 6

Structure of vancomycin obtained after the 700 ns MD simulation. The amino sugar vancosamine (black circle) does not form any hydrogen bonds with the backbone amide hydrogens (indicated by black dots), explaining the absence of scrambling, which is otherwise mediated through the intramolecular migration of hydrons between peptide backbone amides and glycan. MD simulation movies are found in the Supporting Information (SI), Movies S6 and S7.