Protonated α-N-Acetyl Galactose Glycopeptide Dissociation Chemistry

Abstract

We recently provided mass spectrometric, H/D labeling, and computational evidence of pyranose to furanose N-acetylated ion isomerization reactions that occurred prior to glycosidic bond cleavage in both O- and N-linked glycosylated amino acid model systems (Phys. Chem. Chem. Phys.2021232325623266). These reactions occurred irrespective of the glycosidic linkage stereochemistry (α or β) and the N-acetylated hexose structure (GlcNAc or GalNAc). In the present article, we test the generality of the preceding findings by examining threonyl α-GalNAc-glycosylated peptides. We utilize computational chemistry to compare the various dissociation and isomerization pathways accessible with collisional activation. We then interrogate the structure(s) of the resulting charged glycan and peptide fragments with infrared “action” spectroscopy. Isomerization of the original pyranose, the protonated glycopeptide [AT­(GalNAc)­A+H]+, is predicted to be facile compared to direct dissociation, as is the glycosidic bond cleavage of the newly formed furanose form, i.e., furanose oxazolinium ion structures are predicted to predominate. IR action spectra for the m/z 204, C8H14N1O5 +, glycan fragment population support this prediction. The IR action spectra of the complementary m/z 262 peptide fragment were assigned as a mixture of the lowest-energy structures of [ATA+H]+ consistent with the literature. If general, the change to a furanose m/z 204 product ion structure fundamentally alters the ion population available for MS3 dissociation and glycopeptide sequence identification.

Gas-Phase Behavior of Galactofuranosides upon Collisional Fragmentation: A Multistage High-Resolution Ion Mobility Study

“…This questions whether other rearrangements described for protonated species can occur. For example, pyranose-to-furanose isomerization was recently evidenced in protonated peptidoglycans. , …”

“…For example, pyranose-to-furanose isomerization was recently evidenced in protonated peptidoglycans. 43,44 ■ EXPERIMENTAL SECTION Synthesis of the Standards. Gal-Man Disaccharides.…”