Protonated α-N-Acetyl Galactose Glycopeptide Dissociation Chemistry.

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 (Guan et al. Phys. Chem. Chem. Phys., 2021, 23, 23256-23266). These reactions occurred irrespective of the glycosidic linkage stereochemistry (α or ß) 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 Dissociation Chemistry of Deprotonated RGD.

We investigate the structure and dissociation pathways of the deprotonated amphoteric peptide arginylglycylasparic acid, [RGD-H]-. We model the pertinent gas-phase structures and fragmentation chemistry of the precursor anions and predominant sequence-informative bond cleavages (b2+H2O, c2, and z1 peaks) and compare these predictions to our tandem mass spectra and infrared spectroscopy experiments. Formation of the b2+H2O anions requires rate-limiting intramolecular back biting to cleave the second amide bond and generate an anhydride structure. Facile cleavage of the newly formed ester bond with concerted expulsion of a cyclic anhydride neutral generates the product structure. IR spectroscopy supports this b2+H2O anion having structures that are essentially identical to C-terminally deprotonated arginylglycine, [RG-H]-. Formation of the c2 anion is predicted to require concerted expulsion of CO2 from the aspartyl side chain carboxylate and cleavage of the N-Calpha bond to produce a proton-bound dimer of arginylglycinamide and acrylate. Proton transfers within the dimer then enable predominant detection of a c2 anion with the negative charge nominally on the central, glycine nitrogen (amidate structure) as the proton affinity of this structure is predicted to be lower than acrylate by ∼27 kJ mol-1. Alternate means of cleaving the same N-Calpha bond produce deprotonated cis-1,4-dibut-2-enoic acid z1 anion structures. These lowest energy processes involve C-H proton mobilization from the aspartyl side chain prior to N-Calpha bond cleavage consistent with proposals from the literature.

Sodium-cationized carbohydrate gas-phase fragmentation chemistry: influence of glycosidic linkage position.

We investigate the gas-phase structures and fragmentation chemistry of two isomeric sodium-cationized carbohydrates using combined tandem mass spectrometry, hydrogen/deuterium exchange experiments, and computational methods. Our model systems are the glucose-based disaccharide analytes cellobiose (ß-d-glucopyranosyl-(1 → 4)-d-glucose) and gentiobiose (ß-d-glucopyranosyl-(1 → 6)-d-glucose). These analytes show substantially different tandem mass spectra. We characterize the rate-determining barriers to both the glycosidic and structurally-informative cross-ring bond cleavages. Sodiated cellobiose produces abundant Y1 and B1 peaks. Our deuterium labelling and computational chemistry approach provides evidence for 1,6-anhydroglucose B1 ion structures rather than the 1,2-anhydroglucose and oxacarbenium ion structures proposed elsewhere. Unlike those earlier proposals, this finding is consistent with the experimentally observed Bn/Ym branching ratios. In contrast to cellobiose, sodiated gentiobiose primarily fragments by cross-ring cleavage to form various A2 ion types. Fragmentation is facilitated by ring-opening at the reducing end which enables losses of CnH2nOn oligomers. Deuterium labelling and theory enables rationalization of these processes. Theory and experiment also support the importance of consecutive fragmentation processes at higher collision energies.

Cationized Carbohydrate Gas-Phase Fragmentation Chemistry.

We investigate the fragmentation chemistry of cationized carbohydrates using a combination of tandem mass spectrometry, regioselective labeling, and computational methods. Our model system is D-lactose. Barriers to the fundamental glyosidic bond cleavage reactions, neutral loss pathways, and structurally informative cross-ring cleavages are investigated. The most energetically favorable conformations of cationized D-lactose were found to be similar. In agreement with the literature, larger group I cations result in structures with increased cation coordination number which require greater collision energy to dissociate. In contrast with earlier proposals, the B n -Y m fragmentation pathways of both protonated and sodium-cationized analytes proceed via protonation of the glycosidic oxygen with concerted glycosidic bond cleavage. Additionally, for the sodiated congeners our calculations support sodiated 1,6-anhydrogalactose B n ion structures, unlike the preceding literature. This affects the subsequent propensity of formation and prediction of B n /Y m branching ratio. The nature of the anomeric center (α/ß) affects the relative energies of these processes, but not the overall ranking. Low-energy cross-ring cleavages are observed for the metal-cationized analytes with a retro-aldol mechanism producing the 0,2 A 2 ion from the sodiated forms. Theory and experiment support the importance of consecutive fragmentation processes, particularly for the protonated congeners at higher collision energies. Graphical Abstract ᅟ.