Dissection of Fragmentation Pathways in Protonated N-Acetylhexosamines

Abstract

Structural characterization of carbohydrates by mass spectrometry necessitates a detailed understanding of their gas phase behavior, particularly for protonated carbohydrates that can undergo complex structural rearrangements during fragmentation. Here we utilize tandem mass spectrometry, isotopic labeling, gas-phase hydrogen/deuterium exchange, and ion mobility measurements to characterize structures of the various product ions of protonated N-acetylhexosamines. Following the facile loss of the reducing end hydroxyl group, we identify two primary fragmentation pathways. Detailed mapping of each step in the fragmentation pathway provides new insight into the mechanisms that drive collision-induced dissociation of protonated carbohydrates. Several of the smaller fragment ions are mixtures of structural isomers, and the relative distributions of these structures reveals information about the stereochemistry of the precursor molecule.

Evidence of Gas-phase Pyranose-to-furanose Isomerization in Protonated Peptidoglycans

“…Our combined data offer a mechanistic explanation of the dissociation chemistry consistent with Nilsson and co-workers 69,71 conclusion that the m/z 126 and 144 peaks "correspond to entirely different structures, which have followed completely different decomposition pathways" 71 . Consistent with the present dataset, Mookherjee et al 72 recently provided evidence that cast doubt on the mechanism and product ion structure originally proposed by Yu et al 71 for the key m/z 126 peak using model protonated HexNAc analytes rather than peptidoglycans.…”

Linkage Memory in Underivatized Protonated Carbohydrates

“…This is thought to arise from the generation of different isomeric fragment ion structures, whose ensemble is affected by the structure of the precursor. Similar 'memory effects' were also observed by both IM-MS and MS n relative fragmentation propensities even after multi-stage MS 22 . Here we sought to characterize the fragmentation patterns within larger oligosaccharides and the structural disposition of the resulting fragment ions.…”

“…Similarly, the (1-6) linked structure cannot form the O6-C1 bridged structure. Previous mapping of the fragmentation pathways of GlcNAc revealed that the 204 ion dissociates through two competing pathways to generate either: 1) primarily m/z 186 through water loss; or 2) m/z 126 through a ring rearrangement reaction 22 . The O4-C1 bridge structure was proposed to initiate the ring rearrangement to form m/z 126.…”

“…Ion mobility with mass spectrometry (IM-MS) separates ions by their charge, size, and shape due to interactions with an inert gas. IM-MS has shed light on the structural details of carbohydrates in the gas phase 17,22 , and the emerging next-generation of high-resolution IM-MS show much promise at resolving even subtle isomeric variants within larger glycans 20,23,24 . Gas-phase spectroscopy is emerging to define structural variations in carbohydrates and their structures on its own or in combination with IM-MS 18,20,[25][26][27] .…”

Linkage Memory in Underivatized Protonated Carbohydrates

“…1E, F) was performed on all six disaccharides to fragment the GlcNAc (m/z 204) remnant, probing for signature CID fragmentation patterns. In addition to m/z 186, 168, 144 and 138, the MS 4 (384→366→204→) spectra of all disaccharides yield m/z 126 and 84 which are primary and secondary products of m/z 204 22 (Figs. 1E, F).…”