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.

Figure 1.

MSⁿ analysis of HexNAcs. MSⁿ spectra for GlcNAc (top), ManNAc (middle), and GalNAc (bottom) are shown. The numbers above each panel indicate the masses: (A) m/z 204, (B) m/z 186, (C) m/z 168, (D) m/z 144, (E) m/z 138, (F, G) m/z 126, selected for each stage of MSⁿ shown by the arrows. Structures of each monosaccharide are shown on the left.

Figure 2.

IM-MS analysis of HexNAcs using helium (A) or nitrogen (B) as the drift gas. The arrival time distributions (ATDs) for each fragment ion are shown as dots for GlcNAc (blue), ManNAc (green), and GalNAc (red). The lines show the Gaussian fits and numbers within each plot represent the Gaussian peak width (FWHM). Dashed lines are added to help illustrate slight differences in ATDs. C) HDX-MS analysis of HexNAcs. Deuterium uptake for various fragment ions is shown as a function of incubation time with ND₃. The points and error bars represent the average and standard deviations from triplicate measurements. *The m/z 127 ion was mass resolved from the second isotopic peak of the m/z 126 ion.

Figure 3.

Analysis of isotopically labeled GlcNAc at either C(1) (tan), C(2) (green), acetyl carbonyl (blue), or both C(6) and the nitrogen (purple). A) MSⁿ spectra of unlabeled and each isotopically labeled GlcNAc with mass selection steps shown above each panel. The position of ¹³C and ¹⁵N labels are shown on the structure of GlcNAc on the top left. B) IM-MS analysis of the m/z 126, m/z 127, or m/z 128 ions from ¹³C(CO) and ¹³C(6)/¹⁵N GlcNAc shown as described in figure 2.

Figure 4.

Fragmentation pathway of [GlcNAc+H]⁺. After the initial loss of water from the reducing end fragmentation can proceed through two distinct pathways: I) further loss of water and a range of subsequent fragment ions; or II) ring cleavage to form a stable 5-membered ring structure.

Figure 5.

Anomeric effects in HexNAc fragmentation. A) MSⁿ spectra of the m/z 204 ion and m/z 186 ions from the α (left) and β (right) anomers of GlcNAc (blue), ManNAc (green), and GalNAc (red). B) IM-MS analysis of the m/z 204 and m/z 186 ions shown as described in figure 2 from the α (orange dashed lines) and β (blue solid lines) anomers. The LC-MS traces are shown in figure S8.