Populations of metal-glycan structures influence MS fragmentation patterns

Abstract

The structures and collision-induced dissociation (CID) fragmentation patterns of the permethylated glycan Man5GlcNAc2 are investigated by a combination of hybrid ion mobility spectrometry (IMS), mass spectrometry (MS), and MS/MS techniques. IMS analysis of eight metal-adducted glycans ([Man5GlcNAc2 + M](2+), where M = Mn, Fe, Co, Ni, Cu, Mg, Ca, and Ba) shows distinct conformer patterns. These conformers appear to arise from individual metals binding at different sites on the glycan. Fragmentation studies suggest that these different binding sites influence the CID fragmentation patterns. This paper describes a series of separation, activation, and fragmentation studies that assess which fragments arise from each of the different gas-phase conformer states. Comparison of the glycan distributions formed under gentle ionization conditions with those obtained after activation of the gas-phase ions suggests that these conformer binding states also appear to exist in solution.

Fig. 1

Source distributions (black traces) and quasi-equilibrium distributions (red traces) of metal-adducted Man₅GlcNAc₂ glycan ions plotted on a cross section scale. Each panel corresponds to the distribution of the [Man₅GlcNAc₂+M]²⁺ glycan ion, where M refers to the divalent metals of Mn, Fe, Co, Ni, Cu, Mg, Ca, and Ba, as shown on each panel. The dashed lines divide the distributions into three cross section ranges labeled as I, II, and III based on their quasi-equilibrium (QE) distributions. The distributions are obtained by integration of the drift bins across a narrow m/z range corresponding to each of the glycan ions and normalized to the total ion abundance

Fig. 2

CID spectra of the [Man₅GlcNAc₂+M]²⁺ glycan ions. The fragment ions, either doubly-charged or singly-charged, are predominantly formed by glycosidic cleavages at the B₄, Y_3α, and Y_3,4 positions of the glycan structure

Fig. 3

The scatter plot shows the correlations between metal-dependent QE conformations and metal-dependent CID fragmentation patterns. Each data point contains the calculated Pearson correlation coefficients for each pair of metals based on their CID fragmentation patterns (y value) and QE distribution patterns (x value)

Fig. 4

The bar graphs show the comparisons between the peak areas of the three conformations from QE distributions (white bars, representing conformers I, II, and III, from left to right) and the intensities of the three major fragments (grey bars, representing fragments B₄, Y_3α, and Y_3,4, from left to right) for each metallated glycan. Error bars show the standard deviations from triplicate measurements

Scheme 1

Structure of the permethylated Man₅GlcNAc₂ glycan showing the major fragments observed in the collision-induced dissociation (CID) spectra. Cleavages at the non-reducing terminal mannoses lead to three possible fragments Y_4α′, Y_{4α ″}, and Y_3β that are indistinguishable from each other. For succinctness, “Y_3,4” is used to refer to any of these three types of fragments