Why Did Not ECD Dissociate between Glycans in di-<i>N</i>-Glycosylated Peptides? Survey and Solution

In previously reported electron capture dissociation (ECD) mass spectrometry of <i>N-</i>linked glycopeptides, ECD did not provide efficient backbone fragmentation between the two <i>N-</i>glycans in a single peptide, which did not allow the assignment of a correct sugar composition in each glycan. In this study, we found hydrogen bonds and proton-bound interactions between two <i>N</i>-glycans, so the backbone fragments between the two <i>N</i>-glycans were not observed, although the backbone was cleaved. Such behavior is typical for precursor ions produced by conventional electrospray ionization. Supercharging reagent 3-nitrobenzyl alcohol increases the precursor charge state, which leads to the protonation of each <i>N-</i>glycan, and the resulting Coulombic repulsive force is sufficient to quench the noncovalent binding. Highly charged precursors produced using the supercharged reagents yield intense c' and z^· fragments between the <i>N-</i>glycans in ECD analysis. Computational molecular structure calculations and differential mobility spectrometry validated the model of glycan formations in precursors.