Why Did Not ECD Dissociate between Glycans in di--Glycosylated Peptides? Survey and Solution.

In previously reported electron capture dissociation (ECD) mass spectrometry of linked glycopeptides, ECD did not provide efficient backbone fragmentation between the two 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 -glycans, so the backbone fragments between the two -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 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 glycans in ECD analysis. Computational molecular structure calculations and differential mobility spectrometry validated the model of glycan formations in precursors.