<p>Glycoproteomics presents a challenge for analysis by conventional proteomics methods due to the size, complexity, and heterogeneity of glycans. Many published methods use higher-energy collisional dissociation (HCD) that preferentially fragments the glycan, preventing its confident localization on the peptide due to low abundances of peptide backbone fragmentation. However, the glycan fragment ions resulting from collisional dissociation have limited value for determining glycan topologies in part due to rearrangements that occur to protonated glycan groups during vibrational excitation. To overcome this problem, we investigated the use of electron-activated dissociation (ExD) methods that preferentially dissociate the peptide with low levels of vibrational excitation. Hot electron capture dissociation (hECD) has previously been demonstrated for glycosylated peptides using a Fourier-transform ion cyclotron resonance mass spectrometer, but its slower scan speed and lower sensitivity make it ineffective for larger-scale glycoproteomics studies. In this work, we employed an Omnitrap platform integrated with a Q Exactive-class mass spectrometer to perform hECD with enhanced speed and sensitivity. We evaluated a set of higher energy (he) ExD conditions and observed useful glycan fragmentation, including the cross-ring cleavages that define glycan topologies. Using this approach, we detected isomeric glycopeptides differing in glycan topology or glycosylation sites, highlighting the power of on-line LC-MS heExD analysis.</p> Graphical Abstract <p></p>