Optimization of LC-ExD methods for glycopeptides on an Omnitrap-Orbitrap platform

Area of Science:

• Glycoproteomics
• Mass Spectrometry
• Analytical Chemistry

Background:

• Glycoproteomics is challenging due to glycan complexity and heterogeneity, hindering analysis by conventional proteomics.
• Higher-energy collisional dissociation (HCD) fragments glycans preferentially, preventing confident glycopeptide localization.
• Collisional dissociation yields limited glycan structural information due to ion rearrangements.

Purpose of the Study:

• To investigate electron-activated dissociation (ExD) methods for improved glycopeptide analysis.
• To enhance the speed and sensitivity of hot electron capture dissociation (hECD) for large-scale glycoproteomics.
• To enable confident localization and topological characterization of glycopeptides.

Main Methods:

• Utilized an Omnitrap platform integrated with a Q Exactive-class mass spectrometer.
• Employed hot electron capture dissociation (hECD) with enhanced speed and sensitivity.
• Evaluated higher energy (he) ExD conditions for glycopeptide fragmentation.

Main Results:

• Observed useful glycan fragmentation, including cross-ring cleavages crucial for determining glycan topologies.
• Successfully detected isomeric glycopeptides differing in glycan topology or glycosylation sites.
• Demonstrated the power of on-line liquid chromatography-mass spectrometry (LC-MS) with higher energy ExD (heExD) analysis.

Conclusions:

• Electron-activated dissociation (ExD) methods, particularly hECD, offer a superior alternative to HCD for glycopeptide analysis.
• The enhanced hECD approach on the Omnitrap platform significantly improves speed and sensitivity for glycoproteomics.
• This technique enables confident characterization of glycopeptide isomers, advancing glycoproteomics research.