Glycopeptide Identification
Using Liquid-Chromatography-Compatible
Hot Electron Capture Dissociation in a Radio-Frequency-Quadrupole
Ion Trap
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Abstract
We developed a liquid chromatography (LC) compatible
electron capture
dissociation (ECD) mass spectrometer for glycoproteomics, with which
ECD and hot ECD (HECD) experiments can be flexibly switched by quickly
changing the electron energy without further tuning of the mass spectrometer.
Desialylated glycopeptides were dissociated well in both ECD and HECD
experiments. For sialylated glycopeptides, on the other hand, ECD
with electron energy higher than 4 eV showed significantly higher
sequence coverage than that with an electron energy of 0.2 eV. A nano
LC system was coupled to our ECD mass spectrometer to investigate
N-linked glycopeptides from lysylendopeptidase (Lys-C) digests of
human transferrin. ECD spectra at multiple electron energies of 0.2,
5.0, and 9.0 eV were obtained for each targeting precursor ion in
a single LC injection. Glycopeptides with a sialylated bi-, tri-,
or tetra-antennary complex N-glycan were identified with high sequence
coverage by HECD. Glycopeptides with tri- or tetra-antennary N-glycans
have seldom been analyzed by ECD or ETD before this report. We also
found that a preferential dissociation of nonreducing termini of glycans
in glycopeptides by ECD and HECD