PNGaseF-Generated N‑Glycans Adduct onto Peptides in the Gas Phase

Glycoproteomics has recently increased in popularity
due to instrumental
and methodological advances. That said, O-glycoproteomic analysis
is still challenging for various reasons, including signal suppression,
search algorithm limitations, and co-occupancy of N- and O-glycopeptides.
To decrease sample complexity and simplify analysis, most O-glycoproteomic
workflows include PNGaseF digestion, which is an endoglycosidase that
removes most N-glycan structures. Here, we report that N-glycans released
from PNGaseF digestion were identified during data acquisition and
hampered detection of O-glycopeptides. Importantly, we noted instances
where free glycans adducted to unmodified peptides in the gas phase
and were misidentified by search algorithms as O-glycopeptides. We
confirmed the presence of free glycans in other experiments performed
in our laboratory, as well as from data generated by other groups.
To overcome this limitation, we demonstrated that released N-glycans
can be removed using a molecular weight cut off filter prior to (glyco)protease
digestion, which improves O-glycoproteomic coverage.