PNGaseF-generated N-glycans adduct onto peptides in the gas phase

ABSTRACT: 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 and splitting, 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 mammalian N-glycans. Here, we report that N-glycans released from PNGaseF digestion, also referred to as ‘free glycans’, 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 in common O-glycoproteomic workflows, we performed PNGaseF digestion following reduction and alkylation. Here, we demonstrated that released N-glycans can be removed using a molecular weight cut off (MWCO) filter, prior to (glyco)protease digestion, which improved O-glycoproteomic coverage.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Platelet, Blood Plasma, Epithelial Cell, Hela Cell

SUBMITTER: Valentina Rangel-Angarita  

LAB HEAD: Stacy Malaker

PROVIDER: PXD055856 | Pride | 2026-06-29

REPOSITORIES: Pride