Having confirmed the compatibility of PNGase F with S-Trap buffers, we examined three chicken proteins, which all contain at least one previously annotated glycosite in Uniprot. Each protein-albumin from chicken egg white (ovalbumin, Accession ID: P01012), trypsin inhibitor from chicken egg white (ovomucoid, Accession ID: P01005), and conalbumin from chicken egg white (ovotransferrin, Accession ID: P02789)-were successfully deglycosylated using PNGase The cocktail of chicken proteins was deglycosylated and digested simultaneously. For each amino acid in the protein sequence of interest, the area (Fig 2 ) or number of peptide-spectrum matches (PSMs) (SI Fig 2 ) were summed, and the ratio of deglycosylated sample to control sample, or fold-change, was calculated. Ovalbumin has one annotated N-glycosylation site at N293 with heterogeneous neutral glycan attachments. We observed a 129x increase in intensity for the peptides containing that residue, whereas we do not see an increase in peptide intensity at N312, which is part of an N-glycosylation motif but is not an annotated glycosylation site (Fig ). This evidence supports the annotation of N293 but not N312 as N-glycosites. Ovomucoid has five known N-glycosylation sites at N34, N77, N93, N99, and N199, and one unannotated site containing the N-glycosylation motif at N182. Comparing the observed peptide intensity of the deglycosylated sample to the control sample across the amino acid sequence of ovomucoid, we observed a more than 1000x increase at N93 and N99, a 60x increase at N34, and a 50x increase and N77 (Fig ). Ovotransferrin has one known N-glycosylation site at N492, and two unannotated sites at N637 and N691. We observed a 10-fold increase in intensity for peptides containing the N492 site in the deglycosylated sample compared to control (Fig 2C ).