Many proteins identified in the human serum sample exhibit an increase in intensity in peptides containing N-glycan motifs and deamidation modifications in the deglycosylated sample trace compared to the control trace. Follow-up analysis of these peptides yields evidence of previously quantified glycoproteins with known and annotated glycosites. IgG is an abundant protein in human serum that contains an annotated N-glycosite in the Fc domain of the heavy chain with importance in the function of immunoglobulin in both immunity and as drugs. In the deglycosylated samples, we identify peptides containing a deamidated asparagine residue at the known site of N-glycosylation (N180) that were not identified in the control samples (Fig 3C). The area fold-change plot for this protein can be seen in SI Figure . Many other proteins with probable glycosylation sites were identified, include Apolipoprotein (SI Fig 6), Alpha-2-HSglycoprotein (SI Fig 7), and Haptoglobin (SI Fig 8). We conclude that this method of simultaneous N-deglycosylation and proteolytic digestion generates valuable information about potential N-glycosites that can be validated for presence of confirmed glycans. Deglycosylation on S-Trap reveals more potential glycopeptides than N-glyco peptide database searching. MSFragger is a database search software that contains a N-Glyco search module that analyzes tandem mass spectra of intact glycopeptides and then identifies and validates their glycan sites. In comparing three human serum biological replicates from the 0hour time point, 53 unique glycopeptides were identified by MSFragger's N-Glyco search. Using the method described in this manuscript, a total of 138 unique potential N-glycopeptides were identified in the same three biological replicates, a 160% increase (Fig. ). Furthermore, the total number of peptides identified between each identification method differed by no more than 3% (Fig 4B ) indicating that the increase in potential N-glycopeptides is not simply due to an overall increase in peptides from the different search strategies. While we see a dramatic increase in the number of N-glycopeptides using S-Trap deglycosylation, it is important to note that no information about the glycan composition or attached mass is revealed by this approach, unlike MSFragger which identifies glycans from their mass difference. Deglycosylation on S-Trap can be used to generate hypotheses about which proteins contain N-linked glycans and can identify potential glycopeptides missed in N-glycopeptide database searches, but other methods will be necessary for validation. Deglycosylation and digestion on S-Trap combined with the use of N-glyco search modules can provide a robust assay for discovery and validation of glycans on proteins of interest.