We next sought to characterize the suitability of the S-Trap platform to perform the simultaneous Ndeglycosylation and trypsin digestion of more complex samples. Using human serum as a model sample, we investigated three different time intervals (0 h, 2 h and 4 h) between the addition of PNGase F and trypsin (Fig ). Human serum is a well-characterized sample of moderate-to-high complexity containing hundreds of proteins, and it has great clinical relevance as a readily accessible biofluid for diagnosis and prognosis. Across all replicates and timepoints, an average of 72 ± 5% peptides were identified in both the control and deglycosylated samples (n = 9). Over time, we did not observe any trend in the number of peptides identified in the deglycosylated samples and on average, there was less than a 15% change in the number of peptides identified across three biological replicates. Given that there are a comparable number of total peptides identified in both the deglycosylated and control samples, we conclude that PNGase F does not interfere with the digestive activity of trypsin.