Simultaneous N-Deglycosylation and Digestion of Complex Samples on S-Traps Enables Efficient Glycosite Hypothesis Generation

N-linked glycosylation is an important post-transla-tional modification that is difficult to identify and quantify in traditional bottom-up proteomics experiments. Enzymatic degly-cosylation of proteins by peptide:N-glycosidase F (PNGase F) prior to digestion and subsequent mass spectrometry analysis has been shown to improve coverage of various N-linked glycopep-tides, but the inclusion of this step may add up to a day to an already lengthy sample preparation process. An efficient way to integrate deglycosylation with bottom-up proteomics would be a valuable contribution to the glycoproteomics field. Here, we demonstrate a proteomics workflow in which deglycosylation and proteolytic digestion of samples occur simultaneously using suspension trapping (S-Trap). This approach adds no time to standard digestion protocols. Applying this sample preparation strategy to a human serum sample, we demonstrate improved identification of potential N-glycosylated peptides in deglycosylated samples compared with non-deglycosylated samples, identifying 156 unique peptides that contain the N-glycosylation motif (asparagine-X-serine/threonine), the deamidation modification characteristic of PNGase F, and an increase in peptide intensity over a control sample. We expect that this rapid sample preparation strategy will assist in the identification and quantification of both known and potential glycoproteins. Data are available via ProteomeXchange with the identifier PXD037921.

Automated summary

N-linked glycosylation is a challenging modification to analyze in proteomics experiments. This study presents a novel workflow that combines deglycosylation and proteolytic digestion using suspension trapping, without adding extra time to the sample preparation process. The proposed approach improves identification of potential N-glycosylated peptides and can aid in the discovery of known and potential glycoproteins.