PNGase F activity is compatible with S-Trap sample preparation. The direct compatibility of the S-Trap platform with PNGase F and buffers suitable for PNGase F activity has not previously been demonstrated. We investigated whether PNGase F is compatible with S-Trap sample preparation using RNase B as a model glycoprotein containing one known N-glycan that results in a characteristic mass shift upon cleavage. PNGase F is known to be effective at removing glycans in Protein Deglycosylation Mix Buffers 1 and 2, whereas TEAB is a known to be compatible with S-Trap sample preparation and mass spectrometry analysis. To test whether a single buffer system would be compatible with both PNGase F and S-Traps, RNase B was deglycosylated using PNGase F in four different buffer conditions: the two deglycosylation buffers provided with commercially available PNGase F, TEAB, and water. As shown in Figure , PNGase F is found to be effective in all four buffer environments. The ability of PNGase F to function effectively in TEAB eliminates the need for any buffer exchange steps during sample preparation.