TMF–PNGase F or Free PNGase F Deglycosylation of Standard Glycoproteins and Urinary Glycopeptides

Standard glycoprotein RNase B (10 μg/μl) was dissolved in deionized water and heated at 95°C for 10 min to denature. Then, TCEP (10 mM) reduction and CAA (40 mM) alkylation were performed. Urinary N-glycopeptides were obtained according to HILIC Enrichment of Urinary N-Glycopeptides and dissolved in H218O. For TMF–PNGase F deglycosylation, 4 μl of the protein solution was mixed with 1 ml TMF–PNGase F and incubated for 3 min at room temperature. The immobilized PNGase F was retained by a heating-magnet process, and the supernatant was collected for mass spectrometry analysis. The TMF–PNGase F materials can be reused after washing them three times with deionized water (or H218O). For in-solution deglycosylation, PNGase F was introduced to the protein solution at a substrate to enzyme ratio (w/w) of 10:1 and incubated at 37°C for 16 h. No further pre-process was required for in-solution digestion before mass spectrometry analysis.