Endo f3

Approximately 80 μg of glycopeptides of CHO cells were treated with 5 μl of Endo F3 (~5 milliunits/μl, Millipore Sigma) for 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h. For the experiment of 0 min, no enzyme was added to the peptides. The resulting peptides were subjected to LC-MS/MS.

N-linked glycans were digested by Endo F3 (Millipore Sigma) followed by PNGase F (New England BioLabs). First, label-free or TMT-labeled glycopeptides were resuspended in 100 mM ammonium acetate (pH 5.5) and treated with Endo F3 at an enzyme/peptides ratio of 25 milliunits/80 μg of glycopeptides (50 milliunits/80 μg of glycopeptides for HCC tumor and normal tissues). The enzymatic reaction was conducted at 37 °C for 2 hours. The samples were dried in a speed vacuum centrifuge for 5 hours. The dried peptides were redissolved in 100 mM ammonium bicarbonate (pH 8) and treated with PNGase F (New England BioLabs) at an enzyme/peptides ratio of 3000 New England Biolabs units/80 μg of glycopeptides. The reaction solution was incubated at 37 °C for 2 hours. The resulting peptides were desalted with C18 solid-phase extraction.