LC–MS analysis of glycopeptides of recombinant monoclonal antibodies by a rapid digestion procedure

N-glycan analysis of recombinant monoclonal antibodies (mAbs) usually requires the removal of oligosaccharides by PNGase F followed by 2-AB labeling, normal phase high performance liquid chromatography (NP-HPLC) separation and fluorescence detection. Alternatively antibodies can be completely digested by trypsin to generate glycopeptides for analysis by liquid chromatography–mass spectrometry (LC–MS). Here, we report the development of a rapid digestion procedure to generate glycopeptides for quantitative LC–MS analysis. Recombinant monoclonal antibodies were digested using a combination of Lys-C and trypsin at 37 °C for 15 min. The glycan profiles from this rapid digestion procedure are in good agreement with those from LC–MS analysis of glycopeptides from completely digested antibodies and those from NP-HPLC analysis of 2-AB labeled PNGase F released oligosaccharides. This rapid digestion procedure was applied to the comparison of oligosaccharides of two different antibodies. Glycopeptides from the two antibodies were differentially labeled with stable isotopes and analyzed simultaneously after a 1:1 mixing. The combination of the rapid digestion procedure and differential stable isotope labeling significantly reduced the turnaround time.

► A rapid digestion procedure was established to digest antibodies for glycopeptide analysis.
► The rapid digestion procedure can be used in combination with dimethyl labeling.
► In-source fragmentation of glycopeptides was highly dependent on the charge state.