Glycoworks hilic μelution plate

N-glycan of antibodies were released and labeled using GlycoWorks RapiFluor-MS (RFMS) N-glycan kit (Waters Corporation, United States) according to the manufacturer’s protocol. Briefly, 15 μg of dried antibody was reconstituted in 22.8 μL of LCMS grade water and 6 μL of 5% RapiGest solution (Waters Corporation, United States). The solution was incubated at 95°C for 5 min to denature the antibody. N-glycans were released enzymatically by adding 1.2 μL (600 U) of Rapid PNGase F (Waters Corporation, United States) followed by 10 min incubation at 55°C. Released N-glycans were labeled with 12 μL of the RapiFluor-MS Reagent Solution (fluorescence label) at room temperature for 5 min. The solution was diluted in 358 μL of ACN, followed by isolation using a GlycoWorks HILIC μElution Plate (Waters Corporation, United States). Isolated released N-glycans were dried and reconstituted in 9 μL of LCMS grade water, 10 μL of DMF, and 21 μL of ACN sequentially for LC-MS analysis.

The dried protein residues were mixed with 6 μL aliquots of 5% (w/v) solution of RapiGest SF in Rapid Buffer (Waters, ON, Canada) and heated at 90 °C for 3 min. The samples were cooled to ambient temperature, mixed with 1.2 μL of Rapid PNGase F and 22.5 μL of Rapid Buffer (Waters, ON, Canada), incubated at 50 °C for 5 min and cooled to ambient temperature. In each sample, glycans were labeled with RapiFluor-MS reagent and enriched using a GlycoWorks HILIC μElution Plate following the manufacturer’s protocol (Waters, ON, Canada).

N-glycans of meat were released and labelled using GlycoWorks RapiFluor-MS (RFMS) N-glycan kit (Waters Corporation, Milford, MA, USA) according to the manufacturer’s protocol. Briefly, 15 μg of dried protein extracted from meat was reconstituted in 22.8 μL of LCMS grade water and 6 μL of 5% RapiGest solution (final concentration 0.01% RapiGest, Waters Corporation, Milford, MA, USA). The solution was incubated at 95 °C for 5 min to denature the protein extracted from meat. N-glycans were released enzymatically by adding 600 U of recombinant PNGase F (New England Biolabs, Ipswich, MA, USA) followed by 10 min incubation at 55 °C. Released N-glycans were labelled with 12 μL of the RapiFluor-MS Reagent Solution (fluorescence label, 0.07 mg/μL in anhydrous dimethyformamide (DMF), Waters Corporation, Milford, MA, USA) at room temperature for 10 min. The solution was diluted in 358 μL of ACN, followed by clean-up using a GlycoWorks HILIC μElution Plate (Waters Corporation, Milford, MA, USA). Isolated released N-glycans were dried and reconstituted in 9 μL of LCMS grade water, 10 μL of DMF, and 21 μL of ACN sequentially for LC-MS analysis.