Recombinant pngase f

Protein samples were initially diluted to 1.0 mg/mL in PBS (calcium/magnesium-free, pH 7.2) and deglycosylated using recombinant PNGaseF (New England BioLabs, Ipswich, MA, USA). Dithiothreitol (Thermo Fisher Scientific) was added to the reduced samples at a final concentration of 50 mM. Intact and reduced samples were incubated for 2 h at 37 °C and then diluted to 0.25 mg/mL using 0.1% formic acid in water (Thermo Fisher Scientific).

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.