Rapifluor ms reagent solution

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.

Sodium phosphate (Merck) and glycine·HCl (Merck) were purchased from Merck. Tris-HCl and EX-CELL Advanced CHO Fed-batch medium were all purchased from Sigma Aldrich. ACN (Part no: A955-4, Fisher Scientific), PVDF syringe filter (Part no: SLGV013SL, Millex 0.22um PVDF 13 mm sterile syringe filter) were obtained from Merck Millipore (Ireland), whilst centrifugal filters, (Part no: UFC3096, Amicon Ultra Centrifugal Filters) was purchased from Merck Millipore, (USA). Protein A HP Spintrap (28-9031-32) was purchased from GE Healthcare, USA. FAST Glycan Kit (Part no: B94499PTO, SCIEX, USA). Ammonium formate (Part No. 186007081), RapiGest SF (Part No. 186001860), RapiFluor-MS Reagent Solution (Part No. 186008091), and ACQUITY UPLC^® Glycan BEH Amide Column were purchased from Waters Corp.