Hypercarb cartridge

IAC purified mTf or mTf standard (25 g) was reduced with 0.5% 2-mercaptoethanol ( -ME) and 0.5% SDS in 50 mM NH 4 HCO 3 (pH 7.6-7.7) and heated in a thermo block at 100 ºC for 30 min [19] . Once the sample was at room temperature, a volume of 50 mM NH 4 HCO 3 (pH 7.6-7.7) with 1% (v/v) of NP-40 alternative was added to obtain a final concentration of 0.1% SDS and -ME in the sample. To release the N-glycans, 1 L of PNGase F (1 U) solution was added to the mixture, which was afterwards incubated at 37ºC for 18 h. Digestion was stopped by heating the sample in a thermo block at 100ºC for 5 min. Then, released glycans were isolated by solid phase extraction (SPE) using Hypercarb cartridges (25 mg, 1 mL volume, Thermo Fisher Scientific) and, subsequently, purified by ice-cold acetone precipitation following the procedure reported in [19] in both cases. Reduced glycans were diluted with 50:50 H 2 O/ACN with 0.1% FA and directly analyzed by IM-MS in negative ion mode. All the experiments with the standard glycoprotein were carried out in triplicate. Mice derived transferrin glycan analysis was carried out in duplicate, including purification and release of glycans.