N-Glycans were released from serum proteins using PNGase F, as was previously described (32). In summary, 6 µL serum of each sample was denatured by addition of 12 µL of 2% SDS, followed by incubation for 10 min at 60°C. The release step was performed by the addition of 12.6 µL releasing mixture, which consisted of 2.5× PBS containing 2% NP-40 and 0.4 mU PNGase F, followed by 16 h incubation at 37°C. After release, the samples were stored at −20°C or transferred to the robot platform to be used directly.