Fast glycan kit

Water (HPLC-grade), acetonitrile, dithiothreitol (DTT), ammonium acetate, acetic acid, sodium cyanoborohydride (1 M in THF), glycerol, sodium dodecyl sulfate (SDS), and Nonidet P-40 (NP-40) were obtained from Sigma Aldrich (St. Louis, MO, USA). Aminopyrene-1,3,6-trisulfonic acid (APTS), the high-resolution N-linked carbohydrate separation buffer (HR-NCHO), the bracketing standard and the magnetic beads were part of the Fast Glycan Kit (Sciex, Brea, CA, USA). The human serum and blood samples were kindly provided by the Drug Research Center (Balatonfüred, Hungary). PNGase F enzyme was produced in-house (University of Pannonia, Veszprém, Hungary).

All serum samples were collected with the appropriate ethical permissions (approval number: 23580-1/2015/EKU (0180/15)) and informed patient consent at the Department of Pulmonology in the Semmelweis Hospital (Miskolc, Hungary). Serum samples were taken from 17 lung cancer patients before and after tumor removal surgeries; see details in Table 1. Sample preparation protocol included denaturation, glycan release, fluorophore labeling and magnetic bead-mediated cleanup. Serum samples were diluted a hundredfold by HPLC-grade water, followed by denaturation at 70 °C for 10 min by adding 2.0 µL denaturation solution of the Fast Glycan kit (SCIEX). Glycan release was performed by the addition of 1.0 µL of PNGase F enzyme (200 mU) to the reaction mixture and incubated at 60 °C for 20 min to ensure complete deglycosylation. The endoglycosidase digestion reaction was stopped by the addition of the labeling solution containing 1.0 µL of 40 mM 8-aminopyrene-1,3,6-trisulfonic acid (APTS) in HPLC-grade water, 2.0 µL of NaBH₃CN (1 M in THF), 10 µL 50% acetic acid, 8.0 µL THF. The reaction mixture was incubated in a heating block overnight at 37 °C. After the labeling step, the samples were purified by magnetic beads following the Fast Glycan Sample Preparation and Analysis protocol and analyzed by capillary electrophoresis utilizing laser-induced fluorescent detection.

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.