Supplementary DataMaterials and MethodsMaterialsBovine fetuin, human IgG, N-(3-dimethylaminoprophyl)-N’-ethylcarbodiimide hydrochloride (EDC), α-cyano-4-hydroxycinnamic acid (CHCA), dimethyl sulfoxide (DMSO) and 2, 5-dihydroxybenzoic acid (DHB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetohydrazide (Ah) and Girard’s reagent P (GP) was from Tokyo Chemical Industry (Tokyo, Japan). ReadyPrep Proteomics-Grade water was from Bio-Rad Laboratories (CA, USA). Hydrochloric acid (HCl), acetonitrile (ACN), methanol (MeOH) and acetic acid were purchased from Junsei (Tokyo, Japan). Peptide N-glycosidase F (PNGase F) was purchased from Roche (Mannheim, Germany). LudgerTagTM 2-AB glycan labeling kit was obtained from Ludger (Oxfordshire, UK). Human sera samples from healthy people were obtained from Dongguk University (n=6) (Seoul, Korea).Derivatization with 2-AB for UPLC analysisDried human IgG N-glycan was modified by reductive amidation using LudgerTagTM 2-AB glycan labeling kit. N-glycan sample was dissolved with 5 µl of 2-AB labeling reagent prepared following the manufacturer’s instruction. And then, this solution was incubated at 65°C for 3hr. Subsequently, removal of excess labeling reagent was performed by Whatman 3MM chromatography paper (Whatman, UK). 3MM paper cut to 10X2.5 cm2 was washed by ultrapure water for 15min two times and dried. 5 µl of 2-AB labeled N-glycans solution was spotted on dried 3MM paper above 1 cm from the bottom and dried. 3-MM paper was saturated by ACN for 1 hr. After drying, Spot in which 2-AB labeled N-glycans sample was absorbed was identified by UV and then cut. It was put in norm-ject syringe (norm-ject, Germany) and PTFE Millex-LCR filter (Millipore, USA) was fitted in syringe. After 0.5 ml of ultrapure water was added into syringe and kept for 10 min, 2-AB labeled N-glycans were eluted by pushing plunger two times. Finally, the 1 ml of the eluate in effendorf tube was dried in the rotary evaporator.Preparation of AFPs from Huh7 hepatocarcinoma cellsFor AFP purification, an anti-AFP antibody (Boditech Med Inc., Korea) was conjugated to Protein G Sepharose 4 Fast Flow (GE Healthcare Bio-Science, PA, USA) according to the manufacturer’s instructions. Huh7 hepatocarcinoma cells (Korea Cell Line Bank, Cat#60104, Korea) at 80% confluence were cultured in serum-free culture media for 3 days to collect conditioned medium, which contained the secreted AFPs. The collected medium was concentrated and applied to the anti-AFP column and then eluted with IgG elution buffer after extensive washing with PBS. The AFP concentrations were determined by measuring Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc., IL, USA). The purity of the AFPs was confirmed by SDS-PAGE, in conjunction with commassie blue staining.UPLC analysis of human IgG labeled with 2-ABDried sample was dissolved in 60% [v/v] HPLC grade acetonitrile/HPLC grade water and then diluted with same reagent (5mg/ml) for UPLC analysis. 2-AB labeled N-glycans sample was analyzed using ACQUITY UPLCR H-class system (Waters, MA, USA) consisting of quaternary solvent manager, sample manager, column manager and FLR detector and separated by a BEH glycan column (1.7µm particle, 2.1 i.d. × 150mm, Waters, MA, USA). Solvent A was 50 mM ammonium formate at pH4.4 and solvent B was acetonitrile. Initial gradient condition at 30% solvent A was maintained for 2min and then increased from 30% to 80% solvent A for 37 min. Injection volume of sample maintained at 10°C was 2 µl and column temperature was 40°C. Module parameters of FLR detector were 330nm wavelength (excitation) and 420nm wavelength (emission). UPLC is calibrated by dextran ladder for GU assignment (Waters, MA, USA). All acquisition and processing of data were performed by UNIFI software (Waters, MA, USA).

Supplementary Table 1. Comparison between relative intensities of the N-glycans derived from AFP of human cord serum and Auh7 cell labeled with Ah and GP obtained by MALDI-MS

Peak no.

Calculated[M]+

Found[M]+

Human cord serum Huh7 cell

proposed glycan

structure

Compositions

Relative intensity (%)

Relative intensity (%) H HN F NA NG

1 1596.7 1596.6 0.1±0.17 1.6±1.35 3 4 1 0 0

2 1758.8 1758.9 0.2±0.29 1.1±0.98 4 4 1 0 0

3 2121.9(+Ah 1x)

2122.4(+Ah 1x) 12.5±6.98 N.D 5 4 0 1 0

4 2268.0(+Ah 1x)

2267.9(+Ah 1x) 0.8±0.67 16.3±2.81 5 4 1 1 0

5 2414.1(+Ah 1x)

2413.6(+Ah 1x) 11.8±3.48 0.7±0.61 5 4 2 1 0

6 2469.1(+Ah 2x)

2469.4(+Ah 2x) 73.2±4.22 1.4±0.97 5 4 0 2 0

7 2615.1(+Ah 2x)

2615.2(+Ah 2x) 1.4±0.28 78.9±2.81 5 4 1 2 0

Supplementary Figure 1. Determination of limit of detection (LOD) for N-glycans from bovine fetuin using MALDI-QTaG method (0.5 pmol on the MALDI spot)

Supplementary Figure 2. Comparison of relative quantities of N-glycans derived from human IgG using MALDI-MS and UPLC