Blotglyco

AGP preparations were purified from serum samples (n = 10) by an AGP-prep-DOCK^{26 (link)}. N-Glycans of AGP and labeled N-glycans of AGP were prepared and purified as described previously^{26 (link)} using PNGase F (Roche Applied Science, Indianapolis, IN) and BlotGlyco (Sumitomo Bakelite, Co., Tokyo, Japan). Mass spectrometric data were obtained using a MALDI-TOF/TOF ultrafleXtreme (Bruker) and all the primary structures of AGP glycans were assigned with the aid of the AGPAS software^{25 (link),26 (link)}. The relative abundance of α1,3fucosylated tri- and tetraantennary glycans in AGP was defined as FUCAGP^{26 (link)}.

Samples for MALDI-TOF-MS were prepared from 10 μg of purified IgGs from CIA-induced ST6Gal1^f/f AID-Cre, ST6Gal1^f/f, AID-Cre and C57BL/6j mice and total serum IgG from ST6Gal1 KO mice by BlotGlyco (Sumitomo Bakelite Co., Tokyo, Japan) according to the manufacturer's protocol55 (link). Samples were analysed by MALDI-TOF system by using an Autoflex III TOF/TOF mass spectrometer equipped with a reflector and controlled by the FlexControl 3.0 software package (Bruker Daltonics GmbH, Bremen, Germany). Details of the method used for MALDI-TOF-MS analysis are described in Supplementary Methods.

Peroxidase conjugated ABC reagent, Vectastain Elite ABC standard kit was from Vecter Laboratories, Inc. (Burlingame, CA, USA). Antihuman AGP rabbit serum and Protein Block Serum-Free Reagent were from Dako (Carpinteria, CA, USA). Peroxidase conjugated anti-human AGP and Universal HIER antigen retrieval reagent were from Abcam (Cambridge, UK). KPL SureBlue TMB Microwell Peroxidase Substrate was from Sera Care Life Sciences (Milford, MA, USA). Anti-PD-L1 and SignalStain Boost IHC Detection Reagent were obtained from Cell Signaling Technology (Danvers, MA, USA). N-Histofine High Stain HRP (Multi) was from Nichirei Biosciences Inc. (Tokyo, Japan). PNGase F was from Roche Applied Science (Indianapolis, USA). BlotGlyco was obtained from Sumitomo Bakelite, Co. (Tokyo, Japan). Neuraminidase (Arthrobacter ureafaciens, 1U/ml) was purchased from Nacalai Tesque (Kyoto, Japan). Biotinylated Aleuria aurantia lectin (AAL) was kindly provided by Prof. Naohisa Kochibe, Gunma University. Tumor-associated antigens in serum samples measured in this study were carcinoembryonic antigen (CEA) and squamous cell carcinoma (SCC). Levels of each antigen were determined by an ELISA using the Cobas system (Roche for CEA) and the ARCHITECT system (Abbot for SCC) and standard cut-off values were set at 5.0 ng/ml for CEA and 1.5 ng/ml for SCC, respectively.

Release of N-glycans from IgG sample and subsequent derivatization with 2-aminopyridine was performed using BlotGlyco (Sumitomo Bakelite, Tokyo, Japan) under the manufacturer’s instruction with slight modifications. The released N-glycans and peptides were removed using a graphite carbon column (InertSep GC, GL Sciences, Tokyo, Japan), and free 2-aminopyridine was removed from the reaction mixture using MonoFas I Spin Column (GL Sciences, Tokyo, Japan). The resultant pyridylamino (PA)-glycans were then applied on a LaChrom Elite HPLC system (Hitachi, Tokyo, Japan) under conditions reported previously [10] (link), [11] (link) using a TSKgel DEAE-5PW (7.5 mm×7.5 cm, Tosoh Corporation, Tokyo, Japan) and a Shim-pack HRC-ODS column (6.0 mm×15 cm, Shimadzu, Kyoto, Japan). PA-oligosaccharides were detected by fluorescence using excitation and emission at 320 and 400 nm, respectively. Elution times of the individual peaks from the ODS columns were normalized with respect to the PA-derivatized isomalto oligosaccharides with degree of polymerization 3–22 (TaKaRa-Bio, Shiga, Japan), and reported in glucose units (GU). PA-oligosaccharides derived from IVIG were identified by comparison with the GUs of reference PA-oligosaccharides in a web application, GALAXY (http://www.glycoanalysis.info/) [12] . The amount of PA-oligosaccharides was estimated using the peak area in the chromatogram.

Human nasal IgA and recombinant IgG and IgA antibodies were subjected to glycan analysis. Human nasal IgA was purified from nasal wash concentrates using CaptureSelect IgA (Thermo Fisher Scientific), according to the manufacturer’s instructions. Purified antibodies were concentrated using Amicon Ultracell (Merck, Darmstadt, Germany) centrifugation units with a cut-off of 30 kDa. The human nasal wash concentrate samples used here were residual samples collected from a past clinical study [60 (link)]. Antibodies were deglycosylated by PNGase F (Takara Bio, Shiga, Japan) treatment. The released N-glycans were purified and labeled with 2-aminobenzamide using BlotGlyco (Sumitomo Bakelite, Tokyo, Japan), according to the manufacturer's instructions. Liquid chromatography-mass spectrometry (LC-MS) analysis was performed by Sumitomo Bakelite using a LC-MS-IT-TOF apparatus (Shimadzu, Kyoto, Japan). N-glycan structures were predicted using the GlycoMod Tool (http://web.expasy.org/glycomod/) [61 (link)].