Glycoprotein Extraction and N-Glycan Analysis

10⁷ cells per each cell line were collected. Allantoic and amniotic membranes were extracted from 10-day old chicken embryos. All samples were treated as described previously^{37 (link),59 (link)}. Briefly, each cell line was subjected to sonication in the presence of detergent 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS, #10810118001, Roche), reduction in 4 M guanidine-HCl (#24115, Thermo), carboxymethylation, and trypsin (#T0303, Sigma) digestion. The digested glycoproteins were then purified by plus short HLB-Sep-Pak (#186000132, Waters Corp.). N-glycans were released by peptide N-glycosidase F (E.C. 3.5.1.52; #11365177001, Roche) digestion. Released N-glycans were permethylated using the sodium hydroxide procedure and purified by classic short C₁₈-Sep-Pak (#WAT051910, Waters). Permethylated N-glycans were eluted at the 50% acetonitrile fraction.

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Kikuchi C., Antonopoulos A., Wang S., Maemura T., Karamanska R., Lee C., Thompson A.J., Dell A., Kawaoka Y., Haslam S.M, & Paulson J.C. (2023). Glyco-engineered MDCK cells display preferred receptors of H3N2 influenza absent in eggs used for vaccines. Nature Communications, 14, 6178.

Publication 2023

CHAPS guanidine-HCl trypsin peptide N-glycosidase F (

Acetonitrile Allantoic Amniotic membranes Cell line Chaps Chicken Detergent Digestion Embryos Glycans Glycoproteins Guanidine Peptide n glycosidase f Propanesulfonate Sep pak c18 Sodium hydroxide Trypsin

Corresponding Organization :

Other organizations : Scripps Research Institute, Imperial College London, University of Wisconsin–Madison

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Variable analysis

independent variables

Cell lines (allantoic and amniotic membranes extracted from 10-day old chicken embryos)

dependent variables

N-glycan profiles (permethylated N-glycans eluted at the 50% acetonitrile fraction)

control variables

Cell seeding density (10^7 cells per cell line)
Experimental procedures (sonication, detergent treatment, reduction, carboxymethylation, trypsin digestion, glycoprotein purification, PNGase F digestion, permethylation, and purification)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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