For the hinge region digestion of IgA1 we relied on the O-glycopeptidase from Akkermansia muciniphila, OgpA; OpeRATOR®, Genovis, Llund, Sweden). The enzyme docks at O-glycans, by preference non-sialylated core 1 (thus GalNAc-Gal), and then cleaves the protein N-terminally of the glycan. As these O-glycans are unique for IgA1 (and not present in IgA2) exclusively the Fab molecules of IgA1 are cleaved off.
Based on previous analysis of these human milk samples, by MS-based proteomics and ELISA, the expected IgA1 concentrations in the human milk samples were assumed to be 0.2 - 1.4 mg/mL (17 (link)), and therefore contain at least 10 µg in 50 µL. Based on these concentrations we added to each spin column 50 µL PBS containing 40 U SialEXO (a sialidase cocktail to remove sialic acids from the O-glycans) and incubated for 1 h at 37°C with continuous shaking at 750 rpm. Then 1 µL (40 U) of OgpA enzyme was added, and incubation was continued overnight, in and Eppendorf thermal shaker (Eppendorf, The Netherlands). Following overnight digestion with OgpA, 20 µL of pre-washed Ni-NTA agarose slurry (1:1 in PBS) was added to the spin columns in order to capture the His-tagged enzymes. The incubation was continued for 30 more minutes. Then the plug was removed from the column, and the flowthrough, containing the IgA1 Fabs, was collected by centrifugation for 1 min at 500 × g, RT.
Based on previous analysis of these human milk samples, by MS-based proteomics and ELISA, the expected IgA1 concentrations in the human milk samples were assumed to be 0.2 - 1.4 mg/mL (17 (link)), and therefore contain at least 10 µg in 50 µL. Based on these concentrations we added to each spin column 50 µL PBS containing 40 U SialEXO (a sialidase cocktail to remove sialic acids from the O-glycans) and incubated for 1 h at 37°C with continuous shaking at 750 rpm. Then 1 µL (40 U) of OgpA enzyme was added, and incubation was continued overnight, in and Eppendorf thermal shaker (Eppendorf, The Netherlands). Following overnight digestion with OgpA, 20 µL of pre-washed Ni-NTA agarose slurry (1:1 in PBS) was added to the spin columns in order to capture the His-tagged enzymes. The incubation was continued for 30 more minutes. Then the plug was removed from the column, and the flowthrough, containing the IgA1 Fabs, was collected by centrifugation for 1 min at 500 × g, RT.
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