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. 2020 Jan 20;10:671. doi: 10.1038/s41598-020-57510-z

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© The Author(s) 2020, corrected publication 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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New workflow for IgA1 O-glycomic analysis by using the sequential deglycosylation protocol. (a) Protocol for the profiling of IgA1 HR O-glycans. Neuraminidase treatment reduces the types of O-glycan structures from six to two (disaccharide N-acetylgalactosamine [GalNAc]-galactose [Gal] and monosaccharide GalNAc). LC-MS spectrum contains several mass peaks of HR with O-glycans, according to the number of the attached GalNAc and Gal residues. An extracted ion chromatogram (XIC) is generated for each peak and the area under the curve (AUC) of XIC of each O-glycopeptide is determined and used to calculate the relative abundance (RA) of each O-glycoform (RA = percent of total XIC of O-glycoforms detected). (b) Protocol for the analysis of the attachment sites of galactose-deficient (Gd) O-glycan. IgA1 proteins were treated with an IgA-specific protease from Clostridium ramosum AK183. After a sequential enzymatic deglycosylation with neuraminidase and O-glycanase, that ultimately leaves only Gd O-glycans (i.e., GalNAc) attached to the amino-acid backbone, IgA1 is digested by trypsin. Precursor ions corresponding to HR with one to three Gd O-glycans are analyzed by LC-tandem MS with ETD combined with supplemental higher energy collision dissociation (HCD) activation (EThcD) to assign the attachment sites of Gd O-glycans.