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. 2021 Mar 9;11:5505. doi: 10.1038/s41598-021-84771-z

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© The Author(s) 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Analysis of the reaction products of BmGalNAcT. (a) CBB staining, His-tag staining, N-glycoprotein staining, and (b) de-glycosylation analysis of purified BmGalNAcT. Black and white triangles indicate the purified N-glycosylated form and de-glycosylated BmGalNAcTs, respectively. (c) SF- and RP-HPLC analysis of the reaction products. BmGalNAcT reaction was carried out using UDP-GalNAc and GN2M3 as a donor and an acceptor substrate, respectively. The elution position of the product was compared with authentic PA-sugar chains. Numbers at the top represent the elution positions of glucose units on the basis of the elution times of PA-isomalto-oligosaccharides with degrees of polymerization from 3 to 15. Green circles and blue boxes indicate Man and GlcNAc, respectively. (d) MS and MS/MS analysis of the reaction product. The MS signal represents (M + H)⁺ ions of the reaction product. The value of m/z 503 detected in the MS/MS spectra agrees with the calculated mass of HexNAc₂-PA. The mass of the precursor ion, m/z 1801.8, was considered to correspond to HexNAc₄Hex₃HexNAc₂-PA. The black and gray arrows represent N-acetylhexosamine and hexose, respectively. A black diamond indicates the precursor ion of MS/MS fragmentation.