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. 2022 Jun 24;13:3617. doi: 10.1038/s41467-022-31205-7

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© The Author(s) 2022

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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Fig. 4 — a CMP-Neu5Ac’s modified with defined matriglycan polymeric repeats (100 μM) are engineered on HAP1-DAG1^- cells using ST6GAL1 in the presence of C. perfringens neuraminidase. b Detection of matriglycan with 1, 4, 5, and 6 disaccharide repeats on HAP1-DAG1^- cells by flow cytometry. Cells were stained with avidin-AF488 and co-stained with PI to exclude non-viable cells. c Detection of matriglycan with 4 repeats at various concentrations of modified donor. d Binding of IIH6 to HAP1-WT and HAP1-DAG1^- matriglycan modified cells. e Shotgun proteomics analysis of proteins immunoprecipitated from HAP1-DAG1^- cells labeled with Biotin or Biotin+8 disaccharide repeats. Proteins present in the negative control experiment (unlabelled cells), had fewer than 10 spectral counts in the CMP-Neu5Ac-(Biotin) labeling experiment, or known to be localized in intracellular compartments as assessed by UNIPROT annotations, were excluded. Proteins shown are all annotated in UNIPROT to contain sites of N-glycosylation or were manually validated to contain at least one N-X-(S/T) N-glycosylation sequon in the primary sequence. One representative run is shown. Three independent experiments were performed with similar results each time. Source data are provided as a Source Data file.