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. 2018 Jun 7;8:8730. doi: 10.1038/s41598-018-26913-4

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© The Author(s) 2018

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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Preparation of M6PgPs from the glyco-engineered S. cerevisiae. (A) Schematic representation of M6PgP preparation procedure. Cell wall mannoproteins of the glyco-engineered yeast were digested with pronase, which generated short glycopeptides (a). The glycopeptides were uncapped for generation of M6P glycans by MAH (b) and then digested with α(1,2)-mannosidase (c). Finally, the resulting M6PgPs were purified by using TiO₂ column (d). (B) N-glycans of the glycopeptides after each preparation step (a–d) were analyzed in a linear negative ion mode after 2-AA labeling (which adds 121 Da) by using MALDI-TOF mass spectrometry²². For confirmation of phosphate group existence, alkaline phosphatase was treated (+AP). Monosaccharide symbols follow the SNFG (Symbol Nomenclature for Glycans) system, details of which are found at NCBI⁴⁰: green circle, mannose; blue square, GlcNAc; P, phosphate.