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Cellular O‐Glycome Reporter/Amplification (CORA) to Explore O‐Glycans of Living Cells
Author(s) -
Kudelka Matthew R.,
Antonopoulos Aristotelis,
Wang Yingchun,
Duong Duc M.,
Song Xuezheng,
Seyfried Nicholas T.,
Dell Anne,
Haslam Stuart M.,
Cummings Richard D.,
Ju Tongzhong
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.623.1
Subject(s) - glycome , glycan , glycosylation , glycomics , chemistry , biochemistry , fucosylation , computational biology , biology , glycoprotein
O‐glycosylation is present on over 80% of proteins that traverse the secretory apparatus and plays key roles in many biological processes. However, the repertoire of O‐glycans synthesized by cells and thus their function are difficult to determine. Current strategies to evaluate O‐glycans from cells utilize chemical release, such as alkaline β‐elimination, prior to analysis by mass spectrometry or other technologies. However, β‐elimination is inefficient, potentially biased, and results in O‐glycan degradation. To address this challenge, we developed a technology to amplify and profile mucin‐type O‐glycans synthesized by living cells, termed Cellular O‐Glycome Reporter/Amplification (CORA). We developed a chemical O‐glycan precursor that when incubated with live cells crosses the plasma membrane, is taken up by the Golgi Apparatus, and is modified by glycosyltransferases in situ , before being secreted from cells as a variety of modified O‐glycan derivatives, allowing easy purification for analysis by HPLC and mass spectrometry (MS). CORA detected O‐glycans observed by β‐elimination as well as many additional complex structures with ~100–1000‐fold increase in sensitivity over conventional O‐glycan analyses. Furthermore, CORA coupled with computational modeling allowed us to predict the diversity of the human O‐glycome. To our knowledge, CORA is the first technology for glycome amplification and thus could offer new opportunities for understanding the role of glycans in health and disease. Support or Funding Information This work was supported by National Institutes of Health Grant U01CA168930 to TJ and RDC, P41GM103694 to RDC, Georgia Cancer Coalition (now Georgia Research Alliance, GRA) Award to TJ, and by Biotechnology and Biological Sciences Research Council grant BB/K016164/1 (AD and SMH for Core Support for Collaborative Research). AD is supported by a Wellcome Trust Senior Investigator Award. We also acknowledge support from the Emory Integrated Proteomics Core.