Journal
GLYCOBIOLOGY
Volume 28, Issue 4, Pages 214-222Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/glycob/cwy005
Keywords
colorectal cancer; glycomics; mucin-type O-glycans; O-glycans; stable isotope labeling
Categories
Funding
- [3U01CA207821]
- [R01DK107405]
- [U01GM116196]
- [RO1HL128237]
- [GM103490]
- [U01CA168930]
- [P41GM103694]
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Mucin-type O-glycans decorate >80% of secretory and cell surface proteins and contribute to health and disease. However, dynamic alterations in the O-glycome are poorly understood because current O-glycomic methodologies are not sufficiently sensitive nor quantitative. Here we describe a novel isotope labeling approach termed Isotope-Cellular O-glycome Reporter Amplification (ICORA) to amplify and analyze the O-glycome from cells. In this approach, cells are incubated with Ac(3)GalNAc-Bn (Ac(3)GalNAc-[H-1(7)]Bn) or a heavy labeled Ac(3)GalNAc-Bn-D7 (Ac(3)GalNAc-[D-2(7)]Bn) O-glycan precursor (7 Da mass difference), which enters cells and upon de-esterification is modified by Golgi enzymes to generate Bn-O-glycans secreted into the culture media. After recovery, heavy and light Bn-O-glycans from two separate conditions are mixed, analyzed by MS, and statistically interrogated for changes in O-glycan abundance using a semi-automated approach. ICORA enables -100-1000-fold enhanced sensitivity and increased throughput compared to traditional O-glycomics. We validated ICORA with model cell lines and used it to define alterations in the O-glycome in colorectal cancer. ICORA is a useful tool to explore the dynamic regulation of the O-glycome in health and disease.
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