Amplification and Preparation of Cellular O‑Glycomes for Functional Glycomics
journal contributionposted on 2020-07-13, 22:03 authored by Zhonghua Li, Qing Zhang, David Ashline, Yuyang Zhu, Yi Lasanajak, Tatiana Chernova, Vernon Reinhold, Richard D. Cummings, Peng G. Wang, Tongzhong Ju, David F. Smith, Xuezheng Song
Mucin-type O-glycans play key roles in many cellular processes, and they are often altered in human diseases. A major challenge in studying the role of O-glycans through functional O-glycomics is the absence of a complete repertoire of the glycans that comprise the human O-glycome. Here we describe a cellular O-glycome preparation strategy, Preparative Cellular O-Glycome Reporter/Amplification (pCORA), that introduces 4-N3-Bn-GalNAc(Ac)3 as a novel precursor in large-scale cell cultures to generate usable amounts of O-glycans as a potential O-glycome factory. Cultured human non-small cell lung cancer (NSCLC) A549 cells take up the precursor, which is extended by cellular glycosyltransferases to produce 4-N3-Bn-α-O-glycans that are secreted into the culture medium. The O-glycan derivatives can be clicked with a fluorescent bifunctional tag that allows multidimensional HPLC purification and production of a tagged glycan library, representing the O-glycome of the corresponding cells. We obtained ∼5% conversion of precursor to O-glycans and purified a tagged O-glycan library of over 100 O-glycan derivatives, many of which were present in >100 nmol amounts and were sequenced by sequential MS fragmentation (MSn). These O-glycans were successfully printed onto epoxy glass slides as an O-glycome shotgun microarray. We used this novel array to explore binding activity of serum IgM in healthy persons and NSCLC patients at different cancer stages. This novel strategy provides access to complex O-glycans in significant quantities and may offer a new route to discovery of potential diagnostic disease biomarkers.
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NSCLC patientsnon-small cell lung cancernovel strategyglass slides100 O-glycan derivativesO-glycan librarysequential MS fragmentationO-glycome factoryMS ndisease biomarkersO-glycome shotgun microarraynovel arrayBn -α-OFunctional Glycomics Mucin-type O-g...bifunctional tagcancer stagesserum IgM4- N 3binding activitycell cultures549 cellsculture mediumO-glycome preparation strategynovel precursorO-glycan derivativesHPLC purificationglycan library