Session WOC. There are 4 abstracts in this session.

Session: Glycoproteomics in Biology and Medicine, time: 09:50 - 10:15 am

Nutrient Regulation of Transcription & Signaling by O-GlcNAc

Gerald Hart
CCRC, University of Georgia, Athens, GA

O-GlcNAcylation cycles on and off thousands of nucleocytoplasmic proteins and has extensive crosstalk with protein phosphorylation. O-GlcNAc is abundant on nearly all proteins involved in transcription, where it regulates gene expression in response to nutrients. O-GlcNAc also regulates the cycling of the TATA-binding (TBP) protein on and off DNA during the transcription cycle.

Targeted deletion of the O-GlcNAc Transferase in excitatory neurons of adult mice results in a morbidly obese mouse with a satiety defect. Thus, O-GlcNAcylation not only serves as a nutrient sensor in all cells, but also regulates appetite. More than eighty-percent of all human protein kinases are modified by O-GlcNAc, and all kinases that have been tested to-date are indeed regulated by the sugar. Abnormal O-GlcNAcylation of CAMKII contributes directly to diabetic cardiomyopathy and to arrhythmias associated with diabetes. Prolonged elevation of O-GlcNAc, as occurs in diabetes, contributes directly to diabetic complications and is a major mechanism of glucose toxicity. Targeted over-expression of OGT to the heart causes severe heart failure in mice, which is reversed when they are crossed with mice having OGA over-expressed in their hearts. Drugs that elevate O-GlcNAcylation in the brain, which prevents hyperphosphorylation, appear to be of benefit for the treatment of Alzheimer’s disease in animal models. To date, all cancers have elevated O-GlcNAc cycling, which plays a key role regulating the metabolism of cancer cells. Supported by NIH P01HL107153, R01GM116891, and R01DK61671. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU.

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Session: Glycoproteomics in Biology and Medicine, time: 10:15 - 10:40 am

Innovations in Chemistry and Mass Spectrometry Platform Technologies for Quantitative Glycomics

David Muddiman1; Jaclyn Gowen1; James Dodds1; Erin Baker1; James Petitte1; Alison Motsinger-Reif1; Michael MacCoss2; Thomas Montine3
1North Carolina State University, Raleigh, NC; 2University of Washington, Seattle, WA; 3Stanford University, Palo Alto, CA

Mass spectrometry offers the most robust platform to discover and characterize new diagnostic, prognostic, and therapeutic targets for disease.  A detailed characterization of glycans provides mechanistic insights into disease by understanding the role of aberrant glycosylation in disease.  To this end, we have developed bioanalytical tools to characterize structurally challenging analytes that are critical to a systems-level analysis. To increase the electrospray response of N- and O-linked glycans, perform stable-isotope relative quantification, and semi-automated data analysis, we synthesized novel hydrophobic tagging reagents (INLIGHTTM).  While this chemistry is robust and yielded deep glycome coverage, fundamentally we should be able to further increase the ionization of glycans by over 2 orders of magnitude.  Moreover, isomers are also challenging in glycomics (aka the isomer barrier); while difficult to resolve chromatographically, ion mobility spectrometry (gas-phase separations) offers an avenue to effectively address this challenge.  We demonstrate this approach can be realized using nanoLC-IM-TOF-MS of INLIGHT labeled glycans in model and complex samples.  Finally, we are developing new algorithms to analyze these complex data sets and integrate the information with glycopeptide datasets, including the analysis of IM-TOF-MS data.

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Session: Glycoproteomics in Biology and Medicine, time: 10:40 - 10:55 am

Advancing cardiac glycomics: Protein glycosylation in primary and stem cell-derived human cardiomyocytes

Christopher Ashwood1; Matthew Waas1; Ranjuna Weerasekera1; Rebekah L. Gundry1, 2
1Medical College of Wisconsin, Milwaukee, Wisconsin; 2Center for Biomedical Mass Spectrometry, MCW, Milwaukee, WI

Protein glycosylation plays an integral role in cardiomyocyte function, by modulating ion channel localization and function. However, our current view of the human cardiomyocyte glycome is limited. To address this, we applied an optimized PGC-LC-ESI-MS/MS method to characterize and quantify N- and O- glycan structures in primary human heart tissue, primary isolated cardiomyocytes, and human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs; 17 timepoints across days 20-100 of differentiation). These novel libraries, composed of 200 glycan structures quantified over three orders of magnitude, provide coverage of all major N-glycan classes and are expected to benefit future functional analyses of these glycans as well as glycoproteomic analyses.

In the primary tissue homogenate and isolated cardiomyocytes, 103 N-glycan structures were identified. Of the 15 structures which were significantly increased in the isolated cardiomyocytes, high mannose N-glycans were predominant, suggesting their potential cell-type specific expression within the myocardium. In the analysis of hiPSC-CM differentiation, 183 N-glycan structures were identified, including 37 that increase and 51 that decrease over time, demonstrating that glycosylation is dynamic throughout maturation. Specifically, 2 glycan structures that increased exponentially with time were also identified in the primary adult cardiomyocytes. Thus, they may represent novel markers of myocyte maturation. Finally, in addition to the similarities observed between primary cells and hiPSC-CMs, 32 structures were unique to primary cardiomyocytes. This suggests that glycomic analyses will be an important complement to other ‘omic and functional approaches when assessing the accuracy of hiPSC-CMs for disease modeling and drug testing.

Altogether, these data provide the most comprehensive glycomic analysis of primary human cardiomyocytes, providing the first step towards generating a cell-type and chamber-resolved glycomic map of the human heart. Moreover, the discrepancies between primary cardiomyocytes and hiPSC-CMs provide direction for future stem cell engineering efforts to generate hiPSC-CMs that more closely represent primary cardiomyocytes.

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Session: Glycoproteomics in Biology and Medicine, time: 10:55 - 11:10 am

Mapping the O‐glycoproteome using site‐specific extraction of O‐linked glycopeptides (EXoO)

Weiming Yang; Minghui Ao; Yingwei Hu; Qing Kay Li; Hui Zhang
Johns Hopkins University, Baltimore, MD

Protein glycosylation is one of the most abundant post‐translational modifications. However, detailed analysis of O‐linked glycosylation, a major type of protein glycosylation, has been severely impeded by the scarcity of suitable methodologies. Here, a chemoenzymatic method is introduced for the site‐specific extraction of O‐linked glycopeptides (EXoO), which enabled the mapping of over 3,000 O‐linked glycosylation sites and definition of their glycans on over 1,000 proteins in human kidney tissues, T cells, and serum. This large‐scale localization of O‐linked glycosylation sites demonstrated that EXoO is an effective method for defining the site‐specific O‐linked glycoproteome in different types of sample. Detailed structural analysis of the sites identified revealed conserved motifs and topological orientations facing extracellular space, the cell surface, the lumen of the Golgi, and the endoplasmic reticulum (ER). EXoO was also able to reveal significant differences in the O‐linked glycoproteome of tumor and normal kidney tissues pointing to its broader use in clinical diagnostics and therapeutics.

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