Chris Ashwood   Christopher Ashwood is a Postdoctoral Fellow at the Medical College of Wisconsin, USA, as part of the Gundry Lab. He is obtaining his PhD at Macquarie University, Australia under the supervision of Prof. Nicki Packer. He obtained a Master of Research investigating the glycosylation pathway of fungus in 2015 and his Ph.D. focuses on advancing PGC-LC-ESI-MS/MS for glycomics with applications in TLR4-mediated inflammation, submitting this year. Chris has performed oral presentations regarding technical and software applications in the area of glycomics at HUPO in 2016, as part of a Thermo Scientific workshop, and in 2017, as part of the main program.

Applications of Skyline for automated profiling of cellular protein glycosylation

Profiling protein glycosylation is challenging due to the presence of highly similar glycan structures that play diverse roles in cellular physiology. As the anomericity of a single glycosidic bond can influence glycan function, there is a demand for automated methods to discriminate between structurally similar isomers, overcoming a bottleneck in the data analysis of LC-MS-based glycomics.Using internal standards in combination with separation based on glycan structure, we obtain normalised retention times for >200 glycans, covering >95% of glycans released from a human cell lysate. Interrogating similar fragment spectra from structurally related glycan isomers, discriminatory product ions were discovered. Using Skyline, high specificity product ions were validated for automated glycan isomer discrimination. Read More
Finally, we combine both approaches, building an annotated spectral library of >200 glycan structures with normalised retention values. We apply this spectral library in Skyline for automated glycan profiling of cell lines and tissues, achieving confident peak assignment.