Characterization of Maltoheptaose and Complex Glycoprotein Glycan Structure by LC/MS

Characterization of Complex Glycoprotein Glycan Structures by LC/MS Sebastien Cendron, Emily McLaughlin, Christopher N. LaFratta Chemistry Department, Bard College, Annadale-on-Hudson, NY. Hui Zhou, John Froehlich, Children’s Hospital Boston, Boston, MA. The relatively new field of glycomics studies the complete set of glycan structures belonging to a set of proteins, a cell, or an organism. In this work, we seek to isolate and characterize the glycan from ribonuclease B using liquid chromatography and tandem mass spectrometry. The overall procedure involves a reductive alkylation and enzymatic release of the glycans, followed by permethylation of the glycan before LC-MS analysis. A control sample maltoheptose were used to verify the reaction and analysis conditions. Chromatography of methylated maltoheptaose yielded insight into the relative quantities of fully and partially methylated products, a side reaction between the reagents used in permethylation, the presence of α and ß anomers, and stereochemical features of the maltoheptaose-C18 affinity. The cyclic nature of the standard’s reducing end sugar was further confirmed by tandem mass spectrometry. Isolation of glycan from ribonuclease B was then carried out with a reduction and alkylation of the protein, spin filtration, enzymatic deglycosylation, and liquid phase permethylation. Analysis of mass spectral data on the ribonuclease B glycan is only partially complete