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Negative Electron Transfer Dissociation paired with Capillary Electrophoresis-Mass Spectrometry for the investigation of Glycosaminoglycan mixtures

Morgan Stickney
Morgan Stickney
Chemistry Department
University of Georgia
Chemistry Building, Room 400
Analytical Seminar

Sulfated glycosaminoglycan (GAG) carbohydrates are long, linear, acidic polysaccharide chains abundant on the surface of virtually all mammalian cells[1,2]. Non-template driven modifications affect many biological functions through protein-binding interactions[3]. The complex structure and low natural abundance of GAG oligomers remains a significant analytical challenge. Capillary electrophoresis-mass spectrometry (CE-MS) presents an efficient approach for online separation of complex GAG mixtures when paired with a commercial interface that has a low dilution factor. Mixtures of purified GAG standards have been baseline resolved and biological mixtures have been significantly reduced in complexity[4].

Tandem mass spectrometry is essential for characterization of the modification patterns and binding motifs of GAGs. Negative Electron Transfer Dissociation (NETD) is a powerful fragmentation method that can be performed on mass spectrometry platforms capable of ion-ion reactions. Electron based fragmentation methods like NETD cause radical rearrangement reactions that fragment GAG backbones, without stripping sensitive sulfo-modifications, on a time scale amenable to CE separations[5]. By combining a CE-MS separation platform with NETD we can obtain precise structural characterization of diasteriomeric GAG mixtures to determine binding motifs responsible for GAG chain interactions with proteins. Future research will see this platform applied to protein pulldown samples, and automation of sample analysis.

(1) Kailemia, M. J., et al. (2014). "Oligosaccharide Analysis By Mass Spectrometry: A Review Of Recent Developments." Analytical Chemistry 86(1): 196-212.

(2) Laremore, T. N., et al. (2010). "High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis." Analytical Biochemistry 401(2): 236-241.

(3)Aquino, R. S. and P. W. Park (2016). "Glycosaminoglycans and infection." Frontiers in bioscience (Landmark edition) 21: 1260-1277.

(4) Sanderson, P., et al. (2018). "Heparin/heparan sulfate analysis by covalently modified reverse polarity capillary zone electrophoresis-mass spectrometry." Journal of Chromatography A 1545: 75-83.

(5) Leach, F. E., et al. (2017). "Negative Electron Transfer Dissociation Sequencing of Increasingly Sulfated Glycosaminoglycan Oligosaccharides on an Orbitrap Mass Spectrometer." Journal of The American Society for Mass Spectrometry 28(9): 1844-1854.

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