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Biopharmaceutical Glycoanalysis: Comparison of Nonspecific Pronase Digestion and 2-AB Labeled Glycan LC-MS/MS

Published

Author(s)

John E. Schiel, Hua-Jun He, Karen W. Phinney, Jennifer Au

Abstract

Glycosylation, the enzymatic addition of carbohydrates to a protein, is one of the most abundant post-translational modifications found in nature. There is variability in the number, location, and identity of glycans attached. As a result, a glycoprotein consists of a number of glycoforms with different combinations of glycans, potentially resulting in different stability, toxicity, and activity. This is especially important in the biopharmaceutical industry where product consistency and safety are vital. Glycoprotein analysis involves numerous mass spectrometry-based techniques, each of which provides various aspects of characterization. The current paper describes two commonly used analytical techniques for glycoprotein characterization. In one experiment nonspecific proteolysis is combined with a two-tiered mass spectrometry approach (MALDI-TOF and LC-MS/MS) to gain glycosylation site and glycan identity. In a second approach, glycans were enzymatically released, labeled with a fluorescent dye, and analyzed using LC-fluorescence-MS/MS to give glycan identification and relative quantification. The type and degree of information yielded by each method is compared in an effort to identify what types of standard materials may be useful for harmonization of glycoanalysis methods and increased precision in their measurements.
Citation
Analytical and Bioanalytical Chemistry

Keywords

Glycoprotein, Glycan, Mass Spectrometry, Standards

Citation

Schiel, J. , He, H. , Phinney, K. and Au, J. (2011), Biopharmaceutical Glycoanalysis: Comparison of Nonspecific Pronase Digestion and 2-AB Labeled Glycan LC-MS/MS, Analytical and Bioanalytical Chemistry (Accessed June 23, 2024)

Issues

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Created November 29, 2011, Updated February 19, 2017