Research Resource for Integrated Glycotechnology:

Ron Orlando

Development of Mass Spectrometric Techniques for the

Analysis of Glycoprotein Glycans and Oligosaccharides

We have been developing amide exchange mass spectrometry to study protein carbohydrate interactions. Amide hydrogen exchange has been used for over two decades to study protein dynamics and structure. This approach is based upon the rate at which specific amide hydrogens exchange with the solvent, which depends on hydrogen-bonding patterns and the accessibility of each amide hydrogen to the solvent. For example, amide hydrogens on the exterior surface of a protein exchange much faster than those buried in the center of the molecule. While NMR spectroscopy is by far the most common method of studying amide hydrogen exchange, MS has also recently been used in studies of the rate of amide hydrogen exchange.

Amide exchange-MS has not been previously applied to characterize the sites of protein-carbohydrate interaction, and hence, we performed several experiments on the well-studied lysozyme-chitin system to test the ability of amide exchange-MS to characterize this type of interaction. These results clearly show that chitobiose and chitotetraose prevented amide hydrogens on lysozyme from exchange with the solvent. Furthermore, doubling the size of the sugar ligand (biose to tetraose) doubled the number of amide hydrogens protected; approximately 15 were protected by chitobiose, while approximately 30 were protected by chitotetraose. These numbers are in excellent agreement with the structure of these complexes determined by X-ray defraction, which indicate that chitobiose and chitotetraose will protect 16 and 28 amide hydrogens from exchange with the solvent. This data is highly suggestive that the carbohydrate substrate selectively protected amide hydrogens at the site of interaction.