Disulfide Mapping: Searching Stressed Glycoprotein Therapeutics for Disulfide Scrambled Species

摘要

Confirmation of the native disulfide connectivity of GCase was achieved using CAD fragmentation analysis as well as through dual proteolysis in combination with LC-MS. Alternate methods utilizing AspN and pepsin proteases could not efficiently cleave in the region around the cysteines, possibly due to structural hindrances imposed by the disulfide bonds. Thermolysin, contrary to Asp-N and pepsin, could successfully and efficiently digest the disulfide rich region of GCase to generate peptides containing inter-linked disulfides. With the proteolytic combination of Asp-N and thermolysin and the sensitivity from nano LC-MS we can successfully search for scrambled species while confirming the native disulfide connectivity of acid beta-glucocerebrosidase.