Recombinant Nepenthesin II for Hydrogen/Deuterium Exchange Mass Spectrometry

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• 作者：Menglin Yang ; Morgan Hoeppner ; Martial Rey ; Alan Kadek ; Petr Man ; David C. Schriemer
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：July 7, 2015
• 年：2015
• 卷：87
• 期：13
• 页码：6681-6687
• 全文大小：466K
• ISSN：1520-6882

文摘

The pitcher secretions of the Nepenthes genus of carnivorous plants contain a proteolytic activity that is very useful for hydrogen/deuterium exchange mass spectrometry (HX-MS). Our efforts to reconstitute pitcher fluid activity using recombinant nepenthesin I (one of two known aspartic proteases in the fluid) revealed a partial cleavage profile and reduced enzymatic stability in certain HX-MS applications. We produced and characterized recombinant nepenthesin II to determine if it complemented nepenthesin I in HX-MS applications. Nepenthesin II shares many properties with nepenthesin I, such as fast digestion at reduced temperature and pH, and broad cleavage specificity, but in addition, it cleaves C-terminal to tryptophan. Neither enzyme reproduces the C-terminal proline cleavage we observed in the natural extract. Nepenthesin II is considerably more resistant to chemical denaturants and reducing agents than nepenthesin I, and it possesses a stability profile that is similar to that of pepsin. Higher stability combined with the slightly broader cleavage specificity makes nepenthesin II a useful alternative to pepsin and a more complete replacement for pitcher fluid in HX-MS applications.