Recombinant Nepenthesin II for Hydrogen/Deuterium Exchange Mass Spectrometry

被引：53

作者：

Yang, Menglin ^{[1
]}

Hoeppner, Morgan ^{[1
]}

Rey, Martial ^{[1
]}

Kadek, Alan ^{[2
,3
]}

Man, Petr ^{[2
,3
]}

Schriemer, David C. ^{[1
]}

机构：

[1] Univ Calgary, Dept Biochem & Mol Biol, Calgary, AB T2N 1N4, Canada

[2] Acad Sci Czech Republic, Inst Microbiol, Prague 11720, Czech Republic

[3] Charles Univ Prague, Fac Sci, Dept Biochem, CR-11636 Prague, Czech Republic

来源：

ANALYTICAL CHEMISTRY | 2015年 / 87卷 / 13期

基金：

加拿大自然科学与工程研究理事会; 加拿大创新基金会;

关键词：

ASPARTIC PROTEASE NEPENTHESIN-1; UNIQUE MEMBER; STABILITY; PEPSIN; PROTEINASES; SUBFAMILY; DIGESTION; ENZYMES;

D O I：

10.1021/acs.analchem.5b00831

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

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 FIX-MS applications. We produced and characterized recombinant nepenthesin II to determine if it complemented nepenthesin I in FIX-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.

引用

页码：6681 / 6687

页数：7

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