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Recombinant Nepenthesin II for Hydrogen/Deuterium Exchange Mass Spectrometry
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SYSNO ASEP 0457205 Druh ASEP J - Článek v odborném periodiku Zařazení RIV J - Článek v odborném periodiku Poddruh J Článek ve WOS Název Recombinant Nepenthesin II for Hydrogen/Deuterium Exchange Mass Spectrometry Tvůrce(i) Yang, M. (CA)
Hoeppner, M. (CA)
Rey, M. (CA)
Kádek, Alan (MBU-M) RID, ORCID
Man, Petr (MBU-M) RID, ORCID
Schriemer, D. C. (CA)Zdroj.dok. Analytical Chemistry. - : American Chemical Society - ISSN 0003-2700
Roč. 87, č. 13 (2015), s. 6681-6687Poč.str. 7 s. Jazyk dok. eng - angličtina Země vyd. US - Spojené státy americké Klíč. slova ASPARTIC PROTEASE NEPENTHESIN-1 ; UNIQUE MEMBER ; STABILITY Vědní obor RIV CB - Analytická chemie, separace Institucionální podpora MBU-M - RVO:61388971 UT WOS 000357839700040 DOI 10.1021/acs.analchem.5b00831 Anotace 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. Pracoviště Mikrobiologický ústav Kontakt Eliška Spurná, eliska.spurna@biomed.cas.cz, Tel.: 241 062 231 Rok sběru 2016
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