文献类型：期刊文献

英文题名：Identification of the sites of 4-hydroxy-2-nonenal and neprilysin adduction using a linear trap quadrapole Velos Pro-Orbitrap Elite mass spectrometer

作者：Zhou, Li[1];Qian, Jiao[1];Liu, Jianxu[1];Zhao, Ruimin[2];Li, Bowei[1];Wang, Rui[1]

机构：[1]East China Univ Sci & Technol, Sch Pharm, Shanghai Key Lab New Drug Design, Shanghai 200237, Peoples R China;[2]Fourth Peoples Hosp Shenzhen, Dept Emergency, Shenzhen 518033, Guangdong, Peoples R China

年份：2016

卷号：22

期号：3

起止页码：133

外文期刊名：EUROPEAN JOURNAL OF MASS SPECTROMETRY

收录：;EI(收录号：20194907777500);WOS:【SCI-EXPANDED(收录号:WOS:000382300100006)】；

基金：This work was supported by grants from the National Natural Science Foundation of China (81072627), the Shanghai Committee of Science and Technology (12431900901), and the 111 Project of the Chinese Ministry of Education (Grant No. B07023)

语种：英文

外文关键词：4-hydroxy-2-nonenal; LTQ Velos Pro-Orbitrap Elite mass spectrometer; neprilysin; protein modification

摘要：Amyloid-beta (A beta)-degrading enzyme neprilysin (NEP) plays a pivotal role in eliminating A beta. The oxidized modification of NEP by 4-hydroxy2- nonenal (HNE) may reduce the clearance of A beta in cultured cells and Alzheimer's disease (AD) brains. The aim of this research is to study whether HNE could modify the NEP protein and identify the specific sites of HNE-NEP modification using a linear trap quadrapole (LTQ) Velos Pro-Orbitrap Elite mass spectrometer. NEP activity was determined after SH-SY5Y cells had incubated with HNE (20 mu M) for 24 hours. To identify the sites of NEP modification, samples of both native and HNE-modified NEP digested by trypsin were analyzed using a LTQ Velos Pro-Orbitrap Elite mass spectrometer. The NEP peptide-sequence information from the fragment ion masses was used to search for the sites of NEP adduction. HNE-treated cells showed a 60% loss of NEP activity. NEP was covalently adducted at Lys 93, Lys 472 by HNE via Michael addition. Compared to the control group, the sites of modified peptide in NEP showed a consistent 156 Da increased in m/z, which provides sequence information and might contribute to further studies on drug design and the therapeutics of AD.