title: Identification of the sites of 4-hydroxy-2-nonenal and neprilysin adduction using a linear trap quadrapole Velos Pro-Orbitrap Elite mass spectrometer.
abstract: Amyloid-βdegrading enzyme neprilysin (NEP) plays a pivotal role in eliminating Aβ The oxidized modification of NEP by 4-hydroxy-2-nonenal (HNE) may reduce the clearance of Aβ 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 μM) 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.
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Jiao
authors.initials: J
authors.lastname: Qian
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Jianxu
authors.initials: J
authors.lastname: Liu
authors.affiliation: The Department of Emergency, the Fourth People's Hospital of Shenzhen, Shenzhen, Guangdong 518033, China.
authors.forename: Ruimin
authors.initials: R
authors.lastname: Zhao
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Bowei
authors.initials: B
authors.lastname: Li
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China. ruiwang@ecust.edu.cn.
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
mesh_terms: D000447:Aldehydes / Q000032:analysis* / Q000737:chemistry*; D001665:Binding Sites; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D015260:Neprilysin / Q000032:analysis* / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation*
chemical_list: D000447:Aldehydes; D015260:Neprilysin; C027576:4-hydroxy-2-nonenal