Optical Characterization and Monitoring of Enzyme Catalyzed Short Chain Peptides in Cellular Environment

被引:1
|
作者
Anwar, Shahzad [1 ,2 ]
Ovais, Muhammad [2 ,3 ]
Zhang, Xiang [1 ,4 ,5 ]
机构
[1] Chinese Acad Sci, Inst Biophys, CAS Ctr Excellence Biomacromol, Natl Lab Biomacromol, Beijing 100101, Peoples R China
[2] Univ Chinese Acad Sci, Beijing 100049, Peoples R China
[3] Natl Ctr Nanosci & Technol NCNST, CAS Ctr Excellence Nanosci, CAS Key Lab Biomed Effects Nanomat & Nanosafety, Beijing 100190, Peoples R China
[4] Huairou Natl Comprehens Sci Ctr, Phys Sci Lab, Beijing 101400, Peoples R China
[5] Chinese Acad Sci, Inst Biophys, Ctr Biol Instrument Dev, Core Facil Prot Res, Beijing 100101, Peoples R China
关键词
Enzyme catalysis; Optical characterization; Fluorescence; Mass spectrometry; Binding interactions; Electron microscopy; MASS-SPECTROMETRY; FUSION PROTEINS; MALDI; NANOSTRUCTURES; IDENTIFICATION; MOLECULES; DELIVERY; ORIGIN; TARGET;
D O I
10.1007/s10562-020-03110-5
中图分类号
O64 [物理化学(理论化学)、化学物理学];
学科分类号
070304 ; 081704 ;
摘要
Enzyme catalysis is an emerging technique that has been employed to identify the protein of interest in a complex cellular environment. In present article, we have utilized optical techniques for the identification of enzyme mediated catalysis of fusion protein to study the short chain amino acid epitope in both aqueous solution and cellular environment. We characterized enzyme catalysis short chain peptide with electron microscopic techniques, for the study of reaction product and its morphology in aqueous solution. Furthermore, we applied MALDI-TOF-MS technique for the analysis of fusion protein, FLAG-tag peptides and enterokinase enzyme (EK), in complex solutions and cellular environment. The enzyme catalytic reactions were studied HEK-293T cells with confocal fluorescence microscopy. In spectroscopic studies two sharp prominent peaks at 310 nm and 330 nm were appeared in case of Flag-tag peptide and for fusion protein absorption peaks were found at 315 nm and 410 nm with relative increase in intensity level. Furthermore, a linear relation between response unit and binding time (ms) for both fusion protein and EK was observed. Mass spectra reveal the presence of FLAG-tag peptide epitope at mass to charge (m/z) ratio of 2190.023 and 2191.102 in cell lysation with and without enterokinase respectively. We provide here a rapid and accurate trace detection system for enzyme cleaved peptides in fusion protein by taking a snapshot of peptide to identify specific domains based on amino acids and their mass spectrum calculations. [GRAPHICS] .
引用
收藏
页码:2400 / 2408
页数:9
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