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

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Abstract

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.

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Acknowledgements

The author thanks the staff of the Institute of Biophysics (IBP) for providing all the supports and technical facilities. Shahzad Anwar in particular is grateful to Professor Tao Xu from Institute of Biophysics (IBP).

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Authors and Affiliations

  1. National Laboratory of Biomacromolecules, CAS Centre for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China

    Shahzad Anwar & Xiang Zhang

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Shahzad Anwar & Muhammad Ovais

  3. CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience & Technology (NCNST), Beijing, 100190, China

    Muhammad Ovais

  4. Physical Science Laboratory, Huairou National Comprehensive Science Center, Beijing, 101400, China

    Xiang Zhang

  5. Center for Biological Instrument Development, Core Facility for Protein Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China

    Xiang Zhang

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  1. Shahzad Anwar
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  2. Muhammad Ovais
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  3. Xiang Zhang
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SA designed the studies. SA, MO, and ZX wrote the paper and conducted the experiments.

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Correspondence to Shahzad Anwar.

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Anwar, S., Ovais, M. & Zhang, X. Optical Characterization and Monitoring of Enzyme Catalyzed Short Chain Peptides in Cellular Environment. Catal Lett 150, 2400–2408 (2020). https://doi.org/10.1007/s10562-020-03110-5

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  • DOI: https://doi.org/10.1007/s10562-020-03110-5

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