Intensive Protein Digestion Using Cross-Linked Trypsin Aggregates in Proteomics Analysis

Authors

  • Dr. Meng-Fan Wang,

    1. Chemical Engineering Research Center, School of Chemical Engineering and Technology, 92 Weijin Road, Nankai District, Tianjin University (P. R. China), Fax: (+86) 022-27407599
    2. Tianjin Key Laboratory of Membrane Science and Desalination Technology (P. R. China)
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  • Yang-Yang Mu,

    1. Chemical Engineering Research Center, School of Chemical Engineering and Technology, 92 Weijin Road, Nankai District, Tianjin University (P. R. China), Fax: (+86) 022-27407599
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  • Prof. Dr. Wei Qi,

    Corresponding author
    1. Chemical Engineering Research Center, School of Chemical Engineering and Technology, 92 Weijin Road, Nankai District, Tianjin University (P. R. China), Fax: (+86) 022-27407599
    2. State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University (P. R. China)
    3. Tianjin Key Laboratory of Membrane Science and Desalination Technology (P. R. China)
    • Chemical Engineering Research Center, School of Chemical Engineering and Technology, 92 Weijin Road, Nankai District, Tianjin University (P. R. China), Fax: (+86) 022-27407599
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  • Dr. Rong-Xin Su,

    1. Chemical Engineering Research Center, School of Chemical Engineering and Technology, 92 Weijin Road, Nankai District, Tianjin University (P. R. China), Fax: (+86) 022-27407599
    2. State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University (P. R. China)
    3. Tianjin Key Laboratory of Membrane Science and Desalination Technology (P. R. China)
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  • Prof. Dr. Zhi-Min He

    1. Chemical Engineering Research Center, School of Chemical Engineering and Technology, 92 Weijin Road, Nankai District, Tianjin University (P. R. China), Fax: (+86) 022-27407599
    2. State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University (P. R. China)
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Abstract

In this study, cross-linked enzyme aggregates of tissue-culture-grade trypsin (CLEAs-T) were prepared and introduced to the proteomics analysis instead of the expensive proteomics-grade trypsin, which benefits from the protein purification during the preparation of the CLEAs. Bovine serum albumin, lysozyme, bovine hemoglobin, and α-casein were used as model proteins, and three intensive strategies (high-enzyme-concentration, high-temperature, and ultrasound-assisted digestion) were applied to the rapid and efficient digestion of model proteins by CLEAs-T. The peptide fragments were then identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, which revealed the higher sequence coverage and sharply reduced digestion time of these strategies compared with the conventional in-solution 12 h digestion method. Morphology studies demonstrated that the excellent performance of CLEAs-T in proteomics analysis came from the “scaffoldlike” supermolecular structure, which provided a high resistance to the autolysis and denaturation of trypsin under high-enzyme-concentration, high-temperature, and ultrasound-assisted digestion.

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