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Covalent organic framework-coated magnetic graphene as a novel support for trypsin immobilization


Deep and efficient proteolysis is the critical premise in mass spectrometry-based bottom-up proteomics. It is difficult for traditional in-solution digestion to meet the requirement unless prolonged digestion time and enhanced enzyme dosage are employed, which makes the whole workflow time-consuming and costly. The abovementioned problems could be effectively ameliorated by anchoring many proteases on solid supports. In this work, covalent organic framework-coated magnetic graphene (MG@TpPa-1) was designed and prepared as a novel enzyme carrier for the covalent immobilization of trypsin with a high degree of loading (up to 268 μg mg−1). Profiting from the advantages of magnetic graphene and covalent organic frameworks, the novel trypsin bioreactor was successfully applied for the enzymatic digestion of a model protein with dramatically improved digestion efficiency, stability, and reusability. Complete digestion could be achieved in a time period as short as 2 min. For the digestion of proteins extracted from Amygdalus pedunculata, a total of 2833 protein groups were identified, which was slightly more than those obtained by 12 h of in-solution digestion (2739 protein groups). All of the results demonstrate that MG@TpPa-1-trypsin is an excellent candidate for sample preparation in a high-throughput proteomics analysis.

Covalent organic frameworks-coated magnetic graphene was prepared as novel carrier for highly efficient tryptic immobilization

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  1. Huang J, Wang F, Ye M, Zou H. Enrichment and separation techniques for large-scale proteomics analysis of the protein post-translational modifications. J Chromatogr A. 2014;1372:1–17.

    Article  CAS  Google Scholar 

  2. Wiśniewski JR, Mann M. Consecutive proteolytic digestion in an enzyme reactor increases depth of proteomic and phosphoproteomic analysis. Anal Chem. 2012;84(6):2631–7.

    Article  Google Scholar 

  3. Atacan K, Özacar M. Characterization and immobilization of trypsin on tannic acid modified Fe3O4 nanoparticles. Colloids Surf B: Biointerfaces. 2015;128:227–36.

    Article  CAS  Google Scholar 

  4. Cao Y, Wen L, Svec F, Tan T, Lv Y. Magnetic AuNP@ Fe3O4 nanoparticles as reusable carriers for reversible enzyme immobilization. Chem Eng J. 2016;286:272–81.

    Article  CAS  Google Scholar 

  5. Atacan K, Çakıroğlu B, Özacar M. Improvement of the stability and activity of immobilized trypsin on modified Fe3O4 magnetic nanoparticles for hydrolysis of bovine serum albumin and its application in the bovine milk. Food Chem. 2016;212:460–8.

    Article  CAS  Google Scholar 

  6. Fan C, Shi Z, Pan Y, Song Z, Zhang W, Zhao X, et al. Dual matrix-based immobilized trypsin for complementary proteolytic digestion and fast proteomics analysis with higher protein sequence coverage. Anal Chem. 2014;86(3):1452–8.

    Article  CAS  Google Scholar 

  7. Liu S, Bao H, Zhang L, Chen G. Efficient proteolysis strategies based on microchip bioreactors. J Proteomics. 2013;82:1–13.

    Article  CAS  Google Scholar 

  8. Ning W, Bruening ML. Rapid protein digestion and purification with membranes attached to pipet tips. Anal Chem. 2015;87(24):11984–9.

    Article  CAS  Google Scholar 

  9. Wang C, Gao M, Zhang P, Zhang X. Efficient proteolysis of glycoprotein using a hydrophilic immobilized enzyme reactor coupled with MALDI-QIT-TOF-MS detection and μHPLC analysis. Chromatographia. 2014;77(5-6):413–8.

    Article  CAS  Google Scholar 

  10. Qiao J, Kim JY, Wang YY, Qi L, Wang FY, Moon MH. Trypsin immobilization in ordered porous polymer membranes for effective protein digestion. Anal Chim Acta. 2016;906:156–64.

    Article  CAS  Google Scholar 

  11. Li Y, Xu X, Deng C, Yang P, Zhang X. Immobilization of trypsin on superparamagnetic nanoparticles for rapid and effective proteolysis. J Proteome Res. 2007;6(9):3849–55.

    Article  CAS  Google Scholar 

  12. Sun X, Cai X, Wang R-Q, Xiao J. Immobilized trypsin on hydrophobic cellulose decorated nanoparticles shows good stability and reusability for protein digestion. Anal Biochem. 2015;477:21–7.

    Article  CAS  Google Scholar 

  13. Shi C, Deng C, Li Y, Zhang X, Yang P. Hydrophilic polydopamine-coated magnetic graphene nanocomposites for highly efficient tryptic immobilization. Proteomics. 2014;14(12):1457–63.

    Article  CAS  Google Scholar 

  14. Wang S, Bao H, Yang P, Chen G. Immobilization of trypsin in polyaniline-coated nano-Fe3O4/carbon nanotube composite for protein digestion. Anal Chim Acta. 2008;612(2):182–9.

    Article  CAS  Google Scholar 

  15. Silva TR, Rodrigues DP, Rocha JM, Gil MH, Pinto SC, Lopes-da-Silva JA, et al. Immobilization of trypsin onto poly (ethylene terephthalate)/poly(lactic acid) nonwoven nanofiber mats. Biochem Eng J. 2015;104:48–56.

    Article  CAS  Google Scholar 

  16. Jiao F, Zhai R, Huang J, Zhang Y, Zhang Y, Qian X. Hollow silica bubble based immobilized trypsin for highly efficient proteome digestion and buoyant separation. RSC Adv. 2016;6(87):84113–8. doi:10.1039/C6RA12599A.

    Article  CAS  Google Scholar 

  17. Pavlidis IV, Patila M, Bornscheuer UT, Gournis D, Stamatis H. Graphene-based nanobiocatalytic systems: recent advances and future prospects. Trends Biotechnol. 2014;32(6):312–20.

    Article  CAS  Google Scholar 

  18. Jiang B, Yang K, Zhang L, Liang Z, Peng X, Zhang Y. Dendrimer-grafted graphene oxide nanosheets as novel support for trypsin immobilization to achieve fast on-plate digestion of proteins. Talanta. 2014;122:278–84.

    Article  CAS  Google Scholar 

  19. Liang P, Bao H, Yang J, Zhang L, Chen G. Preparation of porous graphene oxide–poly (urea–formaldehyde) hybrid monolith for trypsin immobilization and efficient proteolysis. Carbon. 2016;97:25–34.

    Article  CAS  Google Scholar 

  20. Yin Z, Zhao W, Tian M, Zhang Q, Guo L, Yang L. A capillary electrophoresis-based immobilized enzyme reactor using graphene oxide as a support via layer by layer electrostatic assembly. Analyst. 2014;139(8):1973–9.

    Article  CAS  Google Scholar 

  21. Zhao M, Deng C, Zhang X. The design and synthesis of a hydrophilic core–shell–shell structured magnetic metal–organic framework as a novel immobilized metal ion affinity platform for phosphoproteome research. Chem Commun. 2014;50(47):6228–31.

    Article  CAS  Google Scholar 

  22. Ma W, Xu L, Li Z, Sun Y, Bai Y, Liu H. Post-synthetic modification of an amino-functionalized metal–organic framework for highly efficient enrichment of N-linked glycopeptides. Nanoscale. 2016;8(21):10908–12.

    Article  CAS  Google Scholar 

  23. Xiong Z, Ji Y, Fang C, Zhang Q, Zhang L, Ye M, et al. Facile preparation of core–shell magnetic metal–organic framework nanospheres for the selective enrichment of endogenous peptides. Chem A Eur J. 2014;20(24):7389–95.

    Article  CAS  Google Scholar 

  24. Wen L, Gao A, Cao Y, Svec F, Tan T, Lv Y. Layer-by-layer assembly of metal–organic frameworks in macroporous polymer monolith and their use for enzyme immobilization. Macromol Rapid Commun. 2016;37(6):551–7. doi:10.1002/marc.201500705.

    Article  CAS  Google Scholar 

  25. Zhao M, Zhang X, Deng C. Rational synthesis of novel recyclable Fe3O4@ MOF nanocomposites for enzymatic digestion. Chem Commun. 2015;51(38):8116–9.

    Article  CAS  Google Scholar 

  26. Jeremias F, Fröhlich D, Janiak C, Henninger SK. Water and methanol adsorption on MOFs for cycling heat transformation processes. New J Chem. 2014;38(5):1846–52.

    Article  CAS  Google Scholar 

  27. Kandambeth S, Mallick A, Lukose B, Mane MV, Heine T, Banerjee R. Construction of crystalline 2D covalent organic frameworks with remarkable chemical (acid/base) stability via a combined reversible and irreversible route. J Am Chem Soc. 2012;134(48):19524–7.

    Article  CAS  Google Scholar 

  28. Cheng G, Chen P, Wang Z-G, Sui X-J, Zhang J-L, Ni J-Z. Immobilization of trypsin onto multifunctional meso-/macroporous core-shell microspheres: a new platform for rapid enzymatic digestion. Anal Chim Acta. 2014;812:65–73.

    Article  CAS  Google Scholar 

  29. Yamaura M, Camilo R, Sampaio L, Macedo M, Nakamura M, Toma H. Preparation and characterization of (3-aminopropyl) triethoxysilane-coated magnetite nanoparticles. J Magn Magn Mater. 2004;279(2):210–7.

    Article  CAS  Google Scholar 

  30. Jiang B, Yang K, Zhao Q, Wu Q, Liang Z, Zhang L, et al. Hydrophilic immobilized trypsin reactor with magnetic graphene oxide as support for high efficient proteome digestion. J Chromatogr A. 2012;1254:8–13.

    Article  CAS  Google Scholar 

  31. Liu WL, Wu CY, Chen CY, Singco B, Lin CH, Huang HY. Fast multipoint immobilized MOF bioreactor. Chem A Eur J. 2014;20(29):8923–8.

    CAS  Google Scholar 

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This work was supported by the National Natural Science Foundation of China (21675125, 21606181, 21275159, and 21235001), the National Key Program for Basic Research of China (2013CB911204 and 2016YFA0501403), the National Key Program for Scientific Instrument and Equipment Development (2012YQ12004407, 2011YQ06008408, and 2013YQ14040506). This work was also partly supported by the Amygdalus pedunculata Engineering Technology Research Center of State Forestry Administration and the Key Laboratory of Yulin Desert Plants Resources.

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Correspondence to Yehua Shen, Yangjun Zhang or Xiaohong Qian.

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Wang, H., Jiao, F., Gao, F. et al. Covalent organic framework-coated magnetic graphene as a novel support for trypsin immobilization. Anal Bioanal Chem 409, 2179–2187 (2017).

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  • Covalent organic framework
  • Immobilized trypsin
  • Magnetic graphene