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.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
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.
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.
Atacan K, Özacar M. Characterization and immobilization of trypsin on tannic acid modified Fe3O4 nanoparticles. Colloids Surf B: Biointerfaces. 2015;128:227–36.
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.
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.
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.
Liu S, Bao H, Zhang L, Chen G. Efficient proteolysis strategies based on microchip bioreactors. J Proteomics. 2013;82:1–13.
Ning W, Bruening ML. Rapid protein digestion and purification with membranes attached to pipet tips. Anal Chem. 2015;87(24):11984–9.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Zhao M, Zhang X, Deng C. Rational synthesis of novel recyclable Fe3O4@ MOF nanocomposites for enzymatic digestion. Chem Commun. 2015;51(38):8116–9.
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.
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.
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.
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.
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.
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.
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.
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
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). https://doi.org/10.1007/s00216-016-0163-z
- Covalent organic framework
- Immobilized trypsin
- Magnetic graphene