Analytical and Bioanalytical Chemistry

, Volume 403, Issue 1, pp 239–246

Ion-exchange-membrane-based enzyme micro-reactor coupled online with liquid chromatography–mass spectrometry for protein analysis

  • Zhigui Zhou
  • Youyou Yang
  • Jialing Zhang
  • Zhengxiang Zhang
  • Yu Bai
  • Yiping Liao
  • Huwei Liu
Original Paper


In this article, we developed a membrane-based enzyme micro-reactor by directly using commercial polystyrene–divinylbenzene cation–exchange membrane as the support for trypsin immobilization via electrostatic and hydrophobic interactions and successfully applied it for protein digestion. The construction of the reactor can be simply achieved by continuously pumping trypsin solution through the reactor for only 2 min, which was much faster than the other enzyme immobilization methods. In addition, the membrane reactor could be rapidly regenerated within 35 min, resulting in a “new” reactor for the digestion of every protein sample, completely eliminating the cross-interference of different protein samples. The amount and the activity of immobilized trypsin were measured, and the repeatability of the reactor was tested, with an RSD of 3.2% for the sequence coverage of cytochrome c in ten digestion replicates. An integrated platform for protein analysis, including online protein digestion and peptide separation and detection, was established by coupling the membrane enzyme reactor with liquid chromatography–quadrupole time-of-flight mass spectrometry. The performance of the platform was evaluated using cytochrome c, myoglobin, and bovine serum albumin, showing that even in the short digestion time of several seconds the obtained sequence coverages was comparable to or higher than that with in-solution digestion. The system was also successfully used for the analysis of proteins from yeast cell lysate.


Schemes of the designed ion-exchange-membrane-based enzyme micro-reactor (a) and the online coupling system of the enzyme micro-reactor with LC-QTOF MS (b)


Ion exchange membrane Enzyme micro-reactor Protein analysis Liquid chromatography Mass spectrometry LC-MS 

Supplementary material

216_2012_5812_MOESM1_ESM.pdf (580 kb)
ESM 1(PDF 579 kb)


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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Zhigui Zhou
    • 1
  • Youyou Yang
    • 1
  • Jialing Zhang
    • 1
  • Zhengxiang Zhang
    • 1
  • Yu Bai
    • 1
  • Yiping Liao
    • 1
  • Huwei Liu
    • 1
  1. 1.Beijing National Laboratory for Molecular Sciences, the Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry and Molecular EngineeringPeking UniversityBeijingChina

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