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Preparing a metal-ion chelated immobilized enzyme reactor based on the polyacrylamide monolith grafted with polyethylenimine for a facile regeneration and high throughput tryptic digestion in proteomics

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Abstract

Initially, a poly (glycidyl methacrylate-co-acrylamide-co-methylenebisacrylamide) monolith was prepared in the 100 μm i.d. capillary, and then was grafted with polyethylenimine (Mw, ∼25,000) for adsorbing Cu2+, followed by chelating trypsin. As a result, efficient digestion for BSA (100 ng/μL) was completed within 50 s via such immobilized enzyme reactor (IMER); yielding 47% sequence coverage by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Compared with the conventional method for preparing the metal-ion chelated IMER, the regeneration of such IMER can be achieved facilely by the respective 30 min desorption and re-adsorption of trypsin, and 51% sequence coverage was obtained for 50 s BSA digestion after regeneration. BSA down to femtomole was also efficiently digested by the prepared regenerable IMER. Meanwhile, after the consecutive digestion of myoglobin and BSA, there was not any mutual interference for both during MALDI-TOF MS identification, indicating the low nonspecific adsorption of such regenerable IMER. To test the applicability of regenerable IMER for complex sample profiling, proteins (150 ng) extracted from Escherichia coli were digested within 80 s by the regenerable IMER and further analyzed by nanoreversed phase liquid chromatography–electrospray ionization–mass spectrometry successfully, showing its practicability for the high throughput analysis of complex samples.

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Acknowledgments

The authors are grateful for the financial support from the National Nature Science Foundation (Grants 20935004 and 20775080), National Basic Research Program of China (Grant 2007CB914100) and the Analytical Method Innovation Program of MOST (2010IM030500).

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Authors and Affiliations

  1. Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China

    Shuaibin Wu, Kaiguang Yang, Zhen Liang, Lihua Zhang & Yukui Zhang

  2. State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023, China

    Lei Zhang

  3. Graduate School of Chinese Academy of Sciences, Beijing, 100049, China

    Shuaibin Wu & Lei Zhang

Authors
  1. Shuaibin Wu
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  2. Lei Zhang
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  3. Kaiguang Yang
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  4. Zhen Liang
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  5. Lihua Zhang
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  6. Yukui Zhang
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Correspondence to Kaiguang Yang.

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Wu, S., Zhang, L., Yang, K. et al. Preparing a metal-ion chelated immobilized enzyme reactor based on the polyacrylamide monolith grafted with polyethylenimine for a facile regeneration and high throughput tryptic digestion in proteomics. Anal Bioanal Chem 402, 703–710 (2012). https://doi.org/10.1007/s00216-011-5501-6

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  • DOI: https://doi.org/10.1007/s00216-011-5501-6

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