Trypsin immobilization in ordered porous polymer membranes for effective protein digestion

Analytica Chimica Acta
Juan QiaoMyeong Hee Moon

Abstract

Fast and effective protein digestion is a vital process for mass spectrometry (MS) based protein analysis. This study introduces a porous polymer membrane enzyme reactor (PPMER) coupled to nanoflow liquid chromatography-tandem MS (nLC-ESI-MS/MS) for on-line digestion and analysis of proteins. Poly (styrene-co-maleic anhydride) (PS-co-MAn) was fabricated by the breath figure method to make a porous polymer membrane in which the MAn group was covalently bound to enzyme. Based on this strategy, microscale PPMER (μPPMER) was constructed for on-line connection with the nLC-ESI-MS/MS system. Its capability for enzymatic digestion with bovine serum albumin (BSA) was evaluated with varied digestion periods. The on-line proteolysis of BSA and subsequent analysis with μPPMER-nLC-ESI-MS/MS revealed that peptide sequence coverage increased from 10.3% (digestion time 10 min) to 89.1% (digestion time 30 min). μPPMER can efficiently digest proteins due to the microscopic confinement effect, showing its potential application in fast protein identification and protease immobilization. Applications of on-line digestion using μPPMER with human plasma and urinary proteome samples showed that the developed on-line method yielded equivalent or bette...Continue Reading

References

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Citations

Oct 19, 2016·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Fabian B H RehmBernd H A Rehm
Jun 14, 2017·The Analyst·Jinlan DongMerlin L Bruening
Jan 16, 2018·Advanced Healthcare Materials·Shuangshuang ChenQinghua Lu
Aug 1, 2020·Soft Matter·Jing LiangLixin Wu
Jan 13, 2017·Analytical and Bioanalytical Chemistry·Heping WangXiaohong Qian
Aug 12, 2021·Analytical and Bioanalytical Chemistry·Cynthia NagyAttila Gaspar
Mar 29, 2019·ACS Applied Materials & Interfaces·Juan QiaoLi Qi
Sep 9, 2021·ACS Biomaterials Science & Engineering·Xun LiuKun Qian

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