title: Trypsin immobilization in ordered porous polymer membranes for effective protein digestion.
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 better performance in protein coverage and identified more membrane proteins than the in-solution method. This may be due to easy accommodation of hydrophobic membrane proteins within membrane pores.
authors.affiliation: Beijing National Laboratory for Molecular Sciences; Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190, PR China.
authors.forename: Juan
authors.initials: J
authors.lastname: Qiao
authors.affiliation: Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seoul 120-749, South Korea.
authors.forename: Jin Yong
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Beijing National Laboratory for Molecular Sciences; Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190, PR China.
authors.forename: Yuan Yuan
authors.initials: YY
authors.lastname: Wang
authors.affiliation: Beijing National Laboratory for Molecular Sciences; Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190, PR China. Electronic address: qili@iccas.ac.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Qi
authors.affiliation: Beijing National Laboratory for Molecular Sciences; Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190, PR China.
authors.forename: Fu Yi
authors.initials: FY
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seoul 120-749, South Korea. Electronic address: mhmoon@yonsei.ac.kr.
authors.forename: Myeong Hee
authors.initials: MH
authors.lastname: Moon
mesh_terms: D002853:Chromatography, Liquid; D004800:Enzymes, Immobilized / Q000737:chemistry*; D008567:Membranes, Artificial; D008855:Microscopy, Electron, Scanning; D011108:Polymers; D016062:Porosity; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D011108:Polymers; D011506:Proteins; D014357:Trypsin
keywords: Breath figure method; On-line proteolysis; Porous polymer membrane enzyme reactor