Trypsin immobilization in ordered porous polymer membranes for effective protein digestion

Juan Qiao, Jin Yong Kim, Yuan Yuan Wang, Li Qi, Fu Yi Wang, Myeong Hee Moon

Research output: Contribution to journalArticle

21 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)156-164
Number of pages9
JournalAnalytica Chimica Acta
Volume906
DOIs
Publication statusPublished - 2016 Feb 4

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy

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