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.
Bibliographical noteFunding Information:
We gratefully acknowledge financial support from NSFC (Grants 21475137 , 21575144 , 21375132 , 21175138 , 21205125 , 21135006 and 21321003 ). Also, we appreciate Professor Huwei Liu from Peking University for his kind help in application of μPPMER. M. H. Moon is appreciative of support from the National Research Foundation of Korea ( NRF-2015R1A2A1A01004677 ).
All Science Journal Classification (ASJC) codes
- Analytical Chemistry
- Environmental Chemistry