Novel monolithic enzymatic microreactor based on single-enzyme nanoparticles for highly efficient proteolysis and its application in multidimensional liquid chromatography

• Published in: Journal of Chromatography A 2009-10, Vol.1216 (44), p.7472-7477
• Main Authors: Gao, Mingxia, Zhang, Peng, Hong, Guangfeng, Guan, Xia, Yan, Guoquan, Deng, Chunhui, Zhang, Xiangmin
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Monolithic enzymatic microreactor; Multidimensional liquid chromatography; Proteins; Proteolysis; Proteome
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2009.05.003

In this work, a novel and facile monolithic enzymatic microreactor was prepared in the fused-silica capillary via a two-step procedure including surface acryloylation and in situ aqueous polymerization/immobilization to encapsulate a single enzyme, and its application to fast protein digestion through a direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis was demonstrated. At first, vinyl groups on the protein surface were generated by a mild acryloylation with N-acryloxysuccinimide in alkali buffer. Then, acryloylated enzyme was encapsulated into polyacrylates by free-radical copolymerization with acrylamide as the monomer, N, N′-methylenebisacrylamide as the cross-linker, and N, N, N′, N′-tetramethylethylenediamine/ammonium persulfate as the initiator. Finally, polymers were immobilized onto the activated inner wall of capillaries via the reaction of vinyl groups. Capability of the enzyme-immobilized monolithic microreactor was demonstrated by myoglobin and bovine serum albumin as model proteins. The digestion products were characterized using MALDI-TOF-MS with sequence coverage of 94% and 29% observed. This microreactor was also applied to the analysis of fractions through two-dimensional separation of weak anion exchange/reversed-phase liquid chromatography of human liver extract. After a database search, 16 unique peptides corresponding to 3 proteins were identified when two RPLC fractions of human liver extract were digested by the microreactor. This opens a route for its future application in top–down proteomic analysis.