Novel monolithic enzymatic microreactor based on single-enzyme nanoparticles for highly efficient proteolysis and its application in multidimensional liquid chromatography.
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.
Gao
Mingxia
M
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
Zhang
Peng
P
Hong
Guangfeng
G
Guan
Xia
X
Yan
Guoquan
G
Deng
Chunhui
C
Zhang
Xiangmin
X
D000178:Acrylamides / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000295:instrumentation* / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D005029:Ethylenediamines / Q000737:chemistry; D006801:Humans; D008099:Liver / Q000378:metabolism; D009211:Myoglobin / Q000032:analysis; D053758:Nanoparticles / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000032:analysis; D012822:Silicon Dioxide / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000178:Acrylamides; D004800:Enzymes, Immobilized; D005029:Ethylenediamines; D009211:Myoglobin; D011506:Proteins; C005798:N,N,N',N'-tetramethylethylenediamine; D012710:Serum Albumin, Bovine; D012822:Silicon Dioxide; C021221:N,N'-methylenebisacrylamide