Trypsin entrapped in poly(diallyldimethylammonium chloride) silica sol-gel microreactor coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
An enzyme-immobilized capillary microreactor for rapid protein digestion and proteomics analysis is reported. The inner surface of the fused-silica capillary was coated with poly(diallyldimethylammonium chloride) (PDDA)-entrapped silica sol-gel matrix, followed by assembly of trypsin onto the PDDA-modified surface via electrostatic adsorption. The immobilization parameters such as PDDA content in the sol-gel matrix, trypsin concentration and pH were investigated in detail. Protein samples including beta-casein, myoglobin and cytochrome c could be effectively digested and electrophoretically separated simultaneously in such a modified capillary. Just 2.26 ng (corresponding to 0.10-0.14 picomole) of sample was sufficient for on-line capillary electrophoresis peptide mapping. The efficiency of the digestion was further demonstrated by digestion of a human liver cytoplasm sample and 253 proteins were identified in one unique run.
Xu
Xuejiao
X
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
Wang
Xiaoyan
X
Liu
Yun
Y
Liu
Baohong
B
Wu
Huiling
H
Yang
Pengyuan
P
D000327:Adsorption; D000818:Animals; D002417:Cattle; D004800:Enzymes, Immobilized / Q000032:analysis; D006801:Humans; D008099:Liver / Q000737:chemistry; D008832:Microchemistry; D010449:Peptide Mapping / Q000379:methods*; D044367:Phase Transition; D011095:Polyethylenes / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D055672:Static Electricity; D014357:Trypsin / Q000737:chemistry* / Q000648:ultrastructure
D004800:Enzymes, Immobilized; D011095:Polyethylenes; D000644:Quaternary Ammonium Compounds; C041004:poly-N,N-dimethyl-N,N-diallylammonium chloride; D012710:Serum Albumin, Bovine; D012822:Silicon Dioxide; D014357:Trypsin