Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity.
A novel kind of immobilized trypsin reactor based on organic-inorganic hybrid silica monoliths has been developed. With the presence of cetyltrimethyl ammonium bromide (CTAB) in the polymerization mixture, the hybrid silica monolithic support was prepared in a 100 microm i.d. capillary by the sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, the monolith was activated by glutaraldehyde, and trypsin was covalently immobilized. By monitoring the reaction of a decapeptide, C-myc (EQKLISEEDL), the enzymatic activity of the immobilized trypsin was calculated, and the results showed that the digestion speed was about 6600 times faster than that performed in free solution. The performance of such a microreactor was further demonstrated by digesting myoglobin, with the digested products analyzed by microflow reversed-phase liquid chromatography coupled with tandem mass spectrometry (microRPLC-MS/MS). With a stringent threshold for the unambiguous identification of the digests, the yielding sequence coverage for on-column digestion was 92%, the same as that obtained by in-solution digestion, whereas the residence time of myoglobin in the former case was only 30 s, about 1/1440 of that performed in the latter case (12 h). Moreover, such an immobilized trypsin reactor was also successfully applied to the digestion of a mixture of model proteins and proteins extracted from E. coli.
Ma
Junfeng
J
National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
Liang
Zhen
Z
Qiao
Xiaoqiang
X
Deng
Qiliang
Q
Tao
Dingyin
D
Zhang
Lihua
L
Zhang
Yukui
Y
D002853:Chromatography, Liquid; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D046210:Microfluidic Analytical Techniques; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D009842:Oligopeptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry*; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
D004800:Enzymes, Immobilized; D009211:Myoglobin; D009842:Oligopeptides; D011506:Proteins; D012822:Silicon Dioxide; D014357:Trypsin