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Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling
Wang, Tingting1,2; Ma, Junfeng1,2; Wu, Shuaibin1,2; Yuan, Huiming1; Zhang, Lihua1; Liang, Zhen1; Zhang, Yukui1
KeywordCapillary Isoelectric Focusing Immobilized Enzyme Microreactor Integrated Platform Nanoreversed-phase Liquid Chromatography Trypsin
Source PublicationELECTROPHORESIS
2011-10-01
DOI10.1002/elps.201100030
Volume32Issue:20Pages:2848-2856
Indexed BySCI
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine ; Physical Sciences
WOS SubjectBiochemical Research Methods ; Chemistry, Analytical
WOS Research AreaBiochemistry & Molecular Biology ; Chemistry
WOS KeywordZONE-ELECTROPHORESIS ; PH GRADIENT ; SEPARATION ; SYSTEM ; PRECONCENTRATION ; MONOLITHS ; DIGESTION ; REACTORS ; CE
AbstractAn integrated platform with the combination of protein and peptide separation was established via online protein digestion, by which proteins were first separated by CIEF, online digested by a trypsin immobilized enzyme microreactor, trapped and desalted by two parallel trap columns, separated by nanoreversed-phase and finally identified by MS. In such a platform, two hollow fiber membrane interfaces were used. One was applied to supply catholyte and electric contact, and another to supply adjustment buffer to improve the compatibility of protein separation and tryptic digestion. A poly(octadecyl acrylate-co-ethylene dimethacrylate) monolithic column served as the trap column to capture sample and to remove the ampholytes from CIEF. A hybrid silica monolith-based immobilized trypsin microreactor was used for online protein digestion. To evaluate the performance of such a platform, a 4-protein mixture with a loading amount of only 0.29 mu g, was analyzed, and sequence coverages for BSA, myoglobin, beta-lactoglobulin and ribonuclease A were 8, 26, 10 and 54%, respectively. Furthermore, such an integrated platform was successfully applied for the analysis of proteins extracted from Escherichia coli, and 101 proteins were positively identified. We anticipate that the integrated platform developed herein will provide a promising tool for low-abundance protein identification with the combination of top-down and bottom-up approaches.
Language英语
WOS IDWOS:000297424500011
Citation statistics
Cited Times:21[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://cas-ir.dicp.ac.cn/handle/321008/142719
Collection中国科学院大连化学物理研究所
Affiliation1.Chinese Acad Sci, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Natl Chromatog Res & Anal Ctr, Dalian 116023, Peoples R China
2.Chinese Acad Sci, Grad Univ, Beijing, Peoples R China
Recommended Citation
GB/T 7714
Wang, Tingting,Ma, Junfeng,Wu, Shuaibin,et al. Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling[J]. ELECTROPHORESIS,2011,32(20):2848-2856.
APA Wang, Tingting.,Ma, Junfeng.,Wu, Shuaibin.,Yuan, Huiming.,Zhang, Lihua.,...&Zhang, Yukui.(2011).Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling.ELECTROPHORESIS,32(20),2848-2856.
MLA Wang, Tingting,et al."Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling".ELECTROPHORESIS 32.20(2011):2848-2856.
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