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Online Integration of Multiple Sample Pretreatment Steps Involving Denaturation, Reduction, and Digestion with Microflow Reversed-Phase Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry for High-Throughput Proteome Profiling
Ma, Junfeng1,2; Liu, Jinxiang1,2; Sun, Liangliang1,2; Gao, Liang1,2; Liang, Zhen1; Zhang, Lihua1; Zhang, Yukui1
Source PublicationANALYTICAL CHEMISTRY
2009-08-01
DOI10.1021/ac900971w
Volume81Issue:15Pages:6534-6540
Indexed BySCI
SubtypeArticle
WOS HeadingsScience & Technology ; Physical Sciences
WOS SubjectChemistry, Analytical
WOS Research AreaChemistry
WOS KeywordIMMOBILIZED ENZYME REACTORS ; TRYPSIN DIGESTION ; IDENTIFICATION ; PROTEINS ; MICROREACTOR ; SEPARATION ; MS/MS ; CAPILLARIES ; PROTEOLYSIS ; EFFICIENT
AbstractA facile integrated platform for proteome profiling was established, in which native proteins were online denatured and reduced within a heater, digested with an immobilized trypsin microreactor, and analyzed by microflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry (mu RPLC-ESI-MS/MS). In comparison to the traditional off-line urea denaturation protocol, even more unique peptides were obtained by online heating in triplicate (14 +/- 2 vs 11 +/- 2 for myoglobin and 16 vs 12 +/- 1 for BSA) within a significantly shortened pretreatment time of similar to 3.5 min (including 1 min of thermal denaturation and reduction and similar to 2.5 min of microreactor digestion). Moreover, proteins with concentrations ranging from 50 ng/mL (similar to 6 fmol) to 1 mg/mL (similar to 120 pmol) were positively identified by the online system. Such a platform was further successfully applied for analyzing the soluble fraction of mouse liver extract. Of all the 367 proteins identified from samples pretreated by the urea protocol and online heating, similar to 40% were overlapped, showing the partial complementation of both approaches. All these results demonstrate that the online integrated platform is of great promise for high-throughput proteome profiling and improved identification capacity for low-abundance proteins with a minute sample amount.
Language英语
WOS IDWOS:000268455600073
Citation statistics
Cited Times:40[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://cas-ir.dicp.ac.cn/handle/321008/141597
Collection中国科学院大连化学物理研究所
Affiliation1.Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog Res & Anal Ctr, Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China
2.Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China
Recommended Citation
GB/T 7714
Ma, Junfeng,Liu, Jinxiang,Sun, Liangliang,et al. Online Integration of Multiple Sample Pretreatment Steps Involving Denaturation, Reduction, and Digestion with Microflow Reversed-Phase Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry for High-Throughput Proteome Profiling[J]. ANALYTICAL CHEMISTRY,2009,81(15):6534-6540.
APA Ma, Junfeng.,Liu, Jinxiang.,Sun, Liangliang.,Gao, Liang.,Liang, Zhen.,...&Zhang, Yukui.(2009).Online Integration of Multiple Sample Pretreatment Steps Involving Denaturation, Reduction, and Digestion with Microflow Reversed-Phase Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry for High-Throughput Proteome Profiling.ANALYTICAL CHEMISTRY,81(15),6534-6540.
MLA Ma, Junfeng,et al."Online Integration of Multiple Sample Pretreatment Steps Involving Denaturation, Reduction, and Digestion with Microflow Reversed-Phase Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry for High-Throughput Proteome Profiling".ANALYTICAL CHEMISTRY 81.15(2009):6534-6540.
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