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学科主题物理化学
Biphasic Microreactor for Efficient Membrane Protein Pretreatment with a Combination of Formic Acid Assisted Solubilization, On-Column pH Adjustment, Reduction, Alkylation, and Tryptic Digestion
Zhao, Qun1,2; Liang, Yu1; Yuan, Huiming1; Sui, Zhigang1; Wu, Qi1,2; Liang, Zhen1; Zhang, Lihua1; Zhang, Yukui1; Zhang LH(张丽华)
刊名ANALYTICAL CHEMISTRY
2013-09-17
DOI10.1021/ac402076u
85期:18页:8507-8512
收录类别SCI
合作性质
文章类型Article
部门归属18
项目归属1810
产权排名待补充
WOS标题词Science & Technology ; Physical Sciences
资助者1,1 ; 1,1 ; 1,1 ; 1,1
类目[WOS]Chemistry, Analytical
研究领域[WOS]Chemistry
关键词[WOS]TANDEM MASS-SPECTROMETRY ; STRONG-CATION-EXCHANGE ; PHASE LIQUID-CHROMATOGRAPHY ; INTEGRAL MEMBRANE ; PROTEOMIC ANALYSIS ; IDENTIFICATION TECHNOLOGY ; SHOTGUN PROTEOMICS ; TRYPSIN DIGESTION ; BRAIN PROTEOME ; METHANOL
英文摘要Combining good dissolving ability of formic acid (FA) for membrane proteins and excellent complementary retention behavior of proteins on strong cation exchange (SCX) and strong anion exchange (SAX) materials, a biphasic microreactor was established to pretreat membrane proteins at microgram and even nanogram levels. With membrane proteins solubilized by FA, all of the proteomics sample processing procedures, including protein preconcentration, pH adjustment, reduction, and alkylation, as well as tryptic digestion, were integrated into an "SCX-SAX" biphasic capillary column. To evaluate the performance of the developed microreactor, a mixture of bovine serum albumin, myoglobin, and cytochrome c was pretreated. Compared with the results obtained by the traditional in-solution process, the peptide recovery (93% vs 83%) and analysis throughput (3.5 vs 14 h) were obviously improved. The microreactor was further applied for the pretreatment of 14 mu g of membrane proteins extracted from rat cerebellums, and 416 integral membrane proteins (IMPs) (43% of total protein groups) and 103 transmembrane peptides were identified by two-dimensional nanoliquid chromatography-electrospray ionization tandem mass spectrometry (2D nano-LC-ESI-MS/MS) in triplicate analysis. With the starting sample preparation amount decreased to as low as 50 ng, 64 IMPs and 17 transmembrane peptides were identified confidently, while those obtained by the traditional in-solution method were 10 and 1, respectively. All these results demonstrated that such an "SCX-SAX" based biphasic microreactor could offer a promising tool for the pretreatment of trace membrane proteins with high efficiency and throughput.
语种英语
资助者1,1 ; 1,1 ; 1,1 ; 1,1
原文出处查看原文
WOS记录号WOS:000330017400007
引用统计
被引频次:13[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://cas-ir.dicp.ac.cn/handle/321008/119507
专题中国科学院大连化学物理研究所
通讯作者Zhang LH(张丽华)
作者单位1.Chinese Acad Sci, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Natl Chromatog R&A Ctr, Dalian 116023, Peoples R China
2.Univ Chinese Acad Sci, Beijing 100039, Peoples R China
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GB/T 7714
Zhao, Qun,Liang, Yu,Yuan, Huiming,et al. Biphasic Microreactor for Efficient Membrane Protein Pretreatment with a Combination of Formic Acid Assisted Solubilization, On-Column pH Adjustment, Reduction, Alkylation, and Tryptic Digestion[J]. ANALYTICAL CHEMISTRY,2013,85(18):8507-8512.
APA Zhao, Qun.,Liang, Yu.,Yuan, Huiming.,Sui, Zhigang.,Wu, Qi.,...&张丽华.(2013).Biphasic Microreactor for Efficient Membrane Protein Pretreatment with a Combination of Formic Acid Assisted Solubilization, On-Column pH Adjustment, Reduction, Alkylation, and Tryptic Digestion.ANALYTICAL CHEMISTRY,85(18),8507-8512.
MLA Zhao, Qun,et al."Biphasic Microreactor for Efficient Membrane Protein Pretreatment with a Combination of Formic Acid Assisted Solubilization, On-Column pH Adjustment, Reduction, Alkylation, and Tryptic Digestion".ANALYTICAL CHEMISTRY 85.18(2013):8507-8512.
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