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题名: High sensitivity analysis of water-soluble, cyanine dye labeled proteins by high-performance liquid chromatography with fluorescence detection
作者: Qiao, Xiaoqiang1, 2;  Wang, Li3;  Ma, Junfeng1, 2;  Deng, Qiliang1;  Liang, Zhen1;  Zhang, Lihua1;  Peng, Xiaojun3;  Zhang, Yukui1
关键词: Fluorescence derivatization ;  Protein ;  Sb-cy3-NHS ;  High-performance liquid chromatography
刊名: ANALYTICA CHIMICA ACTA
发表日期: 2009-04-27
DOI: 10.1016/j.aca.2009.03.021
卷: 640, 期:1-2, 页:114-120
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology ;  Physical Sciences
类目[WOS]: Chemistry, Analytical
研究领域[WOS]: Chemistry
英文摘要: A water-soluble sulfo-3H-indocyanine dye, the active N-hydroxysuccinimide ester of 3H-Indolium,1-[(4-carboxyphenyl)methyl]-2-[3-[1-[(4-carboxyphenyl)methyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propenyl]-3,3-dimethyl-5-sulfo-(9Cl) (sb-cy3-NHS), containing two pcarboxybenzyl groups on nitrogen atoms, previously developed by our laboratory, was for the first time used for protein derivatization, followed by HPLC separation and fluorescence detection. With bovine serum albumin (BSA) as a model protein, effects of various experimental conditions, including denaturant concentration, reaction time and temperature, the pH value of buffer, and the molar ratio of fluorescence reagent to protein, on protein derivatization efficiency were systematically investigated. Under the optimal conditions, the limit of detection (LOD) for derivatized BSA was decreased to 12.8 nM, about 100-fold lower than that by UV and fluorescence detection with commercial fluorescein isothiocyanate (FITC) as labeling reagent. For HPLC analysis, an on-column excess fluorescence reagent depletion technique was developed based on the hydrophilicity of sb-cy3-NHS, which could avoid the interference on the analysis of target compounds. In addition, sb-cy3-NHS was applied for the derivatization of a three-protein mixture and egg white proteins. Compared to the results labeled by FITC, more proteins with low concentrations could be labeled by sb-cy3-NHS, resulting in improved detection sensitivity for protein analysis. All these results demonstrated that sb-cy3-NHS might be promising in detecting low abundance proteins, especially in the quantitative analysis of proteins. (c) 2009 Elsevier B.V. All rights reserved.
关键词[WOS]: 2-DIMENSIONAL CAPILLARY-ELECTROPHORESIS ;  SODIUM DODECYL-SULFATE ;  LOW-ABUNDANCE PROTEINS ;  GEL-ELECTROPHORESIS ;  MASS-SPECTROMETRY ;  DERIVATIZATION ;  SEPARATION ;  PRECONCENTRATION ;  PROTEOMICS ;  CYSTEINE
语种: 英语
WOS记录号: WOS:000265763500018
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/141600
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Natl Chromatog Res & Anal Ctr, Dalian 116023, Peoples R China
2.Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China
3.Dalian Univ Technol, State Key Lab Fine Chem, Dalian 116012, Peoples R China

Recommended Citation:
Qiao, Xiaoqiang,Wang, Li,Ma, Junfeng,et al. High sensitivity analysis of water-soluble, cyanine dye labeled proteins by high-performance liquid chromatography with fluorescence detection[J]. ANALYTICA CHIMICA ACTA,2009,640(1-2):114-120.
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