High sensitivity analysis of water-soluble, cyanine dye labeled proteins by high-performance liquid chromatography with fluorescence detection.
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-(9CI) (sb-cy3-NHS), containing two p-carboxybenzyl 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.
CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic Research & Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China.
Xiaoqiang
X
Qiao
Li
L
Wang
Junfeng
J
Ma
Qiliang
Q
Deng
Zhen
Z
Liang
Lihua
L
Zhang
Xiaojun
X
Peng
Yukui
Y
Zhang
D000818:Animals; D002232:Carbocyanines / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006147:Guanine / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000138:chemical synthesis / Q000737:chemistry*; D007700:Kinetics; D011489:Protein Denaturation / Q000187:drug effects; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D013696:Temperature; D014867:Water / Q000737:chemistry*
D002232:Carbocyanines; D005456:Fluorescent Dyes; D007211:Indoles; D011506:Proteins; D014867:Water; D006147:Guanine; D016650:Fluorescein-5-isothiocyanate