High sensitive protein detection by hollow fiber membrane interface based protein enrichment and in situ fluorescence derivatization.
A novel device, composed of a syringe pump for sample loading, a hydrophilic hollow fiber membrane interface for protein concentration and small molecules removal, and a centrifugation tube for buffer exchange, was designed for protein preconcentration and in situ fluorescence derivatization. With the outlet of the interface blocked, denatured proteins were continually introduced. Restricted by the membrane with the molecular weight cutoff (MWCO) of 3000 Da, proteins were concentrated within the membrane. However, denaturant and other small molecules, which might affect the further fluorescence derivatization, were driven out of the membrane. Then, the membrane with proteins restricted inside was directly put into the fluorescence derivatization buffer. Here, the water-soluble sulfo-3H-indocyanine dye, the active N-hydroxysuccinimide of 3H-indolium,1-(5-carboxypentyl)-2-[3-[1-(5-carboxypentyl)-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propenyl]-3,3-dimethyl-5-sulfo-,monopotassium salt (sw-cy3-NHS), synthesized in our lab, was used for protein labeling. By such a method, the detection sensitivity of bovine serum albumin (BSA) was improved by nearly 200 folds, compared to that obtained by direct in-solution derivatization. Through the derivatization of a fraction of E. coli protein separated by reversed phase HPLC, proteins with low concentration were efficiently labeled, which indicated the potential merit of the developed method for the high sensitive detection of low abundance proteins.
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
Liangliang
L
Sun
Li
L
Wang
Yu
Y
Liang
Lihua
L
Zhang
Yichu
Y
Shan
Xiaojun
X
Peng
Zhen
Z
Liang
Yukui
Y
Zhang
D000818:Animals; D002417:Cattle; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000302:isolation & purification; D005453:Fluorescence; D008567:Membranes, Artificial; D011506:Proteins / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000302:isolation & purification
D029968:Escherichia coli Proteins; D008567:Membranes, Artificial; D011506:Proteins; D012710:Serum Albumin, Bovine