Fabrication and fluorescence imaging of human low-density lipoprotein coatings for highly efficient capillary electrophoresis separation of basic proteins.
Here we present a unique fabrication of capillary coatings by using a native protein-phospholipid assembly, human low-density lipoprotein (LDL). By taking advantage of its constituents of apoproteins and phospholipids, two types of LDL coatings (non-covalent and covalent) were constructed. The non-covalent LDL coatings were prepared through physical adsorption of its phospholipids on fused silica, and the covalent coatings were prepared through covalent bonding of its apoproteins with the aminopropyl-modified silica. These coatings were subjected to CE separation of basic proteins (lysozyme, cytochrome c, ribonuclease A, and alpha-chymotrypsinogen A). Although both types of the coatings display efficient separation of the four proteins while effectively inhibiting the undesirable adsorption, the covalent LDL coatings provide higher separation efficiency (0.67-2.01 x 10(5) theoretical plates/m) and longer coating-life. Results also reveal that the LDL coatings can hydrophobically interact with proteins and provide options for tuning the separation selectivity of proteins besides their inherent electrophoretic mobility. By using SYPRO Orange staining and fluorescence imaging, the coverage and homogeneity of the LDL coatings along the capillary were further visually characterized. It was found that the coverage of LDL particles in the covalent coatings was much higher than that in the non-covalent coatings. To the best of our knowledge, this is the first report describing the enhanced CE protein separation by lipoprotein coating. In virtue of the apoproteins-phospholipids structure, the LDL coatings are potentially useful in various LDL-related binding studies and complex bio-analysis.
Yin
Junfa
J
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, PR China.
Song
Yuling
Y
Wang
Zhixin
Z
Wang
Hailin
H
D002413:Cations, Divalent / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D053841:Electroosmosis; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008077:Lipoproteins, LDL / Q000737:chemistry*; D008274:Magnesium / Q000737:chemistry; D000432:Methanol / Q000737:chemistry; D055550:Protein Stability; D011506:Proteins / Q000302:isolation & purification*; D015203:Reproducibility of Results
D002413:Cations, Divalent; D008077:Lipoproteins, LDL; D011506:Proteins; D003300:Copper; D008274:Magnesium; D000432:Methanol