Application of Protein-Liposome Conjugate as a Pseudo-Stationary Phase in Capillary Electrophoresis

Journal of Chromatographic Science, Vol. 45, October 2007 Application of Protein–Liposome Conjugate as a Pseudo-Stationary Phase in Capillary Electrophoresis Jing Jiang*, Lina Geng*, Feng Qu, Aiqin Luo, Hong Li, and Yulin Deng† School of Life Science and Technology, Beijing Institute of Technology, Beijing 100081, China Abstract Liposomes have very similar structure to cell plasma membranes. Using liposomes as stationary phase in liquid chromatography (LC) or micellar electrokinetic chromatography (MEKC) has been demonstrated to be a good, dynamic method for the study of the interaction between cell membranes and important biomolecules. There has been no report on integrating plasma membrane proteins with phospholipids as pseudo-stationary phase in MEKC. In this paper, a novel mode of capillary electrophoresis (CE) is developed, that is, protein–liposome conjugate. This protein–liposome biomimetic membrane is demonstrated for the first time to be applicable as pseudo-stationary phase in MEKC. The protein is able to significantly improve chromatographic performance and stability. The experimental phenomena are further confirmed in terms of specific capacity factors and free binding energy. This new CE mode is used to investigate the interaction between dopamine transporter and dopamine–nomifensine. Introduction Liposomes are self-assembled vesicles commonly consisting of phospholipid bilayers, which have the amphiphilic character of the phospholipid property of encapsulating hydrophobic compounds in the bilayer membrane, or hydrophilic molecules in the internal cavity (1). In addition, depending on phospholipid composition, size, and surface characteristics, liposomes can establish a variety of interactions with molecular species and cell surfaces in the surrounding solution (2). These features enable liposomes to be widely employed as models for biological membranes as well as carriers for drugs and other agents of therapeutic, diagnostic, and cosmetic value (3). The use of phospholipid coating as a stationary phase in liquid chromatography has given rise to the study of solute membrane interactions and, to a lesser extent, in achieving several solute separations (4). * Authors contributed equally. † Author to whom correspondence should be addressed: . Capillary electrophoresis (CE) has a great deal of characteristics: (i) requiring a small amount of liposomes and samples, which is especially appealing when dealing with membrane transport proteins which are difficult to extract and with limited amount; (ii) being convenient and flexible in applying different modes, which are beneficial for complicated analysis; (iii) ability to employ liposomes not only coating as stationary phase, but also acting as a pseudo-stationary phase in a simple manner without the need of immobilization, that might destroy biological native structure; (iv) owning potential of constructing a high throughput and highly efficient screening platform for the evaluation of drug–membrane permeation ability or protein interaction. Liposomes have recently been found to have a variety of applications in CE, involving their use as coating materials (5–8) or carriers (9–13) to achieve solute separation or to investigate solute membrane interactions. Hjerten et al. were the first to use liposomes in CE for studying their interactions with model drugs and two octapeptides (14). Liposome–water partition coefficients for drugs were determined (12–13). Interactions with and transport of small molecules through cell membranes were simulated and studied using CE techniques (15–18). Liposomes have been applied for protein analysis, the separation of proteins (1), protein biopartition, and bioaffinity (19–23). However, to date, there is no such report that proteins are employed as one of the components in liposome microemulsion for micellar electrokinetic chromatography (MEKC) analysis. In this study, it is proposed that protein–liposome conjugate could not only be a good mode for the separation, but also for the investigation of protein interaction. The dopamine transporter (DAT) is a plasma membrane protein expressed exclusively in DA synthesizing neurons. It plays a crucial role in dopaminergic neurotransmission by taking up extracellular DA into nerve cells, terminating DA neurotransmission, and in maintaining DA homeostasis in the central nervous system (24–26). DAT has been taken as the molecular target for therapeutic agents used in the treatment of mental disorders (27). The mechanism that regulates DA uptake is of medicinal importance as potential sites of action for Parkinson’s and other neurological diseases, and also for psychiatric diseases (28). Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission. 587 Journal of Chromatographic Science, Vol. 45, October 2007 In this article, DAT-based liposome conjugate was constructed for the new CE mode. In order to substantiate one of its potential applications in the drug screening, interaction between DAT and DA as well as nomifensine, a specific inhibitor for DAT, was investigated as an example. cessful preparation of DAT from SH-SY5Y cells. SDS–PAGE was performed on 10% polyacrylamide. Gels were stained for 60 min with 0.1% (w/v) Coomassie Brilliant Blue R-250 in methanol–glacial acetic acid–water (40:10:50, v/v/v), then destained for 5 h in methanol–glacial acetic acid–water (10:10:80, v/v/v). The final protein stock solution was stored at –80°C for use. Experimental Preparation of liposome buffer (12) Materials DA, nomifensine (1,2,3,4-tetrahydro-2-methyl-4-phenyl-8isoquinolinamine), Dulbecco’s Modified Eagle Medium (DMEM), and fetal bovine serum (FBS) were purchased from Sigma (St. Louis, MO). Egg phosphatidylcholine was obtained from Beijing Shuangxuan Microorganism Substrates Factory (Beijing, China). Cholesterol and other chemicals were all of analytical grade and purchased from Beijing Chemical Reagents Company (Beijing, China). Phosphate-buffered saline (NaH2PO4–Na2HPO4, PBS, 10mM, pH 6.8) that was close to the physiological environment of human body was chosen as the background electrolyte (BGE) solution in MEKC. All aqueous solutions were prepared using water purified with a Milli-Q purifier system (Millipore, Milford, MA). Preparation of protein The SH-SY5Y cells were the resource for plasma membrane protein preparation. Cells in culture medium of 90% (v/v) DMEM and 10% (v/v) FBS were cultured in a CO2 incubator (Sanyo Electric Co., Osaka, Japan). Culture medium was removed from the cells, which were then washed with cold phosphate buffered saline (PBS). Cells were detached from culture flasks with cold lysis buffer and centrifuged for 20 min at 4°C at 1,000 × g. Cold lysis buffer was removed and cells were then resuspended in ice-cold PBS. The cells were broken up by ultrasonic lysis. After that, the admixture obtained was centrifuged for 30 min at 4°C at 800 × g. The supern (...truncated)