Biomedical subjects

Mohamed Bedair

Publications and source records attributed to Mohamed Bedair.

7 recordsLinked to original sources

PubMed2006

Lectin affinity chromatography using porous polymer monolith assisted nanoelectrospray MS/MS.

An affinity porous polymer monolith is utilized as a nanoelectrospray emitter as well as an online affinity capture column for the preconcentration of glycans. Porous polymer monolith (PPM) assisted electrospray provides a facile methodology for coupling microfluidics to mass spectrometry that is sheathless and with zero dead volume. Affinity PPM was photopolymerized using glycidyl methacrylate/ethylene dimethacrylate utilizing different porogenic solvents based on aliphatic alcohols to provide PPMs with a variety of pore sizes. The use of longer alkyl chain alcohols decreased the pore size of the formed PPM as indicated by the higher flow back pressure generated. The effect of the pore size on the stability of the electrospray was tested showing higher stability of the TIC with lower pore size. A lectin, namely Concanavaline A, was immobilized on glycidyl methacrylate/ethylene dimethacrylate using the Schiff base method to provide an affinity monolith for high mannose glycans. The amount of the lectin immobilized was studied as a function of the porogenic solvent used in the polymerization. The glycopeptides of the glycoprotein Ribonuclease B was preconcentrated on the affinity PPM sprayer and detected by tandem MS.

Chromatography, Affinity↗

PubMed2005

Affinity chromatography with monolithic capillary columns. II. Polymethacrylate monoliths with immobilized lectins for the separation of glycoconjugates by nano-liquid affinity chromatography.

Monolithic capillary columns with surface bound lectin affinity ligands were introduced for performing lectin affinity chromatography (LAC) by nano-liquid chromatography (nano-LC). Two kinds of polymethacrylate monoliths were prepared, namely poly(glycidyl methacrylateco-ethylene dimethacrylate) and poly(glycidyl methacrylate-co-ethylene dimethacrylate-co-[2-(methacryloyloxy)ethyl]trimethyl ammonium chloride) to yield neutral and cationic macroporous polymer, respectively. Two lectins including concanavalin (Con A) and wheat germ agglutinin (WGA) were immobilized onto the monolithic capillary columns. The neutral monoliths with immobilized lectins exhibited lower permeability under pressure driven flow than the cationic monoliths indicating that the latter had wider flow-through pores than the former. Both types of monoliths with immobilized lectins exhibited strong affinity toward particular glycoproteins and their oligosaccharide chains (i.e., glycans) having sugar sequences recognizable by the lectin. Due to the strong binding affinity, the monoliths with surface bound lectins allowed the injection of relatively large volume (i.e., several column volumes) of dilute samples of glycoproteins and glycans thus allowing the concentration of the glycoconjugates and their subsequent isolation and detection at low levels (approximately 10(-8) M). To further exploit the lectin monoliths in the isolation of glycoconjugates, two-dimensional separation schemes involving LAC in the first dimension and reversed-phase nano-LC in the second dimension were introduced. The various interrelated methods established in this investigation are expected to play a major role in advancing the sciences of "nano-glycomics".

Chromatography, Affinity↗

PubMed2004

Affinity chromatography with monolithic capillary columns I. Polymethacrylate monoliths with immobilized mannan for the separation of mannose-binding proteins by capillary electrochromatography and nano-scale liquid chromatography.

Monolithic capillary columns with surface-immobilized mannan have been introduced for affinity-based micro-column separations by nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). Two kinds of polymethacrylate monoliths were prepared, namely poly(glycidyl methacrylate-co-ethylene dimethacrylate) and poly(glycidyl methacrylate-co-ethylene dimethacrylate-co-[2-(methacryloyloxy)ethyl]trimethyl ammonium chloride) to yield neutral and cationic macroporous polymer, respectively. While neutral monoliths with immobilized mannan were only useful for affinity nano-LC, the cationic monoliths with surface-bound mannan were useful in both affinity nano-LC and affinity CEC. The cationic monoliths allowed a relatively high electro-osmotic flow (EOF) when mannan was immobilized to the epoxy monolith via a positively charged spacer arm, triethylenetetramine. The neutral monoliths exhibited lower permeability under pressure-driven flow (PDF) than the cationic monoliths indicating that the latter had wider flow-through pores than the former. Both types of monoliths with immobilized mannan exhibited strong affinity toward mannose-binding proteins (MBP) such as the plant lectins concanavalin A and Lens culinaris agglutinin and a mammalian lectin (e.g. rabbit serum mannose-binding protein). Due to the strong binding affinity, the monoliths with surface bound mannan allowed the injection of large volume of rabbit serum and to isolate in a single run the mannose-binding protein in an amount sufficient to run with it sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), thus demonstrating their capability in "nano-proteomics".

Animals↗

PubMed2004

Recent advances in polymeric monolithic stationary phases for electrochromatography in capillaries and chips.

This review article summarizes the advances made over the last two years in polymeric monoliths for capillary electrochromatography (CEC). It covers the scientific literature in the period extending form the second half of 2002 until the end of first half of 2004. Currently, there is an increasing interest in monolithic stationary phases in CEC as an alternative to particulate packed capillary columns due in major part to the simplicity of the in situ preparation of monolithic stationary phases and the availability of a wide chemistry for surface ligands, which allow for tailoring the chromatographic sorbent needed for solving a given separation problem(s). The various approaches, formats, and chemistries used for the preparation of monolithic stationary phases are described.

Acrylamides↗

PubMed2003

Capillary electrochromatography with monolithic stationary phases. II. Preparation of cationic stearyl-acrylate monoliths and their electrochromatographic characterization.

A novel cationic monolithic stationary phase based on the co-polymerization of pentaerythritol diacrylate monostearate (PEDAS) with a selected quaternary amine acrylic monomer was designed for performing capillary electrochromatography at high flow velocity. While PEDAS functioned as both the ligand provider and the cross-linker, the quaternary amine acrylic monomer was introduced to control the magnitude of the electroosmotic flow (EOF). The fabrication of the cationic stearyl-acrylate monolith (designated as cationic C17 monolith) with controlled porosity was achieved by free radical polymerization using the initiator 2,2'-azobisisobutyronitrile in the presence of a ternary porogenic solvent composed of cyclohexanol, ethylene glycol and water. Four different quaternary amine acrylic monomers were investigated in order to find the optimum monomer for achieving maximum electroosmotic flow (EOF) velocity. Both photo- and thermally-initiated polymerization proved effective in producing the cationic C17 monolith, and the best monolith was achieved when [2-(acryloyloxy)ethyl]trimethyl ammonium methyl sulfate (AETA) was used as the quaternary amine acrylic monomer. Although the zeta potential of the resulting cationic C17 monolith is positive with respect to water, the magnitude and direction of the EOF was markedly affected by the nature of the electrolyte in the mobile phase. Consequently, anodal, zero or cathodal EOF was observed depending on the nature of the electrolyte, and this was attributed to the adsorption of the ionic components of the electrolyte on to the solid stationary phase, which is characterized by its amphiphilic nature consisting of C17 chains, ester functions, hydroxyl groups and quaternary amine moieties. Optimized PEDAS-AETA monoliths yielded columns with high separation efficiency and allowed rapid separations on the time scale of seconds to be achieved with short capillaries.

Acrylates↗

PubMed2003

Capillary electrochromatography with monolithic stationary phases. III. Evaluation of the electrochromatographic retention of neutral and charged solutes on cationic stearyl-acrylate monoliths and the separation of water-soluble proteins and membrane proteins.

This article, which is closely related to part II, is concerned with the evaluation of the retentive properties of cationic stearyl-acrylate monoliths (i.e. cationic C17 monoliths) over a wide range of elution conditions with various uncharged and charged solutes including proteins. The retention parameters for charged solutes including the retention factor k* observed under capillary electrochromatography conditions and the velocity factor k(ep)*, which reflects the electrophoretic process, were measured for weak, moderate and strong basic compounds. These retention parameters allowed the assessment of the respective contributions from electrophoretic and partitioning separation mechanisms. The cationic C17 monoliths exhibited sufficient hydrophobic interactions with relatively weak basic solutes. Moderate and strong bases showed migration behaviors dominated by their relatively strong electrophoretic mobility with marginal chromatographic partitioning. At low pH, the cationic C17 monoliths allowed the separation of proteins with minimum electrostatic interactions between proteins and the cationic sites on the surface of the stationary phase. The utility of the cationic C17 monoliths was demonstrated in the rapid and efficient separation of two crude extracts of membrane proteins, namely galactosyl transferase and cytochrome c reductase. Short capillary columns (8.5 cm effective length) of the cationic C17 monoliths allowed rapid and efficient separations of neutral and charged pesticides and metabolites, phenylthiohydrantoin amino acids and proteins at the time scale of seconds at relatively high flow velocity.

Acrylates↗

PubMed2002

Capillary electrochromatography with monolithic stationary phases: 1. Preparation of sulfonated stearyl acrylate monoliths and their electrochromatographic characterization with neutral and charged solutes.

A novel monolithic stationary phase having long alkyl chain ligands (C17) was introduced and evaluated in capillary electrochromatography (CEC) of small neutral and charged species. The monolithic stationary phase was prepared by the in situ copolymerization of pentaerythritol diacrylate monostearate (PEDAS) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a ternary porogenic solvent consisting of cyclohexanol/ethylene-glycol/water. While AMPS was meant to support the electroosmotic flow (EOF) necessary for transporting the mobile phase through the monolithic capillary, the PEDAS was introduced to provide the nonpolar sites for chromatographic retention. Monolithic columns at various EOF velocities were readily prepared by conveniently adjusting the amount of AMPS in the polymerization solution as well as the composition of the porogenic solvent. The monolithic stationary phases thus obtained exhibited reversed-phase chromatography behavior toward neutral solutes and yielded a relatively strong EOF. For charged solutes (e.g., dansyl amino acids), nonpolar as well as electrostatic interaction/repulsion with the monoliths were observed in addition to electrophoretic migration. Therefore, for charged solutes, selectivity and migration can be readily manipulated by changing various parameters including the nature of the monolith and the composition of the mobile phase (e.g., pH, ionic strength and organic modifier). Ultrafast separation on the time scale of seconds of 17 different charged and neutral pesticides and metabolites were performed using short capillary columns of 8.5 cm x 100 microm ID.

Acrylic Resins↗