Off-line two-dimensional liquid chromatography with maximized sample loading to reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry for shotgun proteome analysis.

We demonstrate a strategy of maximizing the performance of reversed-phase (RP) liquid chromatography (LC) tandem mass spectrometry (MS/MS) for efficient shotgun proteome analysis by optimizing the sample loading to the instrument in an off-line two-dimensional (2D) LC tandem MS platform. To determine the quantity of peptides present in a proteome digest or fractionated peptides from strong-cation exchange (SCX) separation, an automated system based on RPLC with a rapid step solvent gradient for peptide elution and ultraviolet (UV) detection was developed. This system also allowed the purification of the peptides by removing salts and other impurities present in a sample. It was found that controlling the amount of peptides injected into a RPLC MS/MS system was critical to achieve the maximum efficiency in peptide and protein identification. With the use of off-line 2D-LC-MS/MS, peptide fractions from the first dimension of separation were desalted and quantified, followed by injecting the optimal amount of the sample into RPLC-MS/MS for peptide sequencing. The application of this strategy was demonstrated in the proteome profiling of breast cancer MCF-7 cells. From the analysis of 28 SCX fractions with each injecting 1 microg of sample into a 75 mum x 100 mm C18 column interfaced to a quadrupole/time-of-flight mass spectrometer, a total of 2362 unique proteins or protein groups were identified with a false positive peptide identification rate of 0.19%, as determined by target-decoy proteome sequence searches. Replicate 2 h runs of individual fractions with the exclusion of precursor ions of peptides already identified in the first runs resulted in the identification of an additional 549 unique proteins or protein groups with a false positive identification rate of 0.60%. This example illustrated that off-line 2D-LC-MS/MS, with maximal sample injection to the RPLC-MS, is an effective method for shotgun proteome analysis. Finally, the advantages and limitations of this method, compared to other methods, are discussed.

Characterization of enantioselective binding of racemic natural tetrahydropalmatine to DNA by chromatographic methods.

A racemate from natural product, tetrahydropalmatine (THP), was characterized on its enantioselective binding to DNA by the chromatographic methods including microdialysis/HPLC, centrifugal ultrifiltration/HPLC and immobilized DNA affinity chromatography. It was found that its (+)-enantiomer was preferential to binding on B-form duplex DNA including calf thymus DNA, AT and GC sequence oligo DNA, as well as triplex oligo DNA. The binding constants of the THP enantiomers to ct-DNA were determined with the methods of microdialysis/HPLC and frontal affinity chromatography. In addition, the DNA structural preference of either enantiomer was evaluated with the chromatographic methods.

p-tert-Butylcalix[4]arene-1,3-bis(allyloxyethoxy)ether coated capillaries for open-tubular electrochromatography.

The preparation and characterization of p-tert-butylcalix[4]arene-1,3-bis(allyloxyethoxy)ether (CA[4]-BAE) chemically coated capillaries via a free-radical reaction with vinyltriethoxysilane (VTES) that was attached onto the inner wall previously were carried out. IR spectra and decreased electroosmotic flow (EOF) suggested that the capillary was successfully coated with CA[4]-BAE. A slight slope of EOF versus pH at 5 < pH < 9 would help to make the separation reproducible. The CA[4]-BAE-coated capillary showed improved separations of toluidine isomers, naphthol isomers and polycyclic aromatic hydrocarbons (PAHs) compared with an uncoated capillary. The special selectivity indicates that there is a certain extent of host-guest interactions between the solutes and the CA[4]-BAE coating.

Monolithic columns with mixed modes of reversed-phase and anion-exchange stationary phase for capillary electrochromatography.

A capillary electrochromatography (CEC) monolithic column with mixed modes of reversed-phase and anion-exchange stationary phases was prepared by in situ polymerization of 2-(methacryloxy)ethyltrimethylammonium methyl sulfate (MEAMS) and ethylene dimethacrylate (EDMA) in a binary porogenic solvent consisting 1-propanol and 1,4-butanediol. The ammonium groups on the surface of the stationary phase generate an electroosmotic flow (EOF) from cathode to anode, and serve as a strong anion-exchange stationary phase at the same time. The EOF of the stationary phase can be determined by the amount of MEAMS monomer in reaction mixtures during the polymerization. The monolithic stationary phases exhibited reversed-phase chromatographic behavior toward neutral solutes. For charged solutes, hydrophobic as well as electrostatic interaction/repulsion with the monoliths was observed. Separations of aromatic compounds and basic compounds on the prepared column were performed under the mode of CEC. Peak tailing of basic compounds was avoided and the efficient separation of aromatic acids was achieved using neutral mobile phase due to the same direction of EOF and electrophorestic mobility of negatively charged solutes.

Separation of basic and acidic compounds by capillary electrochromatography using monolithic silica capillary columns with zwitterionic stationary phase.

A novel monolithic silica column with zwitterionic stationary phase was prepared by in-situ covalent attachment of phenylalanine to a 3-glycidoxypropyltriethoxysilane-modified silica monolith. Due to the zwitterionic nature of the resulting stationary phase, the density and sign of the net surface charge, and accordingly the direction and magnitude of electroosmotic flow in this column during capillary electrochromatography could be manipulated by adjusting the pH values of the mobile phase. CEC separations of various acidic and basic compounds were performed on the prepared column in anodic and weakly cathodic EOF modes, respectively. The peak tailing of basic compounds in CEC on a silica column could be alleviated at optimized buffer compositions. Besides the electrophoretic mechanism and weak hydrophobic interaction, weak cation- and anion-exchange interactions are also involved in the separations of acids and bases, respectively, on the zwitterionic column.

Hybrid silica monolithic column for capillary electrochromatography with enhanced cathodic electroosmotic flow.

A hybrid silica monolithic stationary phase for RP CEC was prepared by in situ co-condensation of (3-mercaptopropyl)-trimethoxysilane (MPTMS), phenyltriethoxysilane (PTES), and tetraethoxysilane (TEOS) via a sol-gel process. The thiol groups on the surface of the stationary phase were oxidized to sulfonic acids by peroxytrifluoroacetic acid. The introduced sulfonic acid moieties on the monoliths were characterized by a strong and relatively stable EOF in a broad pH range from 2.35 to 7.0 in CEC. Aromatic acids and neutral compounds can be simultaneously separated in this column under cathodic EOF. The CEC column exhibited a typical RP chromatographic mechanism for neutral compounds due to the introduced phenyl groups.

Octadecylated silica monolith capillary column with integrated nanoelectrospray ionization emitter for highly efficient proteome analysis.

An improved strategy for the preparation of octadecylated silica monolith capillary column with high homogeneity was proposed. Column performance was evaluated by nanoscale HPLC. The design for constructing an integrated nanoelectrospray emitter on the octadecylated silica monolith capillary column was first introduced. In comparison with the separated configuration where the emitter is connected to monolithic capillary column by the aid of a zero dead volume union, the integrated capillary column has the inherent advantage of the minimized extracolumn volume thus providing improved separation quality. The performance of the integrated monolithic capillary column was evaluated by separation of BSA tryptic digest, and peak capacity of 313 with a 30-cm column was obtained. The high separation performance allowed highly confident identification of 662 distinct proteins through assignment of 1933 unique peptides by analysis of tryptic digest of 0.5 mug of Saccharomyces cerevisiae proteins. The higher separation efficiency by a 60-cm monolithic capillary column increased the proteome coverage with identification of 1323 proteins through assignment of 5501 unique peptides over 400-min gradient elution.

Monolithic enantiomer-selective stationary phases for capillary electrochromatography.

Monolithic materials have become a well-established format for stationary phases in the field of capillary electrochromatography. Four types of monoliths, namely particle-fixed, silica-based, polymer-based, and molecularly imprinted monoliths, have been utilized as enantiomer-selective stationary phases in CEC. This review summarizes recent developments in the area of monolithic enantiomer-selective stationary phases for CEC. The preparative procedure and the characterization of these columns are highlighted. In addition, the disadvantages and limitations of different monolithic enantiomer-selective stationary phases in CEC are briefly discussed.

Monolithic silica capillary column with coated cellulose tris(3,5-dimethylphenylcarbamate) for capillary electrochromatographic separation of enantiomers.

Monolithic silica capillary columns were prepared by a sol-gel process in fused-silica capillaries with an inner diameter of 50 microm and were modified by coating of cellulose tris(3,5-dimethylphenylcarbamate). Influences of the factors in the modification process on enantiomer separations were investigated. The prepared columns were used to perform enantiomer separations by CEC. Fifteen and two pairs of enantiomers were separated under aqueous and nonaqueous mobile phases, respectively, and most of them were baseline-separated with very high column efficiencies. The Van Deemter curve was found flat under high linear velocity of the mobile phase, which indicated favorable kinetic properties of the prepared columns. Baseline separation of a pair of enantiomers was achieved in 90 s with high-column efficiency by short-end separation under high voltage.

Large-pore mesoporous SBA-15 silica particles with submicrometer size as stationary phases for high-speed CEC separation.

A new mesoporous sphere-like SBA-15 silica was synthesized and evaluated in terms of its suitability as stationary phases for CEC. The unique and attractive properties of the silica particle are its submicrometer particle size of 400 nm and highly ordered cylindrical mesopores with uniform pore size of 12 nm running along the same direction. The bare silica particles with submicrometer size have been successfully employed for the normal-phase electrochromatographic separation of polar compounds with high efficiency (e.g., 210,000 for thiourea), which is matched well with its submicrometer particle size. The Van Deemeter plot showed the hindrance to mass transfer because of the existence of pore structure. The lowest plate height of 2.0 microm was obtained at the linear velocity of 1.1 mm/s. On the other hand, because of the relatively high linear velocity (e.g., 4.0 mm/s) can be generated, high-speed separation of neutral compounds, anilines, and basic pharmaceuticals in CEC with C18-modified SBA-15 silica as stationary phases was achieved within 36, 60, and 34 s, respectively.

Capillary electrochromatography with a neutral monolithic column for classification of analytes and determination of basic drugs in human serum.

Capillary electrochromatography (CEC) using a neutral hydrophobic polymer-based monolithic column has been developed for classification of analytes based on their acidity and charges of group. The monolithic columns were prepared by in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate without any charged monomers in the reaction mixture. The ionic analytes will only be driven by their electrophoretic mobilities and were separated on the basis of their differences in electrophoretic mobility and in hydrophobic interaction with the stationary phase. Only negatively or positively charged analyte (acid or basic) migrated toward detection window in a single run by applying negative or positive voltage. The CEC system was also applied for the analysis of basic drugs in human serum by internal standard method using acidic running buffer. The sample of human serum spiked with basic drugs was directly injected after a simple sample pretreatment. The calibration curves with regression coefficients (0.9995-0.9997) in the range of 0.318-80 microg/mL were obtained with the limits of detection being below 0.15 microg/mL. The intra- and inter-day precisions, determined as relative standard deviations, were less than 4.21%.

Preparation of monolithic silica column with strong cation-exchange stationary phase for capillary electrochromatography.

A monolithic silica based strong cation-exchange stationary phase was successfully prepared for capillary electrochromatography. The monolithic silica matrix from a sol-gel process was chemically modified by treatment with 3-mercaptopropyltrimethoxysilane followed by a chemical oxidation procedure to produce the desired function. The strong cation-exchange stationary phase was characterized by its substantial and stable electroosmotic flow (EOF), and it was observed that the EOF value of the prepared column remained almost unchanged at different buffer pH values and slowly decreased with increasing phosphate concentration in the mobile phase. The monolithic silica column with strong cation-exchange stationary phase has been successfully employed in the electrochromatographic separation of beta-blockers and alkaloids extracted from traditional Chinese medicines (TCMs). The column efficiencies for the tested beta-blockers varied from 210,000 to 340,000 plates/m. A peak compression effect was observed for atenolol with the mobile phase having a low phosphate concentration.

Electrochromatographic evaluation of a silica monolith capillary column for separation of basic pharmaceuticals.

A silica-based monolithic capillary column was prepared via a sol-gel process. The continuous skeleton and large through-pore structure were characterized by scanning electron microscopy (SEM). The native silica monolith has been successfully employed in the electrochromatographic separation of beta-blockers and alkaloids extracted from traditional Chinese medicines (TCMs). Column efficiencies greater than 250 000 plates/m for capillary electrochromatography (CEC) separation of basic compounds were obtained. It was observed that retention of basic pharmaceuticals on the silica monolith was mainly contributed by a cation-exchange mechanism. Other retention mechanisms including reversed-phase and normal-phase mechanisms and electrophoresis of basic compounds also played a role in separation. A comparison of the differences between CEC and capillary zone electrophoresis (CZE) separation was also discussed.

Polar stationary phases for capillary electrochromatography.

This review article summarizes the variety of polar stationary phases that have been employed for capillary electrochromatographic separations. Compared with reversed-phase stationary phases, the polar alternatives provide a completely different retention selectivity towards polar and charged analytes. Different types of polar stationary phases are reviewed, including the possible retention mechanisms. Electrochromatographic separations of polar solutes, peptides, and basic pharmaceuticals on polar stationary phases are presented.

Open-tubular capillary electrochromatography using capillary columns chemically bonded with the new host molecules calix[6]crown, calix[6]arene.

Two host molecules, p-tert-butylcalix[6]-1,4-crown-4 and p-tert-butylcalix[6]arene were prepared and attached onto the inner surfaces of capillaries for open-tubular electrochromatography with the aid of gamma-glycidoxypropyl-trimethoxysilane (KH-560) as bridging agent. The successful bonding was confirmed by infrared (IR) results and greatly decreased electroosmotic flow (EOF). Parameters affecting separation, such as buffer pH and organic modifier were studied. The two novel stationary phases were evaluated by the separation of isomeric toluidines, a mixture of pyridine and isomeric picolines, and isomeric dihydroxybenzenes; comparisons between capillaries coated with the two stationary phases and bare capillary were investigated. The special selectivity of these two novel stationary phases showed a certain extent of supramolecular interactions between stationary phases and solutes.

Determination of basic pharmaceuticals in human serum by hydrophilic interaction capillary electrochromatography.

Hydrophilic interaction capillary electrochromatography (HI-CEC) for the determination of basic pharmaceuticals spiked in human serum is described. The organic modifier content, ionic strength, and pH value of the mobile phase as well as the applied voltage are optimized for separation and elution of these drug analytes. Excellent separation was achieved for drugs using a mobile phase composition of 80% v/v acetonitrile in 100 mM triethylamine phosphate (TEAP) buffer at pH 2.8 with column efficiencies for analytes more than 200,000 plates/m. The samples of human serum spiked with basic drugs were directly injected after a simple acetonitrile treatment. The linear range and reproducibility of these basic drugs using an external and internal standard method were compared. As a result, the reproducibility could be greatly improved by using the internal standard method. Good calibration curves with regression coefficients more than 0.998 in the range of 5-160 microg/mL were observed with the internal standard method. The limits of quantitation, based on standards with acceptable relative standard deviations (RSDs), were below 5 microg/mL. The intra- and inter-day precisions, determined as RSDs, were less than 4.57%.

Monolithic column with zwitterionic stationary phase for capillary electrochromatography.

A capillary electrochromatography (CEC) monolithic column with zwitterionic stationary phases was prepared by in situ polymerization of butyl methacrylate, ethylene dimethacrylate, methacrylic acid, and 2-(dimethyl amino) ethyl methacrylate in the presence of porogens. The stationary phases have zwitterionic functional groups, that is, both tertiary amine and acrylic acid groups, so the ionization of those groups on the zwitterionic stationary phase was affected by the pH values of the mobile phase, and further affects the strength and direction of the electroosmotic flow (EOF). Separations of alkylbenzenes and polycyclic aromatic hydrocarbons based on the hydrophobic mechanism were obtained. Separation of various types of polar compounds, including phenols, anilines, and peptides, on the prepared column were performed under CEC mode with anodic and cathodic EOF, and different separation selectivities of those polar analytes were observed on the monolithic capillary column by using mobile phases with different pH values.