Non-enzymatic posttranslational modifications of bovine serum albumin by oxo-compounds investigated by chromatographic and electrophoretic methods.

Non-enzymatic posttranslational modifications of bovine serum albumin (BSA) by oxo-compounds, particularly glucose, ribose, glyoxal and glutardialdehyde, have been investigated using a set of modern chromatographic and electrophoretic separation methods. High-performance liquid chromatography (HPLC) alternatively with UV spectrophotometric (diode array) or mass spectrometric (MS) detection, polyacrylamide gel electrophoresis (PAGE) with Coomassie brilliant blue staining detection, and capillary zone electrophoresis (CZE) with UV spectrophotometric detection have been employed for the investigation of the chemical and structural changes of BSA caused by its reaction with the above oxo-compounds exhibiting different degree of reactivity. The extent of modifications was found to be dependent on the nature of the oxo-compound used and progressed in the glucose

Comparison of standard capillary and chip separations of sodium dodecylsulfate-protein complexes.

Conditions for converting a set of five standard proteins to electrochemically active sodium dodecylsulfate (SDS) complexes were worked out with the aim of using such complexes for conductivity detection with a a chip electrophoresis system. The results obtained were compared with standard capillary electrophoresis (37 cm (effective length 30 cm) x 75 microm I.D. capillary, 10 kV, negative polarity at the inlet). The chip separations were run at 500 V per chip (100 V/cm) as compared to the standard capillary arrangement, which was run at 266.6 V/cm. For the capillary set-up the protein complexes were prepared in aqueous solution (Milli-Q water) made 10 mM with respect to SDS. If the SDS concentration was increased to 50 mM, the separation in the capillary was incomplete. On the other hand with the chip system both approaches yielded acceptable results. The chip separations were slightly (but not distinctly) shorter and offered better separations than the standard set-up. The concentration of the surfactant used for the preparation the complexes results in alternations of the elution sequence, which is preserved if the chip separation is used instead of the capillary set-up. Apparently the full capacity of protein-SDS binding is not exploited for the preparation of the adducts.

Influencing electroosmotic flow and selectivity in open tubular electrochromatography by tetrakis(pentafluorophenyl)porphyrin as capillary wall modifier.

A physically adsorbed and covalently bonded porphyrin derivative, 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin, H2TPFPP, has been used as a fused-silica capillary wall modifier in open tubular capillary electrochromatography (OT-CEC), and its influence on the electroosmotic flow (EOF) velocity and on the selectivity of OT-CEC separations of a set of model aromatic carboxylic acids has been tested. Whereas most of the coatings of this category bring about an increase in selectivity with a concomitant slow down of the EOF, H2TPFPP coating, depending on pH of the background electrolyte used, resulted both in decreasing of EOF at pH 8.5 by 5% and in increasing of EOF by 10-43% at pH 6 and 5, respectively. The separation efficiency and the resolution of aromatic carboxylic acids separation in coated capillaries, namely in that one with covalent coating, were better than in the bare fused-silica capillary. The perspectives of H2TPFPP as capillary wall modifier are visualized in introducing well defined electroosmotic properties of materials used for miniaturized separation channels preparation in chip-based electromigration devices.

Open tubular capillary electrochromatography of underivatized amino acids using Rh(III) tetrakis(phenoxyphenyl)porphyrinate as wall modifier.

The separation of 17 "common" underivatized amino acids was attempted by open tubular capillary electrochromatography (OT-CEC) in fused-silica capillaries coated with Rh(III) tetrakis(phenoxyphenyl)porphyrinate (Rh(III)TPP(m-OPh)4OAc) using sodium phosphate and Tris-phosphate buffers as background electrolytes (BGEs). The OT-CEC separation of amino acids was compared with that obtained by capillary zone electrophoresis in bare fused-silica capillaries using the same BGEs. The amino acids were not derivatized and the UV-absorption detection was set at 200 nm. Depending on the experimental conditions at least 15 amino acids were separated. The best separations were obtained in a Rh(III)TPP(m-OPh)4OAc-coated capillary in 50 mM Tris-100 mM phosphate buffer at pH 2.25. Separation of the critical triplet Val-Ile-Leu was always at least indicated being better at higher BGE concentrations. Regarding the sensitivity of the method, lower concentration limits of detection (LODs) in the coated capillary were obtained for Thr, Gly, Tyr, and Val; the other amino acids exhibited lower LODs in the uncoated capillary. The separation of acidic amino acids was not achieved.

Application of capillaries with minimized electroosmotic flow to the electrokinetic study of acidic drug-beta-oleoyl-gamma-palmitoyl-L-alpha-phosphatidyl choline liposome interactions.

Interaction of a model set of common drugs varying widely in their polarity as well as in their chemical structure (salicylic acid, acetylsalicylic acid, ketoprofen, phenytoin and propranolol) with beta-oleoyl-gamma-palmitoyl-L-alpha-phosphatidyl choline (POPC) liposomes was investigated by means of capillary electrophoresis. Two phosphate buffers differing in their pH (50 mM, pH 7.5 and 9.2) were used both for liposome reconstitution and as background electrolytes for capillary electrophoresis using capillaries with minimised electroosmotic flow (EOF). The liposomes showed practically no electrophoretic mobility and formed a stable plug in the capillary. At alkaline pH (9.2), the polyimide coated capillary exhibited residual endoosmotic flow (the EOF marker appeared before the detection window around 40 min as compared to 2.2 min in the untreated capillary; attempts to reveal endoosmotic flow at pH 7.5 were unsuccessful). The concentration of the mixture of the test compounds was 50 microg/ml (except for ketoprofen concentration of which was 5 microg/ml due to the lower solubility of the drug), i.e. large enough to exceed the binding capacity of the injected liposome plug at least at the neutral pH (7.5) which consequently resulted in two regions in the electropherogram, namely that which contained the unbound species and that corresponding to the liposome (lipid)-bound fraction. On the other hand in runs done at high pH of the background electrolyte (9.2) the whole amount injected interacted with the liposomes. Acidic drugs and phenytoin were run with negative polarity at the injection site. It was documented that both at pH 7.5 and 9.2 the investigated solutes interacted with POPC liposomes, though at pH 7.5 the equilibrium between the bound and unbound drugs was in favor of the unbound species. On the contrary, at pH 9.2 binding was considerably stronger and only the liposome bound fraction was seen upon electrophoresis. The well-known instability of phenytoin at room temperature resulted in the formation of an acidic hydrolytic product which was strongly bound to liposomes at the higher pH value. While no binding of phenytoin could be established at pH 7.5, at pH 9.2 this compound was degraded (hydrolyzed) and its degradation product was clearly bound to liposomes. It has to be emphasized that binding experiments must be done separately for acidic/neutral and basic drugs; binding of acidic/neutral drugs must be done at reversed polarity, while in order to reveal binding of basic drugs, positive polarity at the injection site must be used.

Affinity electrochromatography of acidic drugs using a liposome-modified capillary.

Liposomes can be effectively deposited on the inner surface of a capillary wall by flushing the electrophoretic system with a liposome suspension followed by air-drying of the capillary and removal of the excess of loosely bound liposomes by a 0.1 M NaOH wash. It was demonstrated that capillaries prepared in this way could be used for studies of analyte (drug)-liposome binding. The results were expressed as free binding energy changes [delta(deltaG0)] relatively to an arbitrarily selected standard (acetylsalicylic acid). The results were compared to [delta(deltaG0)] changes obtained from binding studies effected by capillary electrophoresis using a stable liposome plug in a capillary with minimized endoosmotic flow. Good agreement of data reported in the literature (without correction for the residual endoosmotic flow), our previous data obtained in a similar way (however, after the correction for the residual endoosmotic flow) and data obtained by the immobilized liposome affinity electrochromatography reported in this communication was achieved.

Comparison of the electrophoretic separation of proteins in capillaries with different inner diameter.

Two fused-silica capillaries of considerably different inner diameter (75 and 10 microm) were used for the separation of a set of five standard proteins. The separations were run in acid pH (50 mM phosphate buffer, pH 2.5). Generally better separations (with minor tailing only) were obtained using a standard capillary [27 cm (20 cm effective length)x 75 microm I.D.] in comparison with a narrow bore capillary [27 cm (20 cm effective length)x 10 microm I.D.]. The conditions of the electrophoretic separation were the same for both types of capillaries (25 degrees C; 10 kV; positive polarity at the inlet). The sequence of the proteins was cytochrome c, albumin, transferrin followed by a partly resolved peak of catalase and chymotrypsinogen A. In the narrow bore capillary severe tailing was observed--tailing factor ranged from 2.11 to 5.54 or 1.67 to 2.53 depending on the concentration of the analytes injected (2 or 0.2 mg/ml of each test compound injected). The relative [delta(deltaG(0))] values of the interaction with the capillary wall in the small bore capillary (with cytochrome c taken as initial standard) ranged from -0.74 to -1.04 kJ/mol. The problem of assaying the speed of the endoosmotic flow (EOF) in both capillary types was thoroughly investigated using thiourea and dithiothreitol as EOF markers. It was revealed that if thiourea is used as the EOF marker, the obtained value was dependent on the concentration of the marker injected. Optimum conditions for the EOF determination in acid buffer were specified. The higher speed of the EOF in the narrow bore capillary (10 microm) as compared to the 75 microm I.D. capillary is discussed.

Separation of low-molecular mass peptides by capillary electrophoresis with the use of alkylamines as dynamic coating agents at low pH.

Modification of the silanophilic activity of the inner surface of the capillary wall was studied in a capillary electrophoretic system using alkylamines containing background electrolytes at acid pH. The effect of the following amine additives was investigated: (1) alkyl-alpha,omega-diamines (1,2-diaminoethane, 1,4-diaminobutane, 1,7-diaminoheptane, spermine), (2) polymeric amines (polyethylenimine, polybrene), (3) cationic amine surfactants (cetrimide, hexamethonium bromide). A seven membered test mixture of peptides (Gly-Pro-Ala, Pro-hPro, Gly-Pro-Arg, Gly-Pro-Gln, Lys-Pro-Gly, Asn-Pro-Gly, His-Pro-Gly) possessing one or more amino groups was used for selectivity evaluation. Under optimised concentration of the amine modifiers the selectivity was always improved (except for polybrene), particularly with the fast moving analytes. The best results were obtained with 1,2-diaminoethane and 1,7-diaminoheptane. On the other hand with slowly moving peaks the best separations were obtained with 1,7-diaminoheptane, hexadecyltrimethylammonium bromide and hexamethonium bromide, i.e. with modifiers possessing large aliphatic domains which are likely to be hydrophobically bonded with the separated solutes. The selectivity improvement with fast moving members of the test mixture can be ascribed to the decrease of the electroosmotic flow, while the improved separation with slowly moving peaks appears to reflect the altered interaction with the hydrophobized capillary wall. As expected the endoosmotic flow was in all cases decreased. The practical applicability of using amine based dynamic modifiers of the capillary wall was demonstrated on a natural peptide mixture (bacterial collagenase hydrolysate of collagen types I and III).

Binding of environmental pollutants to the corn protein zein studied by high-performance liquid chromatography.

The interaction of 16 ring-substituted phenols and anilines with the corn protein zein was studied by reversed-phase high-performance liquid chromatography by preparing silica- and alumina-based stationary phases coated with various concentrations of zein. The relationship between the strength of interaction and the physicochemical parameters of solutes was elucidated by principal component analysis followed by the nonlinear mapping technique. The binding of each phenol and aniline derivative to zein has been demonstrated. It was established that the electrostatical parameters of solutes exert the highest influence on the interaction and the involvement of hydrophobic binding forces is of secondary importance. The binding characteristics of phenol and aniline derivatives were different.

Separation of structurally related peptides by open-tubular capillary electrochromatography using (metallo)porphyrins as the adsorbed stationary phase.

Several (metallo)porphyrins, particularly the porphyrin derivative tetraphenylporphyrin, and complexes of porphyrin derivatives with metal ions (Zn2+, Cu2+, Ni2+, Co2+, Co3+) have been employed as the stationary phase physically adsorbed onto the inner fused-silica capillary surface for open-tubular capillary electrochromatography, and applied for the separation of structurally related peptides. Four octapeptides, derivatives of the B23-B30 fragment of the B-chain of human insulin with minor changes in their sequences (presence of lysine or ornithine in position B-29, presence or absence of phenylacetyl protecting group on the amino group of lysine/ornithine or N-terminal amino group of glycine), were studied as model analytes. Separations were performed both in alkaline (pH 9.0) and in acidic (pH 2.25) background electrolytes, and the changes in the migration/retention behaviour of the model set of peptides were investigated with respect to the porphyrin periphery/central metal atom and the charge of the octapeptides modified. The key moment of successful separation of these peptides seems to be the accessibility of functional groups of the peptides to the interaction with the modifiers tested herein.

Capillary electrochromatographic separation of aromatic amino acids possessing peptides using porphyrin derivatives as the inner wall modifiers.

Two different porphyrin derivatives (H2TPP(m-OPh)4 and Rh(III)TPP(m-OPh)4) were investigated with respect to their capability to help resolution of five model aromatic peptides in capillary electrophoresis/open tubular capillary electrochromatography. Though the main separation mechanism was preferentially based on the ionic properties of the separated analytes, involvement of particularly H2TTPP(m-OPh)4-peptide interactions at alkaline pH (8.0) was clearly demonstrated. In combination with Tris-phosphate buffer, a speed up of the separation was observed at pH 2.25 (particularly if Rh(III)TPP(m-OPh)4 was used as capillary coating); in spite of the speed up of the separation the selectivity of the system was sufficient and resulted in a complete separation of the five model peptides. It can be expected that Rh(III)TPP(m-OPh)4 capillary coating in combination with Tris-phosphate buffer can be generally used for a considerable speeding up of lengthy separations of peptides in acidic media with some decrease in the separation power of the system.

Peptide mapping by capillary electrophoresis with Pluronic F127.

Separation of peptides and proteins by capillary zone electrophoresis suffers from the interaction of these solutes with the capillary wall which results in the formation of broad peaks and low resolution. To minimize the protein/peptide-capillary wall interaction we tried to use Pluronic F127, a triblock copolymer of the general formula (polyethylene oxide)(x)(polypropylene oxide)(y)(polyethylene oxide)(z) when x=106, y=70 and z=106 which can be considered a surfactant capable of self-association both into isotropic and anisotropic gels. The analytes studied were enzymatic digests (obtained by trypsin or pepsin treatment) of insoluble matrix proteins from avian eggshell. The best separations were obtained by a system exploiting 10% Pluronic F127 in 20 mmol/l phosphate buffer, pH 2.5. Electrophoretic peptide profiles obtained were very complex owing to the complicated nature of the samples (the exact composition of the proteinous insoluble part of the eggshell is still unknown). The separation in phosphate buffer only offered complex maps of incompletely resolved peaks. The use of Pluronic F127 distinctly improved the separation with a considerably better resolution regarding both the number of peaks obtained and the quality of the separation.

Capillary electrochromatographic study of the interactions of porphyrin derivatives with amino acids and oligopeptides.

Open-tubular capillary electrochromatography (OT-CEC) was used to study the interactions of synthetic (metallo)porphyrin derivatives (immobilized by physical adsorption to the fused-silica capillary wall) with three aromatic amino acids (phenylalanine, tyrosine, tryptophan), three aliphatic amino acids (beta-alanine, proline, valine) and two oligopeptides (diglycine, triglycine). The effective mobilities of amino acids and peptides measured in OT-CEC mode in the acid and alkaline background electrolytes (BGEs) were compared with those obtained by capillary zone electrophoresis (CZE) in the bare fused-silica capillary in the same BGEs. In this way the influence of the peripheral substituents and the character of the central metal atom in porphyrin derivatives on the interactions with amino acids and peptides in the acid and alkaline media was investigated. Three types of noncovalent interactions, axial ligation to the central metal atom, pi-pi stacking and electrostatic repulsion seem to take part in the interactions of analyzed amino acids and peptides with porphyrin derivatives, resulting in a better separation of these analytes by OT-CEC than by CZE.

Plastic substrates based separation channels in electromigration techniques.

Three types of plastic materials (polyester, polyurethane and polymethylmethacrylate) were tested as materials for manufacturing separation columns (polyester and polyurethane capillaries were used) or separation channels (polymethylmethacrylate) in the chip format. A set of 11 fluorescein isothiocyanate amino acid derivatives was used as the test mixture. Using alpha-cyclodextrin additive to the background electrolyte in the case of the chip separation was also tested. The main problem with all plastic separation media was the selectivity of the separation. The best results, practically identical with bare fused silica capillary, were obtained with the polymethylmethacrylate chip, provided that alpha-cyclodextrin in a concentration 40 mmol/l was added to the background electrolyte. An important observation was that in SDS containing background electrolyte all the plastic materials used exhibited a distinct electroosmotic flow, which was ascribe to the sorption of the negatively charged constituents of the background electrolyte to the capillary wall. Regarding the order in which the individual components of the test mixture were brought to the detector only a single change was observed. Histidine migrated in the polystyrene and polymethylmethacrylate separation channels more slowly than in the bare silica or polyurethane based capillaries.

Application of liquid chromatography-electrospray ionization mass spectrometry for study of steroid-converting enzymes.

A high-performance liquid chromatography-atmospheric pressure ionization-electrospray ionization mass spectrometry (HPLC-API-ESI-MS) method was developed for the analysis of steroids in a study of steroid-converting enzymes. Separations ware done on a Zorbax Eclipse XDB-C18 column (eluted with a linear methanol-water-acetic acid gradient) and identification of the steroids involved was done by API-ESI-MS using positive ion mode and extracted ion analysis. The applicability of the present method for studying steroid metabolism was proven in assaying two steroid-converting enzymes (20beta-hydroxysteroid dehydrogenase and 11beta-hydroxysteroid dehydrogenase) in various biological samples (rat and chicken intestine, chicken oviduct).

Simultaneous effect of organic modifier and physicochemical parameters of barbiturates on their retention on a narrow-bore PGC column.

The retention time of 22 barbituric acid derivatives was measured on a narrow-bore porous graphitized carbon (PGC) column using water-dioxane mixtures as mobile phases. The capacity factor (k), theoretical plate number (N), and asymmetry factor (AF) were calculated for each solute in each mobile phase. The relationships between chromatographic characteristics and physicochemical parameters of solutes were elucidated by stepwise regression analysis (SRA). SRA indicated that the binding of barbiturates to the PGC surface is of mixed character electrostatic and apolar interactive forces are equally involved. Sterical correspondence between the surface of the stationary phase and the solutes also exert a significant influence on the retention behavior.

Reversed-phase liquid chromatographic-mass spectrometric determination of microcystin-LR in cyanobacteria blooms under alkaline conditions.

Reversed-phase HPLC coupled to the atmospheric pressure ionization-electrospray ionization (API-ESI) MS was used for microcystin-LR detection and quantitation in samples of dried Microcystis aeruginosa cells. An alkaline linear gradient (20 mmol/l ammonium hydroxide-acetonitrile, pH 9.7) was used for elution of the toxic peptides. Limit of detection was 1 microg/ml (20 ng per injection) in the scan mode of MS and 0.1 microg/ml (2 ng per injection) in the case of selective ion monitoring.

Chromatography in authenticity and traceability tests of vegetable oils and dairy products: a review.

The new applications of various chromatographic techniques such as gas-liquid chromatography, high-performance liquid chromatography and electrophoretic methods employed for the analysis in macro- and micro-components in vegetable oils and dairy products are compiled and critically evaluated. The employment of these methods for authenticity tests and traceability is discussed.

Capillary electrochromatographic study of sapphyrin-organophosphoric acid derivatives interaction.

Interaction of phosphate moiety possessing compounds with sapphyrin was studied using open-tubular electrochromatography in sapphyrin-coated capillaries. It was revealed that phenylthiohydantoin (PTH)-phosphoserine and PTH-phosphothreonine exert such a strong interaction that they can not be eluted from the sapphyrin-coated capillary even at prolonged run times (70 min). Nucleoside polyphosphates show generally strong interaction (but weaker than the above mentioned serine and threonine derivatives) no matter whether they possess one or two bases. Also the number of phosphate residues present in nucleoside polyphosphates tested plays a secondary role only. p-Aminobenzylphosphoric (p-ABPA) acid exhibited an unexpected behavior. It was retained more in the phosphate containing buffer than in borate-acetate. This appears to indicate that other than complexing of the phosphate moiety may be involved in the interaction. As no such effects were observed with the PTH-derivatives of serine and threonine it was concluded that additional interaction (if involved) depends on the nature of the organic part of the molecule.

Insoluble eggshell matrix proteins--their peptide mapping and partial characterization by capillary electrophoresis and high-performance liquid chromatography.

Avian eggshell matrix proteins were studied by two analytical approaches. Peptide mapping was done by trypsin and pepsin followed by collagenase cleavage; analyses were carried out by capillary electrophoresis and reversed-phase high-performance liquid chromatography (HPLC). Comparison of peptide maps obtained by both methods revealed a complex mixture of peptides in the insoluble layers of the eggshell; it was concluded that there are at least three different insoluble protein/peptide layers in the avian eggshell (cuticle, palisade, and mammillary layer). Partial characterization of peptides in each layer was made by HPLC-mass spectrometry analysis. There is an evidence that the eggshell insoluble proteins contain species susceptible to collagenase cleavage, however, the sequences split by this enzyme probably are not those typical for the main triple-helical core of collagenous proteins. It is proposed that the action of collagenase upon eggshell proteins is caused by the side effect of collagenase described previously with synthetic peptides. Some of the proteins present are probably glycosylated. Fatty acid content in the insoluble eggshell layers (after decalcification) was in the range of 2-4% (which reflected both lipid and lipoproteins bound fatty acids). Porphyrin pigments are dominant in the cuticle layer.