Analysis of trypanosomal endocytic organelles using preparative free-flow electrophoresis

In this paper we demonstrate the power of preparative free-flow electrophoresis (FFE) for the study of endocytosis by African trypanosomes. Endocytosis of extracellular macromolecules by these parasites occurs through a specialized region of the parasite called the flagella pocket. The uptake of fluid phase markers such as horseradish peroxidase (HRP) into the various compartments of the endocytic pathway of bloodstream forms of Trypanosoma brucei brucei was manipulated by regulating the external environment (e.g., by altering the temperature of incubation). The various subcellular compartments were then separated by free-flow electrophoresis (FFE) or isopycnic density gradient centrifugation and analyzed for marker uptake. At low temperatures, HRP was found predominantly in the flagellar pocket. Increasing the temperature resulted in a time-dependent uptake of HRP into more positively charged endosomal fractions. However, little HRP activity was detected in lysosomal compartments, suggesting that either HRP had not yet entered the lysosome or was degraded immediately upon entry. Through the use of FFE we were able to identify and analyze compartments of the endosomal pathway that were not possible to identify by density gradient centrifugation alone. Although the differences in FFE separation of the endocytic compartments as seen in HRP uptake were striking, the minor changes seen within the lysosomal system were more subtle, as depicted in the protease profiles. In conclusion, we show that preparative FFE is a powerful technique for the analysis and separation of flagellar pocket-derived membranes from other endosomal and lysosomal compartments of African trypanosomes.     

Humic acid capillary zone electrophoresis adsorption on capillary walls, separation in metal ion supplemented buffer and the fingerprints

Capillary zone electrophoresis (CZE) in quartz tubes is often being used for the separation and characterization of humic acids (HA). A method was found to follow adsorption (and kinetics) of humic acids on a fused-silica capillary wall. It was shown that the adsorption of humic acids on an uncoated capillary wall is high. The effect on sorption of additives to the background electrolyte (BGE) was studied. Sorption can be eliminated by adding magnesium(II) salts (14-50 mM) to the BGE (pH 3.40) with resultant highly reproducible electropherograms as well as detailed and expressive fingerprints for HA of different origin.     

Isolation of peroxisome subpopulations from rat liver by means of immune free-flow electrophoresis

Immune free-flow electrophoresis (IFFE) has been applied to the separation of peroxisomes (PO). IFFE is a modification of antigen-specific electrophoretic cell separation (ASECS), and combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It differs from the latter in the pH of the electrophoresis buffer, which was shifted from the physiological range (ASECS) to the pI of IgG molecules (pH approximately 8.0), thus further decreasing the mobility produced by the binding of a specific antibody. This enhances the mobility differences between IgG-coupled particles and those nondecorated, with resultant improved separation. We have now succeeded in isolating different subpopulations of PO by applying IFFE to heavy, light, and post-mitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The obtained PO subfractions differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic PO.     

Determination of the dissociation constants of ropinirole and some impurities and their quantification using capillary zone electrophoresis

Ropinirole, 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one, is a potent anti-Parkinson's disease drug developed by SmithKline Beecham Pharmaceuticals. Capillary zone electrophoresis (CZE) was used for the determination of the dissociation constants of ropinirole and five structurally related impurities, potentially formed during its synthesis and for separation and quantification of these substances. The dissociation constants obtained from the CZE measurements were confirmed by UV spectrophotometry for some of the test compounds, obtaining a good agreement between the values. Careful optimization of the running buffer composition permitted base-line resolution of the six compounds in a borate buffer containing acetonitrile and magnesium sulfate (a 100 mM borate buffer containing 30 mM MgSO4 and 20 vol.% of acetonitrile). It was shown that CZE can determine the level of these impurities, down to a level of 0.05% of the main component within 15 min.     

Production of anti-Ro/SS-A and anti-La/SS-B autoantibodies is closely coordinated in systemic lupus erythematosus and independent of anti-dsDNA production

Capillary zone electrophoresis (CZE) was utilized to identify a synaptobrevin-thioredoxin fusion protein (TSB-51). TSB-51 is a substrate for cleavage by botulinum toxin B at the Q(76)-F(77) site. TSB-51 was derivatized with a fluorophore, CBQCA [3-(4-carboxy-benzoyl)-2-quinoline-carboxaldehyde], for 4 h at room temperature. Optimal conditions for CZE separation of the TSB-51-CBQCA complex were determined: buffer (sodium borate), pH (9.0), applied voltage (25 kV), temperature (25 degrees C) and forward polarity. SDS-PAGE showed that TSB-51 had a molecular mass of approximately 19 kDa. The protein was transferred to PVDF membrane and sequenced by the Edman degradation method verifying the first twelve amino acids as SDKIIHLTDDSF. TSB-51 was also collected during CZE separation and subsequently sequenced yielding the first three amino acids as SDK. This CZE-LIF method coupled with the CBQCA derivatization, fraction collection and Edman sequencing allowed for identification of the recombinant protein, a fast separation run time and utilization of small volumes of peptide (1.5 ng protein/23.6 nl injection). This method will be used for monitoring the endopeptidase activity of botulinum toxin B on TSB-51.     

Choice of capillary electrophoresis systems for the impurity profiling of drugs

In order to develop a strategy for the impurity profiling of drugs, the possibilities of some capillary electrophoresis systems were investigated. A mixture containing a drug and some of its possible impurities has been used as a model problem. The test compounds were investigated by capillary zone electrophoresis (CZE) and by micellar electrokinetic chromatography (MEKC). The pH of the CZE buffer was varied, but the two stereoisomers could not be separated. Moreover, CZE is not suitable for neutral compounds. In MEKC, two different types of surfactants, sodium dodecyl sulphate (SDS) and cetyltrimethylammonium bromide (CTAB), have been used and the effect of type and concentration modifier on the separation and the elution window was studied. In the SDS system, both the resolution and the elution window could be increased considerably by the addition of modifier. The use of two MEKC systems of different selectivity seems to be a combination with high potential for the impurity profiling of drugs.     

Application of free-flow electrophoresis for isolation and purification of proteins and peptides

Free-flow electrophoresis (FFE) has been applied to the separation and purification of a variety of proteins and polypeptides: bee venom, tumor necrosis factor, interleukin-1beta, interferon-gamma and superoxide dismutase. FFE at constant pH and conductivity of the carrying buffer is shown to be efficient at various separation schemes. In some cases, the method allows us to obtain proteins with a purity of more than 90% at a productivity of 20-30 mg/h. An electrophoretic apparatus with a new, multi-sectional construction of the electrophoretic chamber and a system for cross-displacement of carrying buffer in the chamber is described.     

Differential regulation of the Janus kinase-STAT pathway and biologic function of IL-13 in primary human NK and T cells: a comparative study with IL-4

We describe new methods for analyzing the apolipoproteins (apo) of the high density lipoproteins (HDL) of several species by two modes of capillary electrophoresis: size separation using a molecular sieving buffer, and capillary zone electrophoresis (CZE) using neutral coated capillaries. By either mode HDL apos were resolved within 25 min. Results for apoA-I and apoA-II mass agreed with those by electroimmunoassay; intra-assay coefficients of variation were 1.8-4.2%. The migration times of human, rat, rabbit, and bovine apoA-I during CZE were proportional to their net charge/Mr ratios. This enabled human and rabbit apoA-I to be quantified simultaneously in transgenic rabbit HDLs. CZE also resolved human apoA-I isoforms, deamidated apoA-I, and pro-apoA-I.     

Enantiomeric separation of tramadol hydrochloride and its metabolites by cyclodextrin-mediated capillary zone electrophoresis

The enantiomeric separation of tramadol hydrochloride and its major metabolites, O-demethyltramadol (M1) and N-demethyltramadol (M2) was studied using cyclodextrin (CD)-mediated capillary zone electrophoresis (CZE). Influence of the choice of type and concentration of CD, capillary temperature, length of capillaries, buffer pH and the addition of polymer modifier on the chiral separation of tramadol and its metabolites was evaluated. It was found that the drug and the metabolites can be baseline-separated simultaneously by using 50 mM phosphate buffer (pH 2.5) containing 75 mM methyl-beta-CD, 220 mM urea and 0.05% (w/v) hydroxypropylmethyl cellulose.     

Quantitative analysis of non-steroidal anti-inflammatory drugs by capillary zone electrophoresis

The potential utility of capillary zone electrophoresis (CZE) for the separation and quantitative determination of some non-steroidal anti-inflammatory drugs (NSAIDs) was investigated. The influence of different parameters on migration times, peak symmetry, efficiency and resolution was studied; these parameters included the nature and concentration of the anionic and cationic components of the separation buffer. A buffer consisting of 75 mM glycine adjusted to pH 9.1 with triethanolamine was found to provide a very efficient and stable electrophoretic system for the CZE analysis of NSAIDs, giving RSD values of about 0.1 and 0.5% for the within-day reproducibility of migration times and peak areas, respectively at a concentration of 25 micrograms ml-1 (n = 5). Response was linear from 2-100 micrograms ml-1 for both sulindac and tiaprofenic acid, for which the LOQ values were 2.8 and 1.9 micrograms ml-1, respectively, using UV detection at 280 nm. Accuracy for each drug was 102-103%.     

Evaluation of capillary zone electrophoresis and micellar electrokinetic capillary chromatography with direct injection of plasma for the determination of cefotaxime and its metabolite

Quantitative aspects of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were investigated for the determination of cefotaxime (C) and its deacetyl metabolite (DA) in human plasma in a concentration range of therapeutic interest. For CZE, plasma samples spiked with C and DA were injected after deproteinization with acetonitrile, and analytes were separated in a fused silica capillary using a borate buffer at pH 9.2 as electrolyte; no suitable internal standard was found. For MECC, plasma samples spiked with C, DA, and theobromine as internal standard were directly injected after dilution with water and analyzed using a phosphate buffer, pH 8.00, containing 165 mM SDS as separation electrolyte and a fused silica capillary. Both methods gave satisfactory interday precision with respect to migration times (RSD < 1%) and gave linear responses over the concentration ranges investigated (5-100 mg L-1 C and 5-20 mg L-1 DA). For CZE, intraday RSD (n = 4 graphs) between the slopes of the calibration graphs was acceptable (5.7%) for C. The corresponding figures for interday precision (n = 4 days) were fair (16.1%) in comparison to those obtained with MECC, for which the RSD was 1.49% when theobromine was used as internal standard. A satisfactory interday precision between slopes was also obtained with MECC even without the use of an internal standard (RSD = 4.38%), which demonstrated the ruggedness of this method. Detection limits (S/N = 3) were about 2 mg L-1 (CZE) and 1 mg L-1 in plasma (MECC) for C and DA. MECC was shown to be superior with regard to simplicity, rapidity, precision, and sensitivity.     

Unusual form of recurrent giant cell granuloma of the mandible and lower extremities in a patient with neurofibromatosis type 1

The high resolution of capillary zone electrophoresis/mass spectrometry (CZE/MS) offers a promising technique to characterize biomolecules in pharmaceutical and biotechnology industries. A novel capillary zone electrophoresis/electrospray ionization time-of-flight mass spectrometry (CZE/ESI-TOFMS) interface was designed in this study to successfully integrate ESI-TOFMS, nanospray, and CZE for biomolecular identification. The interface offers a novel way to take advantage of the high resolution separation achieved during CZE and the detection sensitivity of nanospray ESI-MS. The results showed mixtures of peptides were highly resolved within a few minutes (each CZE electropherogram of a peptide is 2-3 seconds). The novel CZE/ESI-TOFMS interface may simultaneously provide sensitivity, data acquisition speed, mass range, and mass resolution while maintaining resolution of the CZE separation.     

Separation of oligonucleotides of identical size, but different base composition, by free zone capillary electrophoresis in strongly acidic, isoelectric buffers

A novel method for analyzing oligonucleotides of the same length, but bearing a single base substitution, is reported, based on free zone capillary electrophoresis (CZE) under rather acidic pH values. For this purpose, a set of four 18-mers of fairly random base composition has been synthesized, bearing, in nucleotide 9, the following bases: T, C, G or A. Theoretical predictions, based on titration curves of single free nucleotides, allowed us to predict that the simultaneous separation of a mixture of all four oligonucleotides could be possible in a pH 3-4 window. In fact, electrophoresis at pH 5.7 gave a single, asymmetric peak, whereas CZE at pH 4.8 could resolve three out of four species (the T9 and G9 oligonucleotides co-migrating into a single zone). A unique separation power could be obtained at pH 3.3 in a buffer comprising an amphoteric species (isoelectric iminodiacetic acid, IDA) and 7 M urea. Although IDA exhibited a pI of 2.23 (for a 100 mM solution), the addition of 7 M urea (necessary to denature the oligonucleotides) raised the apparent pH of the solution to 3.3.     

Characterization and quantification of organic anions with capillary zone electrophoresis using direct and indirect detection

A comparison of the separation and quantification capabilities of capillary zone electrophoresis (CZE) using direct and indirect detection of organic anions was conducted. A conventional CZE separation (normal polarity, electroosmotic flow toward the cathode) of phenol, benzoic acid, and 2,4-dihydroxybenzoic acid utilized direct UV absorption at 215 nM. A separation of myo-inositol 1,4,5-trisphosphate and myo-inositol hexakisphosphate utilizing a reversed polarity and an electroosmotic flow modifier (flow toward the anode) was monitored by indirect UV absorption at 250 nM. The separation buffers utilized in this study consisted of 50 mM borate buffer (pH 8.3) and IonPhor Anion PMA Electrolyte Buffer (pH 7.7) (Dionex Corp., Sunnyvale, CA, U.S.A.) for studies utilizing direct and indirect detection methods, respectively. The effect of separation voltage on the theoretical plate numbers observed for the separations was linear for both the direct and indirect systems. Sample introduction parameters investigated included electromigration injection voltage and duration, and gravity injection duration. The conventional CZE separation using direct detection gave superior precision and better agreement with theoretical predictions than the separation using indirect detection. Both systems were evaluated for quantitative accuracy using electromigration, pressure, and gravity sample introduction modes. The conventional CZE system showed superior performance with regard to sensitivity and limits of detection. Accuracy and precision in the quantitation of known standards was greatest for both systems when the gravity sample introduction mode was used.     

Effects of a mechanical stimulation of localization of annexin-like proteins in Bryonia dioica internodes

Mechanical stimulation exerted by rubbing a young internode of Bryonia dioica plants inhibits its growth. Previous cellular and biochemical studies showed that this growth inhibition is associated with Ca(2+) redistribution and profound modifications of plasma membrane characteristics. We extracted and purified Ca(2+)-dependent phospholipid-binding proteins from B. dioica internodes. Two main proteins, p33 and p35, and other minor bands were isolated and identified as annexin-like proteins because of their biochemical properties and their cross-reactions with antibodies against maize (Zea mays L.) annexins. Rabbit antiserum was obtained by injection of B. dioica p35. This antiserum was used for the immunocytolocalization of annexin-like proteins in internode parenchyma cells. It appeared that the distribution of annexin-like proteins was different before and 30 min after the mechanical stimulation. Western analysis of proteins in membrane fractions after separation by free-flow electrophoresis showed that p35 was present in most fractions, whereas p33 appeared mainly in plasmalemma-enriched fractions after the mechanical stimulation. It is hypothesized that a subcellular redistribution of these proteins might be involved in growth inhibition by mechanical stress.     

Immuno-isolation of highly purified peroxisomes using magnetic beads and continuous immunomagnetic sorting

Immuno-isolation is a powerful technique for the isolation of cells as well as subcellular organelle populations based on their antigenic properties. We have established a method for immuno-isolation of peroxisomes (PO) from both rat liver and the human hepatoblastoma cell line HepG2 using magnetic beads as solid support. A polyclonal antibody raised against the cytoplasmic C-terminal 10 amino acids of the rat 70 kDa peroxisomal membrane protein was covalently bound to magnetic beads (Dynabeads M-450). The coated beads were incubated with a light mitochondrial fraction and the organelle-bead complexes formed were separated by magnetic sorting in a free-flow system without pelleting the complexes during the isolation procedure. Scanning electron microscopy revealed decoration of beads with particles measuring 150-400 nm in diameter. The particles were identified as PO by catalase cytochemistry and biochemically by marker enzyme analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as immunoblotting for specific detection of peroxisomal matrix, core and membrane proteins. The functional significance of PO in man is emphasized by the existence of inherited diseases such as the Zellweger syndrome in which intact PO are lacking, but peroxisomal remnants called "ghosts" are observed instead. Peroxisomal disorders are usually studied using skin fibroblast cell lines derived from afflicted patients and immuno-magnetic separation may prove particularly useful for the investigation of such cultured cells and for further elucidation of the pathogenesis of fatal peroxisomal disorders.     

Analysis of nucleotides by capillary electrophoresis

Capillary electrophoresis is a useful tool for the analysis of nucleotides. Methods have been optimized for both CZE and MECC modes. A variety of CZE buffers, such as borate, carbonate and phosphate were used successfully. The pH of the buffer changes the charge on the nucleotides. Therefore, the selectivity of the analytes can be controlled by the acidity of the buffer solution. CE separations of nucleotides have been performed at all pH levels, in both CZE and MECC modes. SDS was the most commonly used modifier in MECC separations, but other additives have been added to optimize selectivity. In addition, nucleotides have been quantified in different matrices, including tissue and cell extracts and several DNA and RNA sources. This paper summarizes the methods used for the optimization of nucleotides by CE and includes the most recent techniques to improve selectivity, reproducibility and sensitivity. A summary of CE methods is used in analyses of nucleotides in biological matrices is included.     

Multiple sequential fraction collection of peptides and glycopeptides by high-performance capillary electrophoresis

Multiple sequential fraction collection of peptides and glycopeptides by high-performance capillary electrophoresis (HPCE) under applied voltage has been demonstrated from complex tryptic peptide maps. The collection methodology was adapted from a high-resolution glycopeptide mapping procedure and, as such, requires active temperature control of the sample, electrophoresis vials, and collections vials because the electrophoresis buffer system is higher conductive. Resolution was compromised in the preparative HPCE separation due to heavy sample loading and to reduced voltage. The latter was a requirement for this buffer system in order to control Joule heating at the current levels employed; collections were routinely performed at approximately 1.5 W/m. The collection buffer was optimized by the addition of 12% methanol (v/v), thereby improving collection yields. Tryptic non-glycopeptides were group collected; secondary analysis of the HPCE collections agreed with analytical separations with respect to the number of peptides contained in a given fraction. Sequentially collected peptide fractions were analyzed by Edman sequencing and MALDI mass spectrometry to verify peptide identity and sequence. Consistent peptide sequence or mass measurements were obtained for repeat collections. The isolation of the single pure glycopeptide indicates that unique glycopeptide structures can be collected by HPCE and then analyzed by other methods.     

Two-dimensional separations of peptides and proteins by comprehensive liquid chromatography-capillary electrophoresis

Analysis of biological samples is problematic because of their complex composition. Reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC), and, more recently, capillary zone electrophoresis (CZE) are routinely used for the analysis of these samples, but are eventually limited because they are one-dimensional (1-D) methods. As sample complexity increases, the separation efficiency necessary to resolve a large number of sample components in one dimension becomes prohibitively high. A solution to this problem has been to use a two-dimensional (2-D) approach. Each dimension in a 2-D separation relies on a different separating mechanism. By expanding the separation into two dimensions, sample components unresolved in the first dimension can often be separated in the second. This circumvents the requirement for extremely high efficiencies in either dimension. Two-dimensional slab gel electrophoresis has been used successfully in this area, but a more instrumental approach is desired. In this paper we describe three coupled-column approaches to 2-D separations. First, microcolumn SEC-CZE is explored as a means of 2-D protein analysis. Next, RPLC-CZE is investigated for analysis of peptides in tryptic maps. Finally, RPLC is coupled with fast CZE (FCZE), a unique form of CZE analysis, for fast 2-D analysis of peptides. Details of the instrumentation used in these 2-D systems will be presented along with the results of some typical 2-D analyses.     

Free-flow electrophoretic analysis of endosome subpopulations of rat hepatocytes

The separation of functional early and late endosomes from other cellular compartments by free-flow electrophoresis (FFE) has been previously demonstrated in nonpolarized cells. Here, using 125I-labeled anti-secretory component antibodies ([125I]SC Ab) and FITC-labeled asialoorosomucoid (FITC-ASOR) as markers of the transcytotic and lysosomal pathway, respectively, we demonstrate the separation of three distinct endosome subpopulations from polarized rat hepatocytes. Internalization of both markers at 16 degrees C resulted in their accumulation in a common endosome compartment, indicating that both the transcytotic and the lysosomal pathways are arrested in the sorting early endosome at temperatures below 20 degrees C. After chase of the markers from early endosomes into the transcytotic or the degradative route at 37 degrees C, transcytotic endosomes carrying [125I]SC Ab migrated with an electrophoretic motility between early and late endosomes while late endosomes labeled with FITC-ASOR were deflected more towards the anode than early endosomes. These data indicate that in rat hepatocytes, the transcytotic and lysosomal pathways utilize a common (i.e. early endosomes) and two distinct endosome subpopulations (i.e. transcytotic endosomes, late endosomes) prior to delivering proteins for biliary secretion or lysosomal degradation, respectively.