A capillary electrophoresis method to assay catalytic activity of botulinum neurotoxin serotypes: implications for substrate specificity

The potent botulinum neurotoxin inhibits neurotransmitter release at cholinergic nerve terminals, causing a descending flaccid paralysis characteristic of the disease botulism. The currently expanding medical use of the neurotoxin to treat several disorders, as well as the potential misuse of the neurotoxin as an agent in biowarfare, has made understanding of the nature of the toxin's catalytic activity and development of inhibitors critical. To study the catalytic activity of botulinum neurotoxin more thoroughly and characterize potential inhibitors, we have developed a capillary electrophoresis method to measure catalytic activity of different serotypes of botulinum neurotoxin using peptides derived from the native substrates. This assay requires only a minute amount of sample (25 nl), is relatively rapid (15 min/sample), and allows the determination of enzyme kinetic constants for a more sophisticated characterization of inhibitors and neurotoxin catalytic activity. Using this method, we can measure activity of five of the seven serotypes of botulinum neurotoxin (A, B, E, F, and G) with two peptide substrates. Botulinum neurotoxin serotypes C and D did not cleave our peptides, lending insight into potential substrate requirements among the serotypes.     

Plasma methionine determination by capillary electrophoresis-UV assay: application on patients affected by retinal venous occlusive disease

Methionine is an important amino acid involved in protein synthesis and transmethylation reactions. It is also the precursor of homocysteine and cysteine, two important risk factors for cardiovascular diseases. As homocysteine research has gained impulsion, the evaluation of plasma methionine concentrations has acquired importance. Methionine measurement generally has been performed by HPLC after o-phthalaldehyde derivatization. Its separation from other amino acids is time-consuming. We set up a new specific capillary electrophoresis method in which analyte derivatization was avoided by sample concentration before analysis. Methionine was detected by UV absorbance at 204 nm with a detection limit of 0.5 micromol/L. By a capillary with an effective length of 50 cm filled with 125 mmol/L Tris phosphate buffer at pH 2.3, the separation occurred in less than 14 min. Precision tests indicated a good test repeatability for both migration times (coefficient of variation [CV]<0.3%) and areas (CV<2.0%). Moreover, a good reproducibility of intraassay and interassay tests was obtained (CV<2.9% and CV<3.5%, respectively). The Passing-Bablok regression and the Bland-Altman test for methods comparison suggest that the data obtained by our method and by a reference HPLC assay are similar. Assay performance was evaluated measuring methionine concentrations in retinal venous occlusive disease.     

Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence

Quantification of gene expression provides valuable information regarding the response of cells or tissue to stimuli and often is accomplished by monitoring the level of messenger RNA (mRNA) being transcribed for a particular protein. Although numerous methods are commonly used to monitor gene expression, including Northern blotting, real-time polymerase chain reaction, and RNase protection assay, each method has its own drawbacks and limitations. Capillary electrophoresis with laser-induced fluorescence (CE-LIF) can reduce protocol time, eliminate the need for radioactivity, and provide superior sensitivity and dynamic range for quantification of RNA. In addition, CE-LIF can be used to directly determine the amount of an RNA species present, something that is difficult and not normally accomplished using current methods. Gene expression is detected using a fluorescently labeled riboprobe specific for a given RNA species. This direct approach was validated by analyzing levels of 28S RNA and also used to determine the amount of discoidin domain receptor 2 mRNA in cardiac tissue.     

Development of an apparatus for rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in chondroitin sulfate-type proteoglycans

An apparatus, AutoGlycoCutter (AGC), was developed as a tool for rapid release of O-linked-type glycans under alkaline conditions. This system allowed rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in proteoglycans (PGs). After digestion of PGs with chondroitinase ABC, the oligosaccharides at the linkage region were successfully released from the protein core by AGC within 3 min. The reducing ends of the released oligosaccharides were labeled with 2-aminobenzoic acid and analyzed by a combination of capillary electrophoresis (CE) and matrix-assisted laser desorption time-of-flight mass spectrometry. In addition, the unsaturated disaccharides produced by chondroitinase ABC derived from the outer parts of the glycans were labeled with 2-aminoacridone and analyzed by CE to determine the disaccharide compositions. We evaluated AGC as a method for structural analysis of glycosaminoglycans in some chondroitin-sulfate-type PGs (urinary trypsin inhibitor, bovine nasal cartilage PG, bovine aggrecan, bovine decorin, and bovine biglycan). Recoveries of the released oligosaccharides were 57-73% for all PGs tested in the present study. In particular, we emphasize that the use of AGC achieved ca. 1000-fold rapid release of O-glycans compared with the conventional method.     

A capillary zone electrophoresis method for the quantitative determination of hypoxoside in commercial formulations of African potato (Hypoxis hemerocallidea)

Hypoxoside is a norlignan diglucoside present in the corms of African potato, Hypoxis hemerocallidea, used as a popular African traditional medicine for its nutritional and immune boosting properties. A specific analytical method employing capillary zone electrophoresis has been developed and validated for the quantitative determination of this analyte. Sulfafurazole was used as internal standard, and electrophoretic separation of both analytes could be achieved within 12 min. Linearity of the method was established within the range 5-60 microg/mL and provided a high degree of accuracy (100 +/- 3%). The recovery of the method was found to be 100 +/- 5% and the RSDs of the intra- and inter-day precision were better than 5.19 and 2.52%, respectively. The limits of detection and quantification were calculated to be 0.5 and 2 microg/mL, respectively. The described method was used for the analysis and quality control of two commercially available products containing African potato. The method can also be used to determine product stability since it could separate the hypoxoside peak from its degraded products obtained from degradation studies.     

Phenols removal in musts: Strategy for wine stabilization by laccase

The potential of laccase from Trametes versicolor for phenolic removal in must for wine stabilization was evaluated through a combination of an analytical methodology (capillary zone electrophoresis) and kinetics of phenols removal as the total antioxidant potential variation. Total phenolic content, total antioxidant potential and polyphenols were monitored from 0 to 3 h of must treatment. The results indicated that the treatment of a red must with laccase affect mainly the phenolic compounds responsible for the must antioxidant properties. The treatment of white musts with laccase showed higher reduction in total phenol than in the total antioxidant potential. Phenol degradation by laccase was very fast for catechins, and slowly for stilbenes (cis- and trans-resveratrol) and derivatives of cinnamic (ferulic and caffeic) and benzoic (syringic, vanillic, and gallic) acids. It is possible to conclude in this case that the use of laccase in white wines is perfectly feasible. This would allow softer and ecologically correct treatments, which would diminish the cost of processing and avoid deterioration of wines for long storage times.     

Determination of hyaluronan molecular mass distribution in human breast milk

Hyaluronan (HA) in human milk mediates host responses to microbial infection via TLR4- and CD44-dependent signaling. Signaling by HA is generally size specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low-M component. Here we report the size distribution of HA in human milk samples from 20 unique donors. A new method for HA analysis, employing ion exchange (IEX) chromatography to fractionate HA by size and specific quantification of each size fraction by competitive enzyme-linked sorbent assay (ELSA), was developed. When separated into four fractions, milk HA with M ? 20 kDa, M ∼ 20 to 60 kDa, and M ∼ 60 to 110 kDa comprised averages of 1.5, 1.4, and 2.0% of the total HA, respectively. The remaining 95% was HA with M ? 110 kDa. Electrophoretic analysis of the higher M HA from 13 samples showed nearly identical M distributions, with an average M of approximately 440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low-M HA components.     

High-sensitivity TFA-free LC-MS for profiling histones

The analysis of proteins by RPLC commonly involves the use of TFA as an ion‐pairing agent, even though it forms adducts and suppresses sensitivity. The presence of adducts can complicate protein molecular weight assignment especially when protein isoforms coelute as in the case of histones. To mitigate the complicating effects of TFA adducts in protein LC‐MS, we have optimized TFA‐free methods for protein separation. Protein standards and histones were used to evaluate TFA‐free separations using capillary (0.3 mm id) and nanoscale (0.1 mm id) C₈ columns with the ion‐pairing agents, formic acid or acetic acid. The optimized method was then used to examine the applicability of the approach for histone characterization in human cancer cell lines and primary tumor cells from chronic lymphocytic leukemia patients.     

Analysis of amino acids in individual wheat embryonic protoplast

Summary. Amino acids analysis in single wheat embryonic protoplast was performed using capillary electrophoresis equipped with laser-induced fluorescence (CE-LIF), combination with tissue culture technique. Reagent fluorescein isothiocyanate (FITC) was introduced into living protoplasts by electroporation for intracellular derivatization. A special osmotic buffer (0.6 mol/L mannitol, 5 mmol/L CaCl₂) was used to keep the osmotic balance of embryonic protoplasts during the protoplasts derivatization. After completion of the derivatization reaction in the protoplasts, a single protoplast was drawn into the capillary tip by electroosmotic flow. Then a 0.1 M NaOH lysing solution was injected by diffusion. The derivatized amino acids were separated by capillary electrophoresis and detected by laser-induced fluorescence detection after the protoplast was lysed Nine amino acids were quantitatively and qualitatively determined and compared in lysate and single protoplast of wheat embryonic cells respectively, with mean concentrations of amino acids ranging from 2.68×10⁻⁵ mol/L to 18.18×10⁻⁵ mol/L in single protoplast.     

Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes

We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.