Protein fingerprinting of Staphylococcus aureus by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection.

This chapter describes a complete procedure for obtaining protein fingerprints of microorganisms using capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). Staphylococcus aureus, a human pathogen responsible of frequent and resistant infections, is used as model microorganism to show the feasibility of this procedure. Bacteria are grown in different culture media or submitted to temperature or nitrosative stress conditions. After the growth of the bacteria, the protein extracts are obtained by cell lysis using sonication. The water-soluble fraction of these lysates is derivatized on-capillary with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. The fluorescent products are analyzed by CE and detected by LIF. Practical advices for the interpretation of the electropherograms are given. To do so, the variations of the protein fingerprints of the bacteria with the culture conditions, such as growth medium, or the stressing conditions, such as heat shock or nitrosative stress, are used as example.

Protein fingerprinting of Staphylococcus species by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection.

Capillary electrophoresis (CE) coupled with laser-induced fluorescence detection (LIF) has allowed to obtain protein fingerprints, which have demonstrated to be useful in microorganisms characterization. In this work, protein fingerprints of two species of Staphylococcus grown in different culture media and submitted to temperature and nitrosative stress were studied by CE-LIF. After the growth of the bacteria, protein extracts were obtained by cell lysis using sonication. The water-soluble fraction of these lysates was derivatized on-capillary with a fluorogenic dye, 3-(2-furoyl)quinoline-2-carboxaldehyde. The fluorescent products were analyzed by CE using phosphate buffer containing submicellar concentrations of sodium pentanesulfate and detected by LIF. Different protein fingerprints were obtained depending on the bacterial species studied, indicating the usefulness of this method for the identification of different species of the same bacterial genus. It was also demonstrated that the CE protein fingerprints were dependent on the culture conditions, such as growth medium, or on stressing conditions, such as heat shock or nitrosative stress.

On-capillary derivatization and analysis of amino acids in human plasma by capillary electrophoresis with laser-induced fluorescence detection: application to diagnosis of aminoacidopathies.

A capillary electrophoresis with laser-induced fluorescence detection method for the analysis of free amino acids (AA) in human plasma was developed. A mixture of 16 AA was on-capillary derivatized with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and separated inside the capillary in less than 30 min using 70 mM borax-3.5 mM SDS pH 9.3 as running buffer. Four plasma samples from a healthy donor and patients suffering from phenylketonuria, propionic acidemia, and tyrosinemia type II were studied. Repeatabilities calculated as intra-day RSD (n = 3) values for the AA involved in these aminoacidopathies (glycine, phenylalanine, and tyrosine) were in the range of 0.3 to 1.2% for migration time and 3.7 to 8.2% for peak height. Reproducibilities calculated as inter-day RSD (n = 4) values for the same AA were between 0.7 and 1.4% for migration time and 4.7 and 9.1% for peak height. A fast qualitative analysis allowed the identification of the corresponding disease by comparing the electrophoretic profiles from the patient and the healthy donor and noting the increased level of the specific AA accompanying each individual disease. The results of the quantitative analysis for glycine, phenylalanine, and tyrosine in the plasma samples studied using the developed method showed a good agreement with those provided by the Center of Diagnosis of Molecular Diseases using a standard method for AA analysis.

Amino acids determination using capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection.

Free amino acids have been derivatized on-capillary with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and analyzed using a laboratory-made capillary electrophoresis apparatus with laser-induced fluorescence detection. Several parameters that control on-capillary derivatization of amino acids, including pH, mixing time, reaction time, concentration of the derivatization reagents (potassium cyanide and FQ) and solvent of FQ, as well as the temperature of mixing and reaction were optimized. Repeatabilities better than 1.8% for migration time and 7.8% for peak height were obtained. Assay detection limits for the different amino acids ranged from 23 nM for glycine to 50 nM for lysine and glutamic acid. The methods developed were applied to the analysis of several amino acids in pharmaceutical preparations and plasma samples. Results showed a good agreement with those obtained using an amino acid autoanalyzer for the same samples.

Development of a method for quantitative analysis of the major whey proteins by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection.

The main whey proteins have been derivatized on-capillary with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and analyzed using a laboratory-made capillary electrophoresis apparatus provided with a laser-induced fluorescence detector. Several parameters controlling on-capillary derivatization of proteins, including pH, mixing time, reaction time, concentration of the reagents (potassium cyanide and FQ), and reaction temperature, were optimized. Coefficient variations were lower than 1% for migration time and 7% for peak height. Assay detection limits for the different proteins were in the range 5 nM to 10 nM. The method developed was applied to the separation of the major whey proteins in a laboratory-made cheese whey and in an infant food formulated with milk. In addition, the beta-LG content of these samples was quantitated. The results showed good agreement with those given by an RP-HPLC method and with those reported in the literature.

Analysis of trace amounts of bovine beta-lactoglobulin in infant formulas by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection.

Bovine beta-lactoglobulin (betaLG) has been described by several authors as the main allergen present in cow's milk. It can induce allergic reactions even at the low concentration existing in hypoallergenic formulas based on hydrolyzed cow's milk proteins (generally lower than microM). In this paper, the usefulness of a capillary electrophoresis method with on-capillary derivatization and laser-induced fluorescence detection for the analysis of trace amounts of betaLG in a commercial hypoallergenic formula has been demonstrated. To confirm the identity of the peak of betaLG based on migration time, an immunorecognition step employing an anti-betaLG antibody was performed. BetaLG was quantitated in the whey and casein fractions of the hypoallergenic formula. The concentration of betaLG in the whey fraction of the formula was about 3 orders of magnitude lower than the average value present in cow's milk. In the casein fraction of the formula, the concentration of betaLG was about 1 order of magnitude lower than in the whey fraction. The method developed was also used for the quality control of three cereal-based infant foods formulated without milk to test the presence or absence of betaLG as an indicator of milk contamination during the fabrication process. BetaLG in a concentration of 10(-7) M or higher was not observed in any of the cereal-based infant formulas analyzed.