ABSTRACT
Sample preparation and laser-induced fluorescence detection are two key steps of the analytical methodology to determine by capillary electrophoresis low concentrations of proteins in complex sample matrices. In this chapter the options of performing both steps in different ways are shown by detailing the analysis of the allergen ß-lactoglobulin in food products for infants and the analysis of the isoforms of alpha 1-acid glycoprotein, a potential biomarker, in serum and secretome.
Subject(s)
Allergens/analysis , Infant Food/analysis , Lactoglobulins/analysis , Orosomucoid/metabolism , Allergens/chemistry , Chromatography, Affinity , Electrophoresis, Capillary/methods , Electrophoresis, Capillary/standards , Fluorescent Dyes/chemistry , Food Hypersensitivity/prevention & control , Furans/chemistry , Humans , Infant , Lactoglobulins/chemistry , Lasers , Orosomucoid/isolation & purification , Quinolines/chemistry , Reference Standards , Spectrometry, Fluorescence , Staining and LabelingABSTRACT
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.
Subject(s)
Bacterial Proteins/isolation & purification , Staphylococcus aureus/metabolism , Bacterial Proteins/chemistry , Electrophoresis, Capillary/methods , Fluorescent Dyes/chemistry , Furans/chemistry , Lasers , Peptide Mapping/methods , Quinolines/chemistry , Reproducibility of Results , Sonication , Spectrometry, Fluorescence/methods , Staining and Labeling/methods , Staphylococcus aureus/growth & development , Stress, PhysiologicalABSTRACT
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.
Subject(s)
Electrophoresis, Capillary/methods , Fluorescent Dyes/chemistry , Furans/chemistry , Lasers , Peptide Mapping/methods , Quinolines/chemistry , Staphylococcus/metabolism , Oxidative Stress , Spectrometry, Fluorescence , TemperatureABSTRACT
Dairy products can induce allergic reactions even when present at very low levels, such as levels found in involuntary contamination during food manufacturing. beta-Lactoglobulin (betaLG) is the main allergen in cow's milk. The objective of this work was to develop a sensitive method for betaLG detection in baby foods through the optimization of an innovative sample preparation method. Three types of baby foods deliberately contaminated with dairy products or dairy desserts were sterilized to simulate the potential contamination occurring during manufacturing and then used as samples. Different sample preparation methods were compared. The best results were provided by an extraction solution containing beta-mercaptoethanol, guanidine hydrochloride, and a saline solution. An ELISA method was optimized for the detection of betaLG (LOD = 9.7 x 10(-13) M). The developed method allowed detection of even 1 part of dairy product in 100,000 parts of baby food for some of the analyzed foods.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Infant Food/analysis , Lactoglobulins/analysis , Animals , Cattle , Humans , Infant , Milk/chemistryABSTRACT
Beta-lactoglobulin (betaLG) is the main allergenic protein in cow's milk and can cause allergy even when present at very low concentration. The aim of this work is to develop an innovative sample preparation method fully compatible with capillary electrophoresis and laser-induced fluorescence detection for improving the sensitivity when analyzing betaLG. Different types of baby food were on purpose contaminated with diverse dairy desserts and submitted to thermal treatment to simulate potential contamination at production. Sample preparation prior to CE analysis was performed by the classical extraction method and by the innovative one, and the results were compared. Analysis was performed by capillary electrophoresis with laser-induced fluorescence detection. The innovative method permitted to detect contaminations as low as 1 part of yoghurt in 10,000 parts of baby food.
