RESUMO
The protection of allergic consumers is crucial to the food industry. Therefore, accurate methods for the detection of food allergens are required. Targeted detection of selected molecules by MS combines high selectivity with accurate quantification. A confirmatory method based on LC/selected reaction monitoring (SRM)-MS/MS was established and validated for the quantification of milk traces in food. Tryptic peptides of the major milk proteins beta-lactoglobulin, beta-casein, alphaS2-casein, and K-casein were selected as quantitative markers. Precise quantification was achieved using internal standard peptides containing isotopically labeled amino acids. For each peptide, qualifier and quantifier fragments were selected according to Commission Decision 2002/657/EC. A simple sample preparation method was established without immunoaffinity or SPE enrichment steps for food matrixes containing different amounts of protein, such as baby food, breakfast cereals, infant formula, and cereals. Intermediate reproducibility, repeatability, accuracy, and measurement uncertainty were determined for each matrix. LOD values of 0.2-0.5 mg/kg, e.g., for beta-lactoglobulin, were comparable to those obtained with ELISA kits. An LOQ of approximately 5 mg/kg, expressed as mass fraction skim milk powder, was validated in protein-rich infant cereals. The obtained validation data show that the described LC/SRM-MS/MS approach can serve as a confirmatory method for the determination of milk traces in selected food matrixes.
Assuntos
Cromatografia Líquida/métodos , Análise de Alimentos/métodos , Proteínas do Leite/análise , Espectrometria de Massas em Tandem/métodos , Animais , Bovinos , Grão Comestível/química , Humanos , Lactente , Alimentos Infantis/análise , Reprodutibilidade dos Testes , Glycine maxRESUMO
The following study investigates the preparation of human blood plasma for metabolomic profiling analysis by ultrahigh performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC/TOFMS) in a novel two-step design study. Four different organic solvents (acetonitrile, acetone, methanol, and ethanol) were used to assess human blood plasma preparation via protein precipitation. The optimal conditions for sample preparation were investigated, with consideration to the number of extracted markers, data quality/reproducibility, and column lifetime prolongation. Isotopically labeled internal standards were used to monitor data quality/reproducibility. Gel electrophoresis was also used to measure the protein content in the supernatant of the "first design step" allowing assessment of the amount of protein that would be injected/accumulate onto the column after many injections that would be apparent in a global metabolic profiling study. The second design step followed on from the results obtained in step one, with four of the best conditions selected and further investigated, looking at the effects of vortex time and temperature on precipitation/extraction. Two choices of solvent compositions were found to be "optimal" for preparation of plasma for global metabolic profiling analysis; these were "methanol/ethanol" (1:1, v/v) and "methanol/acetonitrile/acetone" (1:1:1, v/v/v) added to plasma (4:1 ratio, 400 microL total volume).
Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Análise Química do Sangue/métodos , Metabolômica/métodos , Métodos Analíticos de Preparação de Amostras/normas , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/química , Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/metabolismo , Coleta de Amostras Sanguíneas , Precipitação Química , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , Análise de Componente Principal , Padrões de Referência , Reprodutibilidade dos Testes , Solventes/química , Coloração e Rotulagem , Temperatura , Fatores de TempoRESUMO
An isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine lysine (Lys), N(epsilon)-fructosyllysine (FL), N epsilon-carboxymethyllysine (CML), and pyrraline (Pyr) in dairy products. The presented approach entails protein cleavage via enzymatic digestion to liberate the aforementioned compounds, which were then quantified using a stable isotope dilution assay. LC-MS/MS analysis was performed by positive electrospray ionization recording two transition reactions per analyte in selected reaction monitoring mode. The CML and Lys values obtained with enzymatic digestion were compared to those acquired with acid hydrolysis HCl (6 mol/L), and the two proteolysis methods yielded comparable quantifications. Allowing for the fact that the investigated compounds are formed during different stages of the glycation process, the method is able to reveal the progress of protein glycation in dairy products.
Assuntos
Laticínios/análise , Produtos Finais de Glicação Avançada/análise , Lisina/análise , Boroidretos , Isótopos de Carbono/análise , Cromatografia Líquida , Indicadores e Reagentes , Lisina/análogos & derivados , Lisina/química , Espectrometria de MassasRESUMO
A common approach for the quantification of 3-nitrotyrosine (NY) in routine analyses relies on the cleavage of peptide bonds in order to release the free amino acids from proteins in tissues or fluids. NY is usually monitored by either GC-MS(/MS) or LC-MS/MS techniques. Various proteolysis methods have been employed to combine digestion efficiency with prevention of artifactual nitration of tyrosine. However, so far, no study was designed to compare the HCl-based hydrolysis method with enzymatic digestion in terms of reliability for the measurement of NY. The present work addresses the digestion efficiency of BSA using either 6M HCl, pronase E or a cocktail of enzymes (pepsin, pronase E, aminopeptidase, prolidase) developed in our laboratory. The HCl-based hydrolysis leads to a digestion yield of 95%, while 25 and 75% are achieved with pronase E and the cocktail of enzymes, respectively. These methods were compared in terms of NY measurement and the results indicate that a prior reduction of the disulfide bonds ensures a reliable quantification of NY. We additionally show that the enzyme efficacy is not altered when the digestion is carried out in the presence of BSA with a high content of NY.
Assuntos
Enzimas/metabolismo , Ácido Clorídrico/metabolismo , Soroalbumina Bovina/metabolismo , Tirosina/análogos & derivados , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Humanos , Hidrólise , Ratos , Fatores de Tempo , Tirosina/análiseRESUMO
The Maillard reaction is commonly encountered during food processing or storage, and also in human nutrition, hence there is a need for analytical methodologies to identify and characterize the modified proteins. This paper reports specific methods using mass spectrometric techniques to localize protein modifications induced by lactose and galactose on beta-lactoglobulin (beta-Lg) under solid-state glycation conditions. The extent of glycation was first determined by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The specific identification of lactose-modified amino acid residues was realized using both NanoESI-MS, NanoESI-MS/MS (neutral loss scanning modes) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) (with and without guanidination of lysine residues) on unfractionated digests. The results indicated that, after 8.25 h of incubation, the lysine residues were the main targets of lactose-induced modification. In addition to the 15 lysine residues, Leu1 (NH2 terminal) and the Arg124 were also found to be modified, thus leading to a total of 17 different modified amino acid residues (versus 15 found by LC/ESI-MS measurement). In a second set of experiments, different strategies consisting of constant neutral loss and precursor ion scanning were compared to characterize galactose-induced modifications. Owing to the high level of beta-Lg glycation, the combined use of these different strategies appeared to be necessary for determining the galactose-modified sites after 8.25 h of incubation. Thus, among the 22 galactose adducts deduced from the LC/ESI-MS measurement, apart from the N-terminal and classical lysine residues, we also observed a few arginine residues (Arg40, Arg124 and Arg148) that were modified, and also dialkylations on specific lysine residues (Lys47, Lys75).
Assuntos
Aminoácidos/análise , Galactose/metabolismo , Lactoglobulinas/metabolismo , Lactose/metabolismo , Espectrometria de Massas/métodos , Aminoácidos/química , Cromatografia Líquida , Glicosilação , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
An analytical method to quantify dityrosine (DiTyr) in milk powder samples is presented. The assay is based on isotope dilution liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (LC-ESIMS/MS). The sample preparation entails acid hydrolysis of milk proteins followed by a solid phase-extraction (SPE) step. Neither artifactual formation nor degradation of DiTyr were observed during the proteolysis step. Mass spectral detection was performed in the positive ion mode by recording five transition reactions for DiTyr, in order to unambiguously confirm the presence of DiTyr by correct ion ratios. Under the analytical conditions used, the limit of detection (LOD) and limit of quantification (LOQ) for DiTyr were estimated at ca. 2 and 6 micromol DiTyr per mol of Tyr (using ca. 500 microg of milk proteins), with a mean recovery of ca. 90%. Quantification was conduced in eight different commercial milk powder samples, and the level of DiTyr ranged from below the LOQ up to 393.0 +/- 9.1 micromol DiTyr per mol of Tyr.
Assuntos
Cromatografia Líquida/métodos , Leite/química , Pós/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Tirosina/análogos & derivados , Tirosina/análise , Animais , Hidrólise , Isótopos , Sensibilidade e EspecificidadeRESUMO
A confirmatory method based on isotope dilution liquid chromatography-electrospray ionization tandem mass spectrometry is described for the determination of the antibiotic chloramphenicol (CAP) in foods. The method is quantitative and entails liquid-liquid extraction followed by a clean-up step on a silica gel solid-phase extraction cartridge. Mass spectral acquisition is done in the negative ion mode applying multiple reaction monitoring of two diagnostic transition reactions for CAP (m/z 321 --> 257 and m/z 321--> 152). In addition, the presence of two chlorine atoms in the CAP molecule provides further analyte certainty by assessing the 37Cl/35Cl ratio using the transition reactions m/z 323 --> 257 and m/z 323 --> 152. Validation of the method in chicken meat is conducted according to the latest European Union criteria for the analysis of veterinary drug residues at levels of 0.05, 0.10, and 0.20 microg/kg, employing [2H5]-chloramphenicol as internal standard. The decision limit and the detection capability were calculated at 0.01 microg/kg and 0.02 microg/kg, respectively. At the lowest fortification level (i.e. 0.05 microg/kg), precision values below 14 and 17% were achieved under repeatability and within-laboratory reproducibility conditions, respectively. The accuracy of the method was within 20, 15, and 5% of the target values at the 0.05, 0.10, and 0.20 microg/kg fortification levels, respectively. The applicability of this procedure was demonstrated by the analysis of other meat (turkey, pork, beef) and seafood (fish, shrimps) products. The method is robust and suitable for routine quality control operations, and more than 200 sample injections were performed without excessive pollution of the mass spectrometer or loss of LC column performance.
Assuntos
Antibacterianos/análise , Cloranfenicol/análise , Cromatografia Líquida de Alta Pressão/métodos , Produtos Pesqueiros/análise , Produtos da Carne/análise , Alimentos Marinhos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Reprodutibilidade dos TestesRESUMO
A fully validated multiple-transition recording isotope dilution liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitative determination of N(epsilon)-carboxymethyllysine (CML) and lysine in dairy products is described. Internal standards were [N-1',2'-(13)C(2)]CML and [1,2,3,4,5,6-(13)C(6)-2,6-(15)N(2)]lysine, and the method was validated by evaluating the selectivity, linearity, precision (repeatability and reproducibility) and trueness, using both powder and liquid products. For liquid dairy products, the repeatability and reproducibility was 2.79% and 11.0%, while 4.85% and 4.92% were determined for powder dairy products, respectively. The trueness of the method ranged from -9.6% to -3.6% for powder and from -0.99% to 6.8% for liquid dairy products. The limit of detection for CML was estimated to be 8 ng CML per mg protein while the limit of quantification was 27 ng CML per mg protein. The method encompasses a proteolytic cleavage mediated by enzymatic digestion to reach a complete release of the amino acids prior to a sample cleanup based on solid phase extraction, and followed by LC-MS/MS analysis of CML and lysine residues. To ensure a suitable performance of the enzymatic digestion, CML measurements were compared to values obtained with an acid hydrolysis-mediated proteolysis. Finally, the method was employed for the analysis of CML in various dairy products. The values compare well to the data available in the literature when similar methods were used, even if some discrepancies were observed upon comparison with the results obtained by other techniques such as enzyme-linked immunosorbent assay and GC-MS.
Assuntos
Cromatografia Líquida/métodos , Produtos Finais de Glicação Avançada/análise , Lisina/análogos & derivados , Leite/química , Espectrometria de Massas em Tandem/métodos , Animais , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Hidrólise , Lisina/análise , Lisina/metabolismo , Leite/metabolismo , Peptídeo Hidrolases/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Extração em Fase Sólida/métodosRESUMO
Ochratoxin A (OTA) is a mycotoxin occurring in a variety of foods. OTA is nephrotoxic and nephrocarcinogenic in rodents. An OTA-mediated increase of the inducible nitric oxide synthase (iNOS) expression was observed in normal rat kidney renal cell line and in rat hepatocyte cultures, suggesting the induction of nitrosative stress. This was associated with an increased nuclear factor kappa-light chain enhancer of activated B cells activity. The potential consequences of iNOS induction were further investigated. A significant increase in the levels of protein nitrotyrosine residues was observed with OTA. In addition, OTA was found to increase the level of DNA abasic sites in both cell cultures system. This end point was used as an indirect measure of 8-nitroguanine formation. Treatment of the cells with L-N(6)-(1-iminoethyl) lysine, a specific inhibitor of iNOS activity, inhibited the OTA-mediated overnitration of proteins but did not reduce the level of DNA abasic sites. It was found previously that nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) activators were able to restore the cellular defense against oxidative stress and could prevent DNA abasic sites in cell cultures. In the present study, pretreatment of the cells with activators of Nrf2 prevented OTA-mediated increase in lipid peroxidation, confirming the potential of Nrf2 activators to confer protection against OTA-mediated oxidative stress. In addition, it was found that Nrf2 activators could also prevent OTA-induced protein nitration and cytotoxicity. In conclusion, the present data further confirm oxidative stress as a key source of OTA-induced DNA damage and provide additional evidence for a role of this mechanism in OTA carcinogenicity. The exact role of nitrosative stress still remains to be established.
Assuntos
Carcinógenos/toxicidade , Dano ao DNA , Ocratoxinas/toxicidade , Espécies Reativas de Nitrogênio/toxicidade , Espécies Reativas de Oxigênio/toxicidade , Aldeídos/farmacologia , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Inibidores de Cisteína Proteinase/farmacologia , Dano ao DNA/efeitos dos fármacos , Ensaio de Desvio de Mobilidade Eletroforética , Heme Oxigenase-1/biossíntese , Masculino , Subunidade p45 do Fator de Transcrição NF-E2/genética , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Advanced glycation endproducts (AGEs) containing carboxymethyllysine (CML) modifications are generally thought to be ligands of the receptor for AGEs, RAGEs. It has been argued that this results in the activation of pro-inflammatory pathways and diseases. However, it has not been shown conclusively that a CML-modified protein can interact directly with RAGE. Here, we have analyzed whether beta-lactoglobulin (bLG) or human serum albumin (HSA) modified chemically to contain only CML (10-40% lysine modification) can (i) interact with RAGE in vitro and (ii) interact with and activate RAGE in lung epithelial cells. Our results show that CML-modified bLG or HSA are unable to bind to RAGE in a cell-free assay system (Biacore). Furthermore, they are unable to activate pro-inflammatory signaling in the cellular system. Thus, CML probably does not form the necessary structure(s) to interact with RAGE and activate an inflammatory signaling cascade in RAGE-expressing cells.
Assuntos
Inflamação/etiologia , Lactoglobulinas/metabolismo , Lisina/análogos & derivados , Receptores Imunológicos/metabolismo , Albumina Sérica/metabolismo , Linhagem Celular , Células Epiteliais , Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/imunologia , Glutationa Transferase/metabolismo , Glicosilação , Glioxilatos/química , Humanos , Inflamação/genética , Interleucina-6/genética , Interleucina-8/genética , Lactoglobulinas/química , Lisina/química , RNA Mensageiro/análise , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Albumina Sérica/química , Fator de Necrose Tumoral alfa/genéticaRESUMO
During industrial treatments, milk proteins could be oxidatively modified, thus leading to the formation of modified/oxidised amino acid residues. The apparition of such modified residues may contribute to the formation of new immunologically reactive structures. Some of these adducts could, in an advanced stage, lead to cross-linked protein species whose proteolytic susceptibility would be drastically decreased. Such protein species, that are resistant to digestion, could also constitute major food allergens. Therefore, these oxidative protein modifications tend to increase the natural allergenicity of milk proteins. For these reasons, monitoring milk protein oxidative modifications could be very useful regarding both product quality and allergenicity issues. In the present paper, we highlight, using different analytical approaches, the preferential carbonylation of beta-lactoglobulin (beta-Lg) during industrial treatments of milk. This result is particularly interesting since native beta-Lg represents one of the major milk allergens.
Assuntos
Carbono/química , Proteínas do Leite/análise , Proteínas do Leite/química , Aldeídos/química , Alérgenos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Proteínas Alimentares , Manipulação de Alimentos , Hipersensibilidade Alimentar , Temperatura Alta , Imunoensaio , Lactoglobulinas/química , Espectrometria de Massas , Leite/química , Hipersensibilidade a Leite/diagnóstico , Dados de Sequência Molecular , Oxigênio/química , Proteínas/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria , Raios UltravioletaRESUMO
Infant formula powders were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) to assess the whey proteins quality, which may be altered by the heat treatment used during the processing conditions. Lactosylation was found to be the major chemical modification occurring in whey proteins. In parallel, a two-dimensional (2-D) gel electrophoresis was performed on the milk sample and the entire protein patterns were analyzed by nano-ESI-MS after cutting the different gel spots and in-gel trypsin digestion. A highly selective and specific tandem MS technique has been developed to characterize and localize up to ten lactosylation sites in beta-lactoglobulin (beta-Lg) and alpha(S2)-casein. alpha-Lactalbumin (alpha-La), with five lactosylated peptides, was found to be an interesting protein marker in the milk powder sample to detect chemical modification induced by the processing/storage conditions.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Alimentos Infantis/análise , Lactose/química , Proteínas do Leite/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Caseínas/química , Humanos , Lactente , Lactalbumina/química , Lactoglobulinas/química , Nanotecnologia , Pós , Proteínas do Soro do LeiteRESUMO
Glycation of beta-lactoglobulin (beta-Lg) with either lactose or galactose in a solid-state medium was monitored using gel electrophoresis techniques and liquid chromatography coupled to electrospray ionisation mass spectrometry (LC/ESI-MS). The kinetics of glycation monitored by SDS polyacrylamide gel electrophoresis showed a molecular weight increase over time of the beta-Lg bands for both sugars, but no significant amounts of aggregated proteins were observed. The isoelectric point of the protein, observed by isoelectric focusing gel electrophoresis, was dramatically affected by galactosylation. LC/MS measurements of beta-Lg variants A and B, over the whole glycation reaction time, showed a larger extent of glycation with galactose (from 4 up to 22 adducts) as compared with lactose (from 0 up to 14 adducts), and confirmed that early Maillard reaction products were the main species observed. Based on the relative abundances obtained from the deconvoluted mass spectra after a 8 h 15 min incubation time at 60 degrees C, the mean values of lactose and galactose molecules bound to the protein species were calculated to be 10.4 and 17.9, and 10.5 and 18.6, for variants A and B, respectively. Furthermore, the charge state distribution data obtained by ESI-MS was studied using different methanol percentages, and indicated that adduct formation with lactose, but more significantly galactose, tends to improve the stability properties of the native protein towards denaturation.