RESUMO
Bacillus are aerobic spore-forming bacteria that are known to lead to specific diseases, such as anthrax and food poisoning. This study focuses on the characterization of these bacteria by the detection of lipids extracted from 33 well-characterized strains from the Bacillus and Brevibacillus genera, with the aim to discriminate between the different species. For the purpose of analysing the lipids extracted from these bacterial samples, two rapid physicochemical techniques were used: matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography in conjunction with mass spectrometry (LC-MS). The findings of this investigation confirmed that MALDI-TOF-MS could be used to identify different bacterial lipids and, in combination with appropriate chemometrics, allowed for the discrimination between these different bacterial species, which was supported by LC-MS. The average correct classification rates for the seven species of bacteria were 62.23 and 77.03 % based on MALDI-TOF-MS and LC-MS data, respectively. The Procrustes distance for the two datasets was 0.0699, indicating that the results from the two techniques were very similar. In addition, we also compared these bacterial lipid MALDI-TOF-MS profiles to protein profiles also collected by MALDI-TOF-MS on the same bacteria (Procrustes distance, 0.1006). The level of discrimination between lipids and proteins was equivalent, and this further indicated the potential of MALDI-TOF-MS analysis as a rapid, robust and reliable method for the classification of bacteria based on different bacterial chemical components. Graphical abstract MALDI-MS has been successfully developed for the characterization of bacteria at the subspecies level using lipids and benchmarked against HPLC.
Assuntos
Bacillus/classificação , Técnicas de Tipagem Bacteriana , Brevibacillus/classificação , Lipídeos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Bacillus/química , Bacillus/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/isolamento & purificação , Brevibacillus/química , Brevibacillus/metabolismo , Cromatografia Líquida , Conjuntos de Dados como Assunto , Lipídeos/classificação , FilogeniaRESUMO
The present research is focused on the evaluation of a recently developed high-resolution time-of-flight mass spectrometer (HR TOF MS), under the challenging conditions of a flow-modulation comprehensive two-dimensional gas chromatography (FM GC × GC) experiment. The HR TOF MS instrument was operated at a spectral generation frequency of 30 Hz and a mass resolution of ≥25,000 (fwhm). The effluent exiting the second-dimension column was in the range 6-8 mL/min, with part directed to waste to avoid exceeding the maximum pumping capacity of the MS system. An FM GC × GC-HR TOF MS method was developed for the untargeted and targeted analysis of a sample of high complexity, namely, heavy gas oil. With regard to the untargeted results, these were satisfactory in relation to MS database searching and mass accuracies. Considering the targeted data, the high selectivity of the MS system was highlighted by the use of accurate mass extracted-ion-chromatograms with narrow mass windows (±5 and ±1 ppm), for specific classes of polyaromatic sulfur heterocycles (PASHs), namely, benzothiophenes and dibenzothiophenes. Finally, the instrumental performance was also evaluated through the injection of standard solutions of four classes of PASHs.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Compostos Heterocíclicos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Compostos de Enxofre/análiseRESUMO
The main focus of the present research is the analysis of the unsaponifiable lipid fraction of human plasma by using data derived from comprehensive two-dimensional gas chromatography with dual quadrupole mass spectrometry and flame ionization detection. This approach enabled us to attain both mass spectral information and analyte percentage data. Furthermore, gas chromatography coupled with high-resolution time-of-flight mass spectrometry was used to increase the reliability of identification of several unsaponifiable lipid constituents. The synergism between both the high-resolution gas chromatography and mass spectrometry processes enabled us to attain a more in-depth knowledge of the unsaponifiable fraction of human plasma. Additionally, information was attained on the fatty acid and triacylglycerol composition of the plasma samples, subjected to investigation by using comprehensive two-dimensional gas chromatography with dual quadrupole mass spectrometry and flame ionization detection and high-performance liquid chromatography with atmospheric pressure chemical ionization quadrupole mass spectrometry, respectively.
Assuntos
Cromatografia Gasosa/métodos , Ácidos Graxos/sangue , Cromatografia Gasosa-Espectrometria de Massas/métodos , Triglicerídeos/sangue , HumanosRESUMO
A chemical characterization of major lipid components, namely, triacylglycerols, fatty acids and the unsaponifiable fraction, in a Quinoa seed lipids sample is reported. To tackle such a task, non-aqueous reversed-phase high-performance liquid chromatography with mass spectrometry detection was employed. The latter was interfaced with atmospheric pressure chemical ionization for the analysis of triacylglycerols. The main triacylglycerols (>10%) were represented by OLP, OOL and OLL (P = palmitoyl, O = oleoyl, L = linoleoyl); the latter was present in the oil sample at the highest percentage (18.1%). Furthermore, fatty acid methyl esters were evaluated by gas chromatography with flame ionization detection. 89% of the total fatty acids was represented by unsaturated fatty acid methyl esters with the greatest percentage represented by linoleic and oleic acids accounting for approximately 48 and 28%, respectively. An extensive characterization of the unsaponifiable fraction of Quinoa seed lipids was performed for the first time, by using comprehensive two-dimensional gas chromatography with dual mass spectrometry/flame ionization detection. Overall, 66 compounds of the unsaponifiable fraction were tentatively identified, many constituents of which (particularly sterols) were confirmed by using gas chromatography with high-resolution time-of-flight mass spectrometry.
RESUMO
Inflammatory Bowel Disease (IBD) comprises a heterogeneous group of chronic inflammatory conditions of the gastrointestinal tract that include ulcerative colitis (UC) and Crohn's disease. Although the etiology is not well understood, IBD is characterized by a loss of the normal epithelium homeostasis that disrupts the intestinal barrier of these patients. Previous work by our group demonstrated that epithelial homeostasis along the colonic crypts involves a tight regulation of lipid profiles. To evaluate whether lipidomic profiles conveyed the functional alterations observed in the colonic epithelium of IBD, we performed matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) analyses of endoscopic biopsies from inflamed and non-inflamed segments obtained from UC patients. Our results indicated that lipid profiling of epithelial cells discriminated between healthy and UC patients. We also demonstrated that epithelial cells of the inflamed mucosa were characterized by a decrease in mono- and di-unsaturated fatty acid-containing phospholipids and higher levels of arachidonic acid-containing species, suggesting an alteration of the lipid gradients occurring concomitantly to the epithelial differentiation. This result was reinforced by the immunofluorescence analysis of EPHB2 and HPGD, markers of epithelial cell differentiation, sustaining that altered lipid profiles were at least partially due to a faulty differentiation process. Overall, our results showed that lipid profiling by MALDI-MSI faithfully conveys molecular and functional alterations associated with the inflamed epithelium, providing the foundation for a novel molecular characterization of UC patients.
Assuntos
Diferenciação Celular , Colo , Humanos , Colo/metabolismo , Colo/patologia , Masculino , Feminino , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Pessoa de Meia-Idade , Adulto , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Lipidômica/métodos , Enterócitos/metabolismo , Enterócitos/patologia , Metabolismo dos Lipídeos , Inflamação/metabolismo , Inflamação/patologia , Lipídeos/análise , Células Epiteliais/metabolismo , Células Epiteliais/patologiaRESUMO
The present investigation is focused on the development of a comprehensive two-dimensional GC (GC × GC) method, with dual MS/FID detection, for the qualitative and quantitative analysis of the entire unsaponifiable fraction of vegetable oils. The unsaponifiable fraction forms a minor, highly specific part of a vegetable oil, and can be used as an indicator of genuineness. The column set used consisted of a low-polarity first dimension, and a medium-polarity secondary one, both characterized by a high thermal stability. The use of dual detection enabled the attainment of both mass spectral information and relative % FID data. The complexity of the fingerprint, generated by the unsaponifiable fraction, fully justified the employment of the two-dimensional GC technology. Furthermore, two other GC × GC benefits contributed greatly to the attainment of promising results, namely sensitivity enhancement and the formation of group-type patterns. The method herein proposed could potentially open a new opportunity for the more in-depth knowledge of the unsaponifiable fraction of vegetable oils.
Assuntos
Misturas Complexas/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Fitosteróis/análise , Óleos de Plantas/química , Saponinas , Sensibilidade e EspecificidadeRESUMO
The fatty acids (FAs) composition of lipids extracted from Rhodobacter sphaeroides 2.4.1 was investigated by gas chromatography-mass spectrometry (GC-MS) analysis of the corresponding FA methyl esters (FAMEs), obtained through trans-esterification of the original lipid species. A GC stationary phase based on a highly polar ionic liquid (IL) was selected, aimed to enhance the separation of isomeric FAMEs with particular emphasis on positional and geometrical isomers of monounsaturated 16:1 and 18:1 fatty acyl chains. The occurrence of 18:1 cis-Δ(9) (oleic) acid, a positional isomer of the well-known and most predominant 18:1 cis-Δ(11) (cis-vaccenic) acid, has been demonstrated here for the first time. Furthermore a methyl branched 18:1 FA was also identified and its structure tentatively assigned as 11-methyl-Δ(12)-octadecenoic acid (most likely as trans isomer). The unprecedented observation about 18:1 cis-Δ(9) FA occurrence in R. sphaeroides 2.4.1 is, even indirectly, supported by a biosynthetic pathway postulated with the aid of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The concurrent presence of 16:1 cis-Δ(7) and 18:1 cis-Δ(9) FAs suggested the existence of parallel and/or complementary processes to those invoked for the formation of most common 16:1 cis-Δ(9) and 18:1 cis-Δ(11) FAs. A further route was hypothesized for the trans FAs biosynthesis in wild-type cells of R. sphaeroides.
Assuntos
Ácidos Graxos/análise , Rhodobacter sphaeroides/química , Cromatografia Gasosa-Espectrometria de Massas , Líquidos Iônicos/química , Metilação , Ácido Oleico/análiseRESUMO
The present investigation is focused on the use of a comprehensive two-dimensional GC (GC×GC) method, with dual mass spectrometry/flame ionization detection (MS/FID), for the qualitative and quantitative analysis of the unsaponifiable fraction of milk lipids (cow butter, buffalo, ewe, and goat milks). The structure of many constituents (particularly sterols) was confirmed by using GC-high resolution time-of-flight MS. The GC×GC column set used consisted of a low-polarity first dimension, and a medium-polarity secondary one, both characterized by a high thermal stability. The use of dual detection enabled the attainment of both mass spectral information and relative % FID data. The complexity of the fingerprint, generated by the unsaponifiable fraction, justified the employment of the two-dimensional GC technology. However, it was two other GC×GC characteristics that contributed most to the attainment of promising results, namely sensitivity enhancement and the formation of group-type patterns. Because many milk lipid constituents were not contained in the MS databases employed, exact mass information proved to be valuable for identification purposes.