Proteometabolomic Analysis Reveals Molecular Features Associated with Grain Size and Antioxidant Properties amongst Chickpea (Cicer arietinum L.) Seeds Genotypes.

Legumes are an essential source of nutrients that complement energy and protein requirements in the human diet. They also contribute to the intake of bioactive compounds such as polyphenols, whose content can vary depending on cultivars and genotypes. We conducted a comparative proteomics and metabolomics study to determine if there were significant variations in relevant nutraceutical compounds in the five genotypes of Kabuli-type chickpea grains. We performed an isobaric tandem mass tag (TMT) couple to synchronous precursor selection (SPS)-MS3 method along with a targeted and untargeted metabolomics approach based on accurate mass spectrometry. We observed an association between the overproduction of proteins involved in starch, lipid, and amino acid metabolism with gibberellin accumulation in large grains. In contrast, we visualized the over-accumulation of proteins associated with water deprivation in small grains. It was possible to visualize in small grains the over-accumulation of some phenolics such as vanillin, salicylic acid, protocatechuic acid, 4-coumaric acid, 4-hydroxybenzoic acid, vanillic acid, ferulic acid, and kaempferol 3-O-glucoside as well as the amino acid l-phenylalanine. The activated phenolic pathway was associated with the higher antioxidant capacity of small grains. Small grains consumption could be advantageous due to their nutraceutical properties.

Biochemical diagnosis of aromatic-L-amino acid decarboxylase deficiency (AADCD) by assay of AADC activity in plasma using liquid chromatography/tandem mass spectrometry.

Aromatic l-amino acid decarboxylase (AADC, EC 4.1.1.28) deficiency is a rare genetic disorder characterized by developmental delay, oculogyric crises, autonomic dysfunction and other problems, caused by biallelic mutations in the DDC gene leading to deficient activity of aromatic l-amino acid decarboxylase, an enzyme involved in the formation of important neurotransmitters, such as dopamine and serotonin. A clinical development program of gene therapy for AADC deficiency is ongoing. An important step for the success of this therapy is the early and precise identification of the affected individuals, but it has been estimated that around 90% of the cases remain undiagnosed. The availability measurement of the AADC activity is mandatory for an accurate biochemical diagnosis. Based on these statements, our objectives were to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method suitable for the determination of the AADC activity, and to evaluate its capacity to confirm the deficiency of AADC in potential patients in Brazil. The AADC activities were measured in plasma samples of seven AADC deficient patients and 35 healthy controls, after enzymatic reaction and LC-MS/MS analysis of dopamine, the main reaction product. The results obtained showed clear discrimination between confirmed AADC deficient patients and healthy controls. The method presented here could be incorporated in the IEM laboratories for confirmation of the diagnosis of when a suspicion of AADC deficiency is present due to clinical signs and/or abnormal biomarkers, including when an increased level of 3-O-methyldopa (3-OMD) is found in dried blood spots (DBS) samples from high-risk patients or from newborn screening programs.

Modern Approaches in the Identification and Quantification of Immunogenic Peptides in Cereals by LC-MS/MS.

Celiac disease (CD) is an immunogenic disorder that affects the small intestine. It is caused by the ingestion of gluten, a protein network formed by prolamins and glutelins from cereals such as wheat, barley, rye and, possibly, oats. For predisposed people, gluten presents epitopes able to stimulate T-cells causing symptoms like nausea, vomiting, diarrhea, among others unrelated to the gastrointestinal system. The only treatment for CD is to maintain a gluten-free diet, not exceeding 20 mg/kg of gluten, what is generally considered the safe amount for celiacs. Due to this context, it is very important to identify and quantify the gluten content of food products. ELISA is the most commonly used method to detect gluten traces in food. However, by detecting only prolamins, the results of ELISA tests may be underestimated. For this reason, more reliable and sensitive assays are needed to improve gluten quantification. Because of high sensitivity and the ability to detect even trace amounts of peptides in complex matrices, the most promising approaches to verify the presence of gluten peptides in food are non-immunological techniques, like liquid chromatography coupled to mass spectrometry. Different methodologies using this approach have been developed and described in the last years, ranging from non-targeted and exploratory analysis to targeted and specific methods depending on the purpose of interest. Non-targeted analyses aim to define the proteomic profile of the sample, while targeted analyses allow the search for specific peptides, making it possible to quantify them. This review aims to gather and summarize the main proteomic techniques used in the identification and quantitation of gluten peptides related to CD-activity and gluten-related allergies.

Electrospray ionization tandem mass spectrometry of labdane-type acid diterpenes.

Copaifera (Leguminoseae) species produce a commercially interesting oleoresin that displays several biological activities, including antimicrobial and anti-inflammatory properties. Labdane-type diterpenes are the main chemical constituents of these oleoresins, and copalic acid is the only compound that has been detected in all Copaifera oleoresins. In this study, we investigate some aspects of the gas-phase fragmentation reactions involved in the formation of the product ions from the deprotonated compounds (-)-ent-copalic acid (1), (-)-ent-3ß-hydroxy-copalic acid (2), (-)-ent-3ß-acetoxy-copalic acid (3), and (-)-ent-agathic acid (4) by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and multiple stage mass spectrometry (MSn ). Our results reveal that the product ion with m/z 99 is common to all the analyzed compounds, whereas the product ion with m/z 217 is diagnostic for compounds 2 and 3. Moreover, only compound 4 undergoes CO2 (44 u) and acetic acid (60 u) elimination from the precursor ion. Thermochemical data obtained by computational chemistry at the B3LYP/6-31G(d) level of theory support the proposed ion structures. These data helped us to identify these compounds in a crude commercial Copaifera langsdorffii oleoresin by selective multiple reaction monitoring (MRM). Finally, a precursor ion scan (PIS) strategy aided screening of labdane-type acid diterpenes other than 1 to 4 in the same Copaifera oleoresin sample and led us to propose the structures of 8,17-dihydro-ent-agathic acid (5) and 3-keto-ent-copalic acid (6), which have not been previously reported in Copaifera oleoresins.

Fingerprint and multi-ingredient quantitative analyses for quality evaluation of hawthorn leaves and Guang hawthorn leaves by UPLC-MS

ABSTRACT This paper aimed to evaluate the quality of hawthorn leaves and Guang hawthorn leaves by an UPLC-MS method from two aspects, fingerprint analysis and multi-ingredient quantification. Chromatographic separation was carried out on an UPLC system, the standardized characteristic fingerprints was established by Similarity Evaluation System for chromatographic fingerprinting of traditional Chinese medicine and cluster analysis. Eight components were simultaneously determined by mass spectrometry in multiple reaction-monitoring mode. The method was validated in terms of linearity (R2 > 0.9971), intraday and interday precision (RSD < 2.0%), repeatability (RSD < 2.3%), stability (RSD < 2.5%) and recovery (96.2-103.8%). The developed method was successfully applied to the quality evaluation between hawthorn leaves and Guang hawthorn leaves, and there were differences in the component and the content, hawthorn leaves and Guang hawthorn leaves cannot substitute each other in clinical medication.

Versatility of tandem mass spectrometry for focused analysis of oxylipids.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring (MRM) scan mode has been the primary MS method applied for the target identification of specific and minor oxylipids in complex matrices, such as eicosanoids and docosanoids, which are potent lipid mediators derived from polyunsaturated fatty acid oxygenation. However, the high specificity of MRM can limit the detection of species with m/z MRM transitions not covered by the method. In addition to MRM, tandem-quadrupole mass analyzers enable other experiments to be conducted, by fragmenting ions via collision-induced dissociation process (CID). This paper presents the potential of tandem mass spectrometry for the focused analysis of oxylipids. We have successfully developed an LC-MS/MS method for the identification of precursor ions of m/z 115, a diagnostic product ion of 5-hydroxy- and 5-epoxy-fatty acids. As a proof of concept, the developed method was used to discover several oxylipids oxidized at C5 derived from arachidonic acid (C20 : 4) oxygenation in a hypothalamus rat extract that were not identified using the target MRM methodology. The proposed focused MS/MS-based approach in a tandem mass analyzer has proven to be a powerful strategy to accelerate the identification of oxylipids with structural similarities and assist the field of lipidomic research.