Literatura científica selecionada sobre o tema "Semi-targeted profiling"

Abstract Background: Fish oils, which are rich in health-promoting polyunsaturated fatty acids (PUFA), have emerged as promising functional foods in the global health and wellness food market. Their source regarding the fish type, season, and location of harvesting might influence the nutritional value of such bioactive oils and determine their market price. The differences in price among such oils often lead to economically motivated mislabeling and adulteration. Objective: In this study, our objective was to demonstrate how a qualitative targeted shotgun lipid profile workflow using an electrospray ionization–quadrupole-linear ion trap MS (QTrap) could differentiate fish oils originating from two different species. Methods: Five samples each of sardine (Sardinella longiceps) oil and shark (Echinorhinus brucus) liver oil were diluted to a concentration of 80 µg/mL in chloroform–methanol (1 + 2, v/v) with 5 mM ammonium acetate. These samples were directly infused into a QTrap MS. The data were acquired for 23 precursor ion and 4 neutral loss scan experiments in the positive ionization mode and compared. Results: We identified the following major lipid classes: cholesteryl ester, diacyl glycerol, triacylglycerol, monoalkyldiacylglycerol, and phophatydyl choline. The relative peak areas of the identified lipid species, when subjected to supervised multivariate analysis, could effectively distinguish the sardine oil and shark liver oil. Conclusions: The approach will be useful in establishing authenticity of fish oil and to support the regulatory agencies in dispute resolution. It can also be extended to establish authenticity in other agricultural and food commodities. Highlights: This paper reports a proof of concept for authenticating PUFA-rich fish supplements. A shotgun targeted lipidomics profile and chemometrics modeling successfully discriminated sardine oil and shark liver oil.

The ascomycete Erysiphe necator is a serious pathogen in viticulture. Despite the fact that some grapevine genotypes exhibit mono-locus or pyramided resistance to this fungus, the lipidomics basis of these genotypes’ defense mechanisms remains unknown. Lipid molecules have critical functions in plant defenses, acting as structural barriers in the cell wall that limit pathogen access or as signaling molecules after stress responses that may regulate innate plant immunity. To unravel and better understand their involvement in plant defense, we used a novel approach of ultra-high performance liquid chromatography (UHPLC)-MS/MS to study how E. necator infection changes the lipid profile of genotypes with different sources of resistance, including BC4 (Run1), “Kishmish vatkhana” (Ren1), F26P92 (Ren3; Ren9), and “Teroldego” (a susceptible genotype), at 0, 24, and 48 hpi. The lipidome alterations were most visible at 24 hpi for BC4 and F26P92, and at 48 hpi for “Kishmish vatkhana”. Among the most abundant lipids in grapevine leaves were the extra-plastidial lipids: glycerophosphocholine (PCs), glycerophosphoethanolamine (PEs) and the signaling lipids: glycerophosphates (Pas) and glycerophosphoinositols (PIs), followed by the plastid lipids: glycerophosphoglycerols (PGs), monogalactosyldiacylglycerols (MGDGs), and digalactosyldiacylglycerols (DGDGs) and, in lower amounts lyso-glycerophosphocholines (LPCs), lyso-glycerophosphoglycerols (LPGs), lyso-glycerophosphoinositols (LPIs), and lyso-glycerophosphoethanolamine (LPEs). Furthermore, the three resistant genotypes had the most prevalent down-accumulated lipid classes, while the susceptible genotype had the most prevalent up-accumulated lipid classes.

Using a semi-targeted approach, we have investigated the effect of acetaminophen on circulating bile acid profiles in rats, including many known bile acids and potential isomeric structures, as well as glucuronide and sulfate conjugates. The chromatographic separation was based on an optimized reverse-phase method exhibiting excellent resolution for a complex mix of bile acids using a solid-core C18 column, coupled to a high-resolution quadrupole time-of-flight system. The semi-targeted workflow consisted of first assigning all peaks detectable in samples from 46 known bile acids contained in a standard mix, as well as additional peaks for other bile acid isomers. The presence of glucuronide and sulfate conjugates was also examined based on their elemental formulae and detectable peaks with matching exact masses were added to the list of features for statistical analysis. In this study, rats were administered acetaminophen at four different doses, from 75 to 600 mg/kg, with the highest dose being a good model of drug-induced liver injury. Statistically significant changes were found by comparing bile acid profiles between dosing levels. Some tentatively assigned conjugates were further elucidated using in vitro metabolism incubations with rat liver fractions and standard bile acids. Overall, 13 identified bile acids, 23 tentatively assigned bile acid isomers, and 9 sulfate conjugates were found to increase significantly at the highest acetaminophen dose, and thus could be linked to drug-induced liver injury.

Background: Kidney transplantation is the therapy of choice for patients with advanced chronic kidney disease; however, predicting graft outcomes remains a significant challenge. Early identification of reliable biomarkers could enhance post-transplant management and improve long-term outcomes. This study aimed to identify metabolomic biomarkers within the first week after kidney transplantation that predict renal function at six months. Methods: We conducted a prospective study involving 50 adult patients who received deceased donor kidney transplants. Plasma samples collected one week after transplant were analyzed using liquid chromatography–mass spectrometry in a semi-targeted metabolomic approach. A Partial Least Squares-Discriminant Analysis (PLS-DA) model identified metabolites associated with serum creatinine > 1.5 mg/dL at six months. Metabolites were selected based on a Variable Importance in Projection (VIP) score > 1.5, which was used to optimize model performance. Results: The PLS-DA model demonstrated strong predictive performance with an area under the curve (AUC) of 0.958. The metabolites negatively associated with serum creatinine > 1.5 mg/dL were 3-methylindole, guaiacol, histidine, 3-indolepropionic acid, and α-lipoic acid. Conversely, the metabolites positively associated with worse kidney graft outcomes included homocarnosine, 5-methylcytosine, xanthosine, choline, phenylalanine, kynurenic acid, and L-kynurenine. Conclusions: Early metabolomic profiling after transplantation shows promise in predicting renal function. Identifying metabolites with antioxidant and anti-inflammatory properties, as well as those that are harmful and could be targeted therapeutically, underscores their potential clinical significance. The link between several metabolites and the tryptophan pathway suggests that further specific evaluation of this pathway is warranted. These biomarkers can enhance patient management and graft survival.

The diversity and activity of sulfate-reducing bacteria (SRB) in the camel gut remains largely unexplored. An abundant SRB community has been previously revealed in the feces of Bactrian camels (Camelus bactrianus). This study aims to combine the 16S rRNA gene profiling, sulfate reduction rate (SRR) measurement with a radioactive tracer, and targeted cultivation to shed light on SRB activity in the camel gut. Fresh feces of 55 domestic Bactrian camels grazing freely on semi-arid mountain pastures in the Kosh-Agach district of the Russian Altai area were analyzed. Feces were sampled in early winter at an ambient temperature of −15 °C, which prevented possible contamination. SRR values measured with a radioactive tracer in feces were relatively high and ranged from 0.018 to 0.168 nmol S cm−3 day−1. The 16S rRNA gene profiles revealed the presence of Gram-negative Desulfovibrionaceae and spore-forming Desulfotomaculaceae. Targeted isolation allowed us to obtain four pure culture isolates belonging to Desulfovibrio and Desulforamulus. An active SRB community may affect the iron and copper availability in the camel intestine due to metal ions precipitation in the form of sparingly soluble sulfides. The copper-iron sulfide, chalcopyrite (CuFeS2), was detected by X-ray diffraction in 36 out of 55 analyzed camel feces. In semi-arid areas, gypsum, like other evaporite sulfates, can be used as a solid-phase electron acceptor for sulfate reduction in the camel gastrointestinal tract.

Optic nerve head (ONH) and retina (RET) are the main sites of damage in neurodegenerative optic neuropathies including glaucoma. Up to date, little is known about the molecular interplay between these two adjoining ocular components in terms of proteomics. To close this gap, we investigated ONH and RET protein extracts derived from porcine eyes (n = 12) (Sus scrofa domestica Linnaeus 1758) using semi-quantitative mass spectrometry (MS)-based proteomics comprising bottom-up LC–ESI MS/MS and targeted SPE-MALDI-TOF MS analysis. In summary, more than 1600 proteins could be identified from the ONH/RET tissue complex. Moreover, ONH and RET displayed tissue-specific characteristics regarding their qualitative and semi-quantitative protein compositions. Gene ontology (GO)-based functional and protein–protein interaction analyses supported a close functional connection between the metabolic-related RET and the structural-associated ONH subproteomes, which could be affected under disease conditions. Inferred from the MS findings, stress-associated proteins including clusterin, ceruloplasmin, and endoplasmin can be proposed as extracellular mediators of the ONH/ RET proteome interface. In conclusion, ONH and RET show obvious proteomic differences reflecting characteristic functional features which have to be considered for future protein biomarker profiling studies.

Advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs), particularly carboxymethyl-lysine (CML), have been largely proposed as factors involved in the establishment and progression of heart failure (HF). Despite this evidence, the current literature lacks the comprehensive identification and characterization of the plasma AGEs/ALEs involved in HF (untargeted approach). This work provides the first ex vivo high-resolution mass spectrometry (HR-MS) profiling of AGEs/ALEs occurring in human serum albumin (HSA), the most abundant protein in plasma, characterized by several nucleophilic sites and thus representing the main protein substrate for AGE/ALE formation. A set of AGE/ALE adducts in pooled HF-HSA samples was defined, and a semi-quantitative analysis was carried out in order to finally select those presenting in increased amounts in the HF samples with respect to the control condition. These adducts were statistically confirmed by monitoring their content in individual HF samples by applying a targeted approach. Selected AGEs/ALEs proved to be mostly CML derivatives on Lys residues (i.e., CML-Lys12, CML-Lys378, CML-Lys402), and one deoxy-fructosyl derivative on the Lys 389 (DFK-Lys 389). The nature of CML adducts was finally confirmed using immunological methods and in vitro production of such adducts further confirmed by mass spectrometry.

Metabolite profiling of blood plasma, by proton nuclear magnetic resonance (1H-NMR) spectroscopy, offers great potential for early cancer diagnosis and unraveling disruptions in cancer metabolism. Despite the essential attempts to standardize pre-analytical and external conditions, such as pH or temperature, the donor-intrinsic plasma protein concentration is highly overlooked. However, this is of utmost importance, since several metabolites bind to these proteins, resulting in an underestimation of signal intensities. This paper describes a novel 1H-NMR approach to avoid metabolite binding by adding 4 mM trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP) as a strong binding competitor. In addition, it is demonstrated, for the first time, that maleic acid is a reliable internal standard to quantify the human plasma metabolites without the need for protein precipitation. Metabolite spiking is further used to identify the peaks of 62 plasma metabolites and to divide the 1H-NMR spectrum into 237 well-defined integration regions, representing these 62 metabolites. A supervised multivariate classification model, trained using the intensities of these integration regions (areas under the peaks), was able to differentiate between lung cancer patients and healthy controls in a large patient cohort (n = 160), with a specificity, sensitivity, and area under the curve of 93%, 85%, and 0.95, respectively. The robustness of the classification model is shown by validation in an independent patient cohort (n = 72).

Les globules rouges suscitent un intéret clinique particulier en raison de leur importance biologique. Il a été récemment démontré que certaines pathologies érythrocytaires pouvaient être associées à une composition lipidique anormale. Parmi ces pathologies figure la maladie de Gaucher, un trouble lysosomal rare causé par une déficience en β-glucocérébrosidase, enzyme dont l'absence entraîne une accumulation excessive de certains sphingolipides dans l'organisme. Cette surcharge lipidique altère les propriétés morphologiques et rhéologiques des globules rouges, provoquant des anomalies hématologiques et vasculaires.Dans cette thèse, nous avons étudié le lien entre la composition lipidique des globules rouges et son implication dans la maladie de Gaucher. En particulier, nous nous sommes concentrés sur deux catégories de lipides : les sphingolipides et les phospholipides, qui jouent un rôle clé dans les altérations érythrocytaires observées. Deux approches complémentaires ont été utilisées : la première utilise la spectrométrie de masse dans le but d'identifier de nouveaux biomarqueurs. La seconde exploite des techniques d'imagerie infrarouge à différentes échelles pour étudier la répartition des lipides dans les globules rouges.Nous avons mis au point une méthode rapide de préparation d'échantillons en ligne avec la chromatographie liquide couplée à la spectrométrie de masse (SPE-HPLC-MS/MS), pour doser 30 sphingolipides et phospholipides en moins de 15 min. Par la suite cette méthode a été appliquée pour quantifier les 30 espèces lipidiques dans le plasma et le culot globulaire de sujets contrôles et de patients Gaucher. Parmi ces espèces lipidiques, certaines étaient surexprimées et n'étaient pas associées à la maladie de Gaucher, ouvrant de nouvelles perspectives pour le diagnostic et le suivi thérapeutique.A l'issue de ce dosage nous nous sommes intéressés à l'exploration du lipidome du globule rouge sain pour pouvoir le comparer à celui du globule rouge Gaucher. Pour cela une approche semi-ciblée a été menée en utilisant un analyseur triple quadripolaire. Ceci nous a permis d'identifier plus de 266 espèces moléculaires réparties sur 12 classes lipidiques différentes.Ces résultats ont contribué à une meilleure connaissance du lipidome du globule rouge sain et sont prometteurs pour l'identification de nouveaux biomarqueurs et la compréhension de diverses pathologies affectant le lipidome érythrocytaire.En complément de cette approche, nous avons étudié la morphologie et la composition chimique des globules rouges en utilisant des techniques d'imagerie vibrationnelle infrarouge à différentes résolutions : à l'échelle microscopique avec la microspectroscopie infrarouge à transformée de Fourier (μ-FTIR), à l'échelle submicronique avec l'imagerie optique thermique (O-PTIR) et à l'échelle nanométrique grâce à l'AFM-IR.Cette étude a été réalisée en trois étapes principales : (1) l'élaboration d'un protocole de préparation permettant la fixation des globules rouges, (2) l'évaluation de leur stabilité chimique et morphologique, et enfin (3) une analyse multi-échelles comparant des globules rouges sains à ceux affectés par la maladie de Gaucher.Grâce à notre protocole de fixation, nous avons pu préserver les globules rouges sans altération pendant 10 jours, ce qui nous a permis de mener à bien l'analyse multi-échelle. L'AFM-IR s'est révélée être la seule technique capable de mettre en évidence des différences significatives entre les globules rouges sains et Gaucher. L'AFM-IR a permis de visualiser les différentes morphologies des cellules Gaucher et de comparer la distribution des lipides à travers des cartographies chimiques, révélant des zones d'intérêt au niveau de la membrane érythrocytaire
Red blood cells attract particular clinical interest due to their biological significance. It has recently been demonstrated that certain erythrocyte pathologies may be associated with abnormal lipid composition. Among these pathologies is Gaucher's disease, a rare lysosomal disorder caused by a deficiency in β-glucocerebrosidase, an enzyme whose absence leads to excessive accumulation of certain sphingolipids in the body. This lipid overload alters the morphological and rheological properties of red blood cells, causing hematological and vascular abnormalities.In this thesis, we studied the link between the lipid composition of red blood cells and its implication in Gaucher's disease. In particular, we focused on two categories of lipids: sphingolipids and phospholipids, which play a key role in the erythrocyte alterations observed. Two complementary approaches were used: the first uses mass spectrometry with the aim of identifying new biomarkers. The second uses infrared imaging techniques at different scales to study the distribution of lipids in red blood cells.We developed a rapid sample preparation method compatible with liquid chromatography coupled to mass spectrometry (SPE-HPLC-MS/MS) to quantify 30 sphingolipids and phospholipids in less than 15 minutes. This method was then applied to quantify the 30 lipid species in the plasma and red blood cell pellet of control subjects and Gaucher patients Among these lipid species, some were overexpressed and were not associatedAfter this quantification, we focused on exploring the lipidome of healthy red blood cells and comparing it to that of Gaucher red blood cells. To achieve this, a semi-targeted approach was conducted using a triple-quadrupole analyzer, which allowed us to identify more than 266 molecular species spread across 12 different lipid classes.These results have contributed to a better understanding of the lipidome of healthy red blood cells and are promising for the identification of new biomarkers and the diagnosis of various pathologies affecting the erythrocyte lipidome.As a complement to this approach, we focused on the morphological and chemical study of red blood cells using vibrational infrared imaging techniques at different resolutions: microscopic with Fourier-transform infrared spectroscopy (FTIR), submicron with optical photothermal infrared imaging (O-PTIR), and nanometric with atomic force microscopy (AFM-IR).Our study was structured around three steps: (1) developing a preparation protocol allowing the fixation of red blood cells, (2) evaluating their chemical and morphological stability, and finally (3) conducting a multi-scale analysis comparing healthy red blood cells with those affected by Gaucher's disease.Due to our fixation protocol, we were able to preserve red blood cells without degradation for 10 days, which enabled us to successfully conduct the multi-scale analysis. AFM-IR proved to be the only technique capable of highlighting significant differences between healthy and Gaucher red blood cells. AFM-IR allowed us to visualize the distinct morphologies of Gaucher cells and to compare lipid distribution through chemical mapping, revealing areas of interesting in the erythrocyte membrane