Project description:Cassava serves as a primary staple food for over one billion people worldwide. The quality of cassava flour is markedly affected by the oxidation and deterioration of lipids during storage. Despite its significance, the lipid composition of cassava flour and its alterations throughout storage periods have not been extensively studied. This study offers a comprehensive lipidomic analysis of cassava flour over storage periods using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The results showed that 545 lipids from five classes and 27 subclasses were identified in cassava flour, including key substances such as free fatty acids (36 species), diglycerides (DGs) (31 species), and triglycerides (TGs) (259 species). Using Metware Cloud for statistical analysis, significant variations were observed in 50 lipid species over long-term storage, reflecting changes in lipid profiles due to storage. These lipids correlate with seven metabolic pathways, among which glycerolipid metabolism is the most affected. The metabolites associated with these pathways can differentiate cassava flour based on the length of storage. This study provides a theoretical basis and storage technology parameters for lipid changes during cassava flour storage.
Project description:Consumers have shown more and more interest in high-quality and healthy dairy products and buffalo milk is commercially more viable than other milks in producing superior dairy products due to its higher contents of fat, crude protein, and total solids. Metabolomics is one of the most powerful strategies in molecular mechanism research however, little study has been focused on the milk metabolites in different buffalo species. Therefore, the aim of this study was to explore the underlying molecular mechanism of the fatty synthesis and candidate biomarkers by analyzing the metabolomic profiles. Milk of three groups of buffaloes, including 10 Mediterranean, 12 Murrah, and 10 crossbred buffaloes (Murrah × local swamp buffalo), were collected and UPLC-Q-Orbitrap HRMS was used to obtain the metabolomic profiles. Results showed that milk fatty acid in Mediterranean buffalo was significantly higher than Murrah buffalo and crossbred buffalo. A total of 1837/726 metabolites was identified in both positive and negative electrospray ionization (ESI±) mode, including 19 significantly different metabolites between Mediterranean and Murrah buffalo, and 18 different metabolites between Mediterranean and crossbred buffalo. We found 11 of the different metabolites were both significantly different between Mediterranean vs. Murrah group and Mediterranean vs crossbred group, indicating that they can be used as candidate biomarkers of Mediterranean buffalo milk. Further analysis found that the different metabolites were mainly enriched in fat synthesis related pathways such as fatty acid biosynthesis, unsaturated fatty acid biosynthesis, and linoleic acid metabolism, indicating that the priority of different pathways affected the milk fat content in different buffalo species. These specific metabolites may be used as biomarkers in the identification of milk quality and molecular breeding of high milk fat buffalo.
Project description:Purine metabolites have been implicated as clinically relevant biomarkers of worsening or improving Parkinson's disease (PD) progression. However, the identification of purine molecules as biomarkers in PD has largely been determined using non-targeted metabolomics analysis. The primary goal of this study was to develop an economical targeted metabolomics approach for the routine detection of purine molecules in biological samples. Specifically, this project utilized LC/MS/MS and LC/QTOF/MS to accurately quantify levels of six purine molecules in samples from cultured N2a murine neuroblastoma cells. The targeted metabolomics workflow was integrated with automated label-free digital microscopy, which enabled normalization of purine concentration per unit cell in the absence of fluorescent dyes. The established method offered significantly enhanced selectivity compared to previously published procedures. In addition, this study demonstrates that a simple, quantitative targeted metabolomics approach can be developed to identify and quantify purine metabolites in biological samples. We envision that this method could be broadly applicable to quantification of purine metabolites from other complex biological samples, such as cerebrospinal fluid or blood.
Project description:An additional class of endogenous lipid amides, N-arachidonoyl amino acids (Ara-AAs), is growing in significance in the field of endocannabinoids. The development, validation, and application of a sensitive and selective method to simultaneously monitor and quantify the level of Ara-AAs along with anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in mouse brain has been established. The linearity of the method over the concentration ranges of 0.2-120 pg/μl for the standards of N-arachidonoyl amino acids, N-arachidonoyl alanine (NAAla), serine (NASer), γ-aminobutyric acid (NAGABA), and glycine (NAGly); 0.7-90 pg/μl for AEA-d(0)/d(8); and 7.5-950 pg/μl for 2-AG was determined with R(2) values of 0.99. Also the effects of the FAAH inhibitor URB 597 on the endogenous levels of these analytes were investigated. AEA and NASer brain levels exhibit a dose-dependent increase after systemic administration of URB 597, whereas NAGly and NAGABA were significantly decreased after treatment. NAAla and 2-AG were not altered after URB 597 treatment. The potential benefit of establishing this assay extends beyond the quantification of the Ara-AAs along with AEA and 2-AG in mouse brain, to reveal a variety of pharmacological effects and physiological roles of these analytes.
Project description:Hepatitis C virus (HCV) infection of the liver is a global health problem and a major risk factor for the development of hepatocellular carcinoma (HCC). Sensitive methods are needed for the improved and earlier detection of HCC, which would provide better therapy options. Metabolic profiling of the high-risk population (HCV patients) and those with HCC provides insights into the process of liver carcinogenesis and possible biomarkers for earlier cancer detection. Seventy-three blood metabolites were quantitatively profiled in HCC (n = 30) and cirrhotic HCV (n = 22) patients using a targeted approach based on LC-MS/MS. Sixteen of 73 targeted metabolites differed significantly (p < 0.05) and their levels varied up to a factor of 3.3 between HCC and HCV. Four of these 16 metabolites (methionine, 5-hydroxymethyl-2'-deoxyuridine, N2,N2-dimethylguanosine, and uric acid) that showed the lowest p values were used to develop and internally validate a classification model using partial least squares discriminant analysis. The model exhibited high classification accuracy for distinguishing the two groups with sensitivity, specificity, and area under the receiver operating characteristic curve of 97%, 95%, and 0.98, respectively. A number of perturbed metabolic pathways, including amino acid, purine, and nucleotide metabolism, were identified based on the 16 biomarker candidates. These results provide a promising methodology to distinguish cirrhotic HCV patients, who are at high risk to develop HCC, from those who have already progressed to HCC. The results also provide insights into the altered metabolism between HCC and HCV.
Project description:Spebrutinib (SBT) is a Bruton's tyrosine kinase inhibitor. SBT is currently in phase II and phase I clinical trials for the management of rheumatoid arthritis and chronic lymphocytic leukaemia, respectively. We developed and validated a liquid chromatography tandem mass spectrometry analytical method to quantify SBT and investigate its metabolic stability. SBT and the naquotinib as internal standard were isocratically eluted on a C18 column. The linearity of the developed method is 5-500 ng ml-1 (r2 ≥ 0.9999) in the human liver microsomes (HLMs) matrix. Good sensitivity was approved by the very low limit of detection (0.39 ng ml-1). Inter- and intra-assay accuracy values of -1.41 to 12.44 and precision values of 0.71% to 4.78%, were obtained. SBT was found to have an in vitro half-life (82.52 min) and intrinsic clearance (8.4 µl min-1 mg-1) as computed following its incubation with HLMs. The latter finding, hypothesize that SBT could be slowly excreted from the body unlike other related tyrosine kinase inhibitors. So, drug plasma level and kidney function should be monitored because of potential bioaccumulation. To the best of our knowledge, this is considered the first analytical method for SBT quantification using LC-MS/MS with application to metabolic stability evaluation.
Project description:The unbiased identification of cytokine-induced, secreted proteins from cells cultured in serum-containing medium is challenging. Here, we describe an experimental and bioinformatics workflow to label interleukin-1α-regulated proteins in living cells with the methionine analogue L-homopropargylglycine. We detail their purification and identification by means of CLICK-chemistry-based biotinylation followed by nanoHPLC-MS/MS. A side-by-side comparison of enriched proteins and their ontologies to serum-free conditions demonstrates the sensitivity and specificity of this approach to study the inducible secreted proteomes of epithelial cells.
Project description:Liquid chromatography-mass spectrometry (LC-MS/MS) based approaches are widely used for the identification and quantitation of specific metabolites, and are a preferred approach towards analyzing cellular metabolism. Most methods developed come with specific requirements such as unique columns, ion-pairing reagents and pH conditions, and typically allow measurements in a specific pathway alone. Here, we present a single column-based set of methods for simultaneous coverage of multiple pathways, primarily focusing on central carbon, amino acid, and nucleotide metabolism. We further demonstrate the use of this method for quantitative, stable isotope-based metabolic flux experiments, expanding its use beyond steady-state level measurements of metabolites. The expected kinetics of label accumulation pertinent to the pathway under study are presented with some examples. The methods discussed here are broadly applicable, minimize the need for multiple chromatographic resolution methods, and highlight how simple labeling experiments can be valuable in facilitating a comprehensive understanding of the metabolic state of cells.
Project description:Warfarin is a narrow therapeutic index anticoagulant drug and its use is associated with infrequent but significant adverse bleeding events. The international normalized ratio (INR) is the most commonly used biomarker to monitor and titrate warfarin therapy. However, INR is derived from a functional assay, which determines clotting efficiency at the time of measurement and is susceptible to technical variability. Protein induced by vitamin K antagonist-II (PIVKA-II) has been suggested as a biomarker of long-term vitamin K status, providing mechanistic insights about variation in the functional assay. However, the currently available antibody-based PIVKA-II assay does not inform on the position and number of des-carboxylation sites in prothrombin. The assay presented in this paper provides simultaneous quantification of carboxy and des-carboxy prothrombin that are essential for monitoring early changes in INR and, thus, serves as the superior tool for managing warfarin therapy. Additionally, this assay permits the quantification of total prothrombin level, which is affected by warfarin treatment. Prothrombin recovery from plasma was 95% and the liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was linear (r2 = 0.98) with a dynamic range of 1-100 µg/mL. The assay interday precision was within 20%. A des-carboxy peptide of prothrombin (GNLER) was negatively correlated with active prothrombin (Pearson r = 0.99, P < 0.0001), whereas its association was positively linked with INR values (Pearson r = 0.75, P < 0.015). This novel LC-MS/MS assay for active and inactive prothrombin quantification can be applied to titrate anticoagulant therapy and to monitor the impact of diseases, such as hepatocellular carcinoma on clotting physiology.
Project description:Sialic acid storage disease (SASD) is an inborn error resulting from defects in the lysosomal membrane protein sialin. The SASD phenotypical spectrum ranges from a severe presentation, infantile sialic acid storage disease (ISSD) which may present as hydrops fetalis, to a relatively mild form, Salla disease. Screening for SASD is performed by determination of free sialic acid (FSA) in urine or amniotic fluid supernatant (AFS). Subsequent diagnosis of SASD is performed by quantification of FSA in cultured fibroblasts and by mutation analysis of the sialin gene, SLC17A5. We describe simple quantitative procedures to determine FSA as well as conjugated sialic acid in AFS, and FSA in cultured fibroblasts, using isotope dilution ((13)C(3)-sialic acid) and multiple reaction monitoring LC-ESI-MS/MS. The whole procedure can be performed in 2-4 h. Reference values in AFS were 0-8.2 μmol/L for 15-25 weeks of gestation and 3.2-12.0 μmol/L for 26-38 weeks of gestation. In AFS samples from five fetuses affected with ISSD FSA was 23.9-58.9 μmol/L demonstrating that this method is able to discriminate ISSD pregnancies from normal ones. The method was also validated for determination of FSA in fibroblast homogenates. FSA in SASD fibroblasts (ISSD; 20-154 nmol/mg protein, intermediate SASD; 12.9-15.1 nmol/mg, Salla disease; 5.9-7.4 nmol/mg) was clearly elevated compared to normal controls (0.3-2.2 nmol/mg). In conclusion, we report simple quantitative procedures to determine FSA in AFS and cultured fibroblasts improving both prenatal diagnostic efficacy for ISSD as well as confirmatory testing in cultured fibroblasts following initial screening in urine or AFS.