Evaluation of Two Simultaneous Metabolomic and Proteomic Extraction Protocols Assessed by Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry
Project description:Untargeted multi-omics analysis of plasma is an emerging tool for the identification of novel biomarkers for evaluating disease prognosis, and for developing a better understanding of molecular mechanisms underlying human disease. The successful application of metabolomic and proteomic approaches relies on reproducibly quantifying a wide range of metabolites and proteins. Herein, we report the results of untargeted metabolomic and proteomic analyses from blood plasma samples following analyte extraction by two frequently-used solvent systems: chloroform/methanol and methanol-only. Whole blood samples were collected from participants (n = 6) at University Hospital Sharjah (UHS) hospital, then plasma was separated and extracted by two methods: (i) methanol precipitation and (ii) 4:3 methanol:chloroform extraction. The coverage and reproducibility of the two methods were assessed by ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). The study revealed that metabolite extraction by methanol-only showed greater reproducibility for both metabolomic and proteomic quantifications than did methanol/chloroform, while yielding similar peptide coverage. However, coverage of extracted metabolites was higher with the methanol/chloroform precipitation.
Project description:Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g. candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches.
Project description:Mycotoxins are secondary metabolites of fungi species widely known for their potentially toxic effects on human health. Considering their frequent presence in crops and their processed food, monitoring them on food-based matrices is now an important topic. Within such a context, the sample preparation step is usually mandatory before the chromatographic analysis, due to the complexity of matrices such as nuts, cereals, beverages, and others. For these reasons, we herein present the evaluation of two greener setups, based on the automation and miniaturization of the sample preparation step for mycotoxin analysis in different beverages. Firstly, we describe an analytical method based on a multidimensional assembly, coupling a lab-made microextraction column (508 µm i.d. × 100 mm) to a UPLC-MS/MS for the analysis of ochratoxin A in beverages. This configuration used a synthesized sorbent phase containing C18-functionalized graphene-silica particles, which exhibited excellent extraction performance, as well as being reusable and cheaper than commercially available extractive phases. Sequentially, a second setup, based on a multidimensional capillary LC coupled to MS/MS, was assessed for the same purpose. In this case, a graphene oxide-based capillary extraction column (254 µm i.d. × 200 mm) was used as the first dimension, while a C18 analytical capillary column performed the mycotoxin separation in beverages. Although this second one has similarities with the first, we focused mainly on the benefits related to the link between a miniaturized/automated sample preparation device with a capillary LC-MS/MS system, which made our analysis greener. Additionally, the chromatographic efficiency could even be enhanced.
Project description:Untargeted multi-omics analysis of plasma is an emerging tool for the identification of novel biomarkers for evaluating disease prognosis and for a better understanding of molecular mechanisms underlying human disease. The successful application of metabolomic and pro-teomic approaches relies on reproducibly quantifying a wide range of metabolites and proteins. Herein, we report the results of untargeted metabolomic and proteomic analyses from blood plasma samples following analyte extraction by two frequently used solvent systems: chloro-form/methanol and methanol-only. Whole blood samples were collected from participants (n=6) at University Hospital Sharjah (UHS) hospital, then plasma was separated and extracted by two methods i. methanol precipitation and, ii. 4:3 methanol:chloroform extraction. The coverage and reproducibility of the two methods were assessed by ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). The study revealed that metabolite extraction by methanol-only showed greater reproducibility for both metabolomic and proteomic quantifications than did methanol/chloroform, while yielding similar peptide coverage. However, coverage of extracted metabolites was higher with the methanol/chloroform precipitation.
Project description:Co-exposure to tobacco and marijuana has become common in areas where recreational marijuana use is legal. To assist in the determination of the combined health risks of this co-exposure, an analytical method capable of simultaneously measuring tobacco and marijuana metabolites is needed to reduce laboratory costs and the required sample volume. So far, no such analytical method exists. Thus, we developed and validated a method to simultaneously quantify urinary levels of trans-3'-hydroxycotinine (3OH-COT), cotinine (COT), and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (COOH-THC) to assess co-exposure to tobacco and marijuana. Urine (200 µL) was spiked with labelled internal standards and enzymatically hydrolyzed to liberate the conjugated analytes before extraction using solid-supported liquid-liquid extraction (SLE) with ethyl acetate serving as an eluent. The target analytes were separated on a C18 (4.6 × 100 mm, 5 μm) analytical column with a gradient mobile phase elution and analyzed using tandem mass spectrometry with multiple reaction monitoring of target ion transitions. Positive electrospray ionization (ESI) was used for 3OH-COT and COT, while negative ESI was used for COOH-THC. The total run time was 13 min. The extraction recoveries were 18.4-23.9 % (3OH-COT), 65.1-96.8 % (COT), and 80.6-95.4 % (COOH-THC). The method limits of quantification were 5.0 ng/mL (3OH-COT) and 2.5 ng/mL (COT and COOH-THC). The method showed good accuracy (82.5-98.5 %) and precision (1.22-6.21 % within-day precision and 1.42-6.26 % between-day precision). The target analytes were stable for at least 144 h inside the autosampler (10 °C). The analyses of reference materials and 146 urine samples demonstrated good method performance. The use of a 96-well plate for preparation makes the method useful for the analysis of large numbers of samples.
Project description:BackgroundLiquid chromatography-mass spectrometry (LC-MS) and tandem LC-MS (LC-MS/MS) are increasingly used in toxicology laboratories as a complementary method to gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-ultraviolet detection (LC-UV) for comprehensive drug screening (CDS). This study was designed to characterize the sensitivity and specificity of three LC-MS(/MS) vendor-supplied methods for targeted CDS and identify the current limitations associated with the use of these technologies.MethodsFive methods for broad spectrum CDS, including LC-UV (REMEDi), full scan GC-MS, LC-MS (ZQ-Mass Detector with MassLynx-software), LC-QTRAP-MS/MS (3200-QTRAP with Cliquid-software) and LC-LIT-MS/MS (LXQ Linear Ion Trap with ToxID-software) were evaluated based on their ability to detect drugs in 48 patient urine samples.ResultsThe tandem MS methods identified 15% more drugs than the single stage MS or LC-UV methods. Use of two broad spectrum screening methods identified more drugs than any single system alone. False negatives and false positives generated by the LC-MS(/MS) software programs were identified upon manual review of the raw data.ConclusionsThe LC-MS/MS methods detected a broader menu of drugs; however, it is essential to establish manual data review criteria for all LC-MS(/MS) drug screening methods. Use of an EI-GC-MS and ESI-LC-MS/MS combination for targeted CDS may be optimal due to the complementary nature of the chromatographic and ionization techniques.
Project description:The diverse characteristics and large number of entities make metabolite separation challenging in metabolomics. To date, there is not a singular instrument capable of analyzing all types of metabolites. In order to achieve a better separation for higher peak capacity and accurate metabolite identification and quantification, we integrated GC × GC-MS and parallel 2DLC-MS for analysis of polar metabolites. To test the performance of the developed system, 13 rats were fed different diets to form two animal groups. Polar metabolites extracted from rat livers were analyzed by GC × GC-MS, parallel 2DLC-MS (-) and parallel 2DLC-MS (+), respectively. By integrating all data together, 58 metabolites were detected with significant change in their abundance levels between groups (p≤ 0.05). Of the 58 metabolites, three metabolites were detected in two platforms and two in all three platforms. Manual examination showed that discrepancy of metabolite regulation measured by different platforms was mainly caused by the poor shape of chromatographic peaks resulting from low instrument response. Pathway analysis demonstrated that integrating the results from multiple platforms increased the confidence of metabolic pathway assignment.
Project description:Polyphenols are secondary metabolites of plants and used as effective antioxidants in dietary supplements, whose main sources are fruits, vegetables, and grains. To clarify the content and distribution of polyphenols in different fruit species samples accurately, a rapid and sensitive ultrahigh-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method combining dispersive liquid-liquid microextraction (DLLME) was developed for quantitative determination of fifteen polyphenol compounds in fruit juice. In this method, the targets were first extracted from 1 g of fruit juice sample using 10 mL of 80% ethanol solution by ultrasonic-assisted extraction (UAE). Then, 1.0 mL of UAE extracted solution, 60 μL of n-octanol and 2.0 mL of H2O were performed in the following DLLME procedure. A C18 reversed-phase column, ZORBAX SB (100 × 4.6 mm, 3.5 μm), was proposed under gradient elution with 0.1% formic acid aqueous solution and methanol mobile phases for the determination of 15 polyphenols, allowing us to obtain polyphenolic profiles in less than 23.0 min. Under the optimum conditions, the enrichment factors ranged from 162 to 194. The results showed that the 15 polyphenols had linear correlation coefficients (R 2) more than 0.99. The limits of detection (LODs) were between 18.3 and 103.5 ng/g, and the average recoveries were between 96.9 and 116.3% with interday relative standard deviations (RSDs) ranging from 4.4 to 8.2% in all cases. The method was successfully applied to the analysis of real fruit juice samples and presented itself as a simple, rapid, practical, and environment-friendly technique.
Project description:IntroductionEndodontic infections are very prevalent and have a polymicrobial etiology characterized by complex interrelationships between endodontic microorganisms and the host defenses. Proteomic analysis of endodontic infections can provide global insights into the invasion, pathogenicity mechanisms, and multifactorial interactions existing between root canal bacteria and the host in the initiation and progression of apical periodontitis. The purpose of this study was to apply proteomic techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the identification of proteins of bacterial origin present in endodontic infections.MethodsEndodontic specimens were aseptically obtained from seven patients with root canal infections. Protein mixtures were subjected to tryptic in-solution digestion and analysed by reverse-phase nano-LC-MS/MS followed by a database search.ResultsProteins, mainly of cell wall or membrane origin, from endodontic bacteria especially Enterococcus faecalis, Enterococcus faecium, Porphyromonas gingivalis, Fusobacterium nucleatum, and Treponema denticola were identified from all the samples tested. Identified proteins included adhesins, autolysins, proteases, virulence factors, and antibiotic-resistance proteins.ConclusionsLC-MS/MS offers a sensitive analytical platform to study the disease processes in the root canal environment. The array of proteins expressed in endodontic infections reflects the complex microbial presence and highlights the bacterial species involved in the inflammatory process.
Project description:It is usually a tedious task to profile the chemical composition of a given herbal medicine (HM) using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) due to the time-consuming sample preparation and laborious post-acquisition data processing procedures. Even worse, some labile compounds may face degradation risks when exposed to organic solvents for a relatively long period. As one of the most popular HMs, the promising therapeutic benefits of Epimedii Herba (Chinese name: Yinyanghuo) are well defined; however, the chemical profile, and in particular those flavonoids that have been claimed to be responsible for the efficacy, remains largely unknown. Attempts are devoted here to achieve direct LC-MS measurement and efficient post-acquisition data processing, and chemome comparison among three original sources of Epimedii Herba, such as Epimedium sagittatum (Esa), E. pubescens (Epu), and E. koreanum (Eko) was employed to illustrate the strategy utility. A home-made online liquid extraction (OLE) module was introduced at the front of the analytical column to comprehensively transfer the compounds from raw materials onto the LC-MS instrument. A mass defect filtering approach was programmed to efficiently mine the massive LC-MS dataset after which a miniature database was built involving all chemical information of flavonoids from the genus Epimedium to draw a pentagonal frame to rapidly capture potential quasi-molecular ions (mainly [M-H]-). A total of 99 flavonoids (66 in Esa, 84 in Eko, and 66 in Epu) were captured, and structurally annotated by summarizing the mass fragmentation pathways from the mass spectrometric data of authentic compounds and an in-house data library as well. Noteworthily, neutral loss of 144 Da was firstly assigned to the neutral cleavage of rhamnosyl residues. Significant species-differences didn't occur among their chemical patterns. The current study proposed a robust strategy enabling rapid chemical profiling of, but not limited to, HMs.