bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒06‒28
fourteen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Anal Chem. 2020 Jun 26.
      Untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS) is currently the gold-standard technique to determine the full chemical diversity in biological samples. However, this approach still has many limitations; notably, the difficulty of accurately estimating the number of unique metabolites profiled among the thousands of MS ion signals arising from chromatograms. Here, we describe a new workflow, MS-CleanR, based on the MS-DIAL/MS-FINDER suite, which tackles feature degeneracy and improves annotation rates. We show that implementation of MS-CleanR reduces the number of signals by nearly 80% while retaining 95% of unique metabolite features. Moreover, the annotation results from MS-FINDER can be ranked according to the database chosen by the user, which enhance identification accuracy. Application of MS-CleanR to the analysis of Arabidopsis thaliana grown in three different conditions fostered class separation resulting from multivariate data analysis and led to annotation of 75% of the final features. The full workflow was applied to metabolomic profiles from three strains of the leguminous plant Medicago truncatula that have different susceptibilities to the oomycete pathogen Aphanomyces euteiches. A group of glycosylated triterpenoids overrepresented in resistant lines were identified as candidate compounds conferring pathogen resistance. MS-CleanR is implemented through a Shiny interface for intuitive use by end-users (available at:
  2. J Chromatogr Sci. 2020 Jun 24. pii: bmaa028. [Epub ahead of print]
      It has been suggested that thiol-containing amino acids could be used as biomarkers for diseases associated with oxidative stress. We investigated the thiol-containing amino acids, homocysteine (Hcy), cysteine (Cys), glutathione (GSH) and γ-glutamylcysteine (γ-GluCys), in commercial human serum by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after precolumn derivatization with 4-fluoro-7-sulfobenzofurazan. This method was applied to determine the composition of thiol-containing amino acids in exosomes prepared from the serum. Hcy, Cys, GSH and γ-GluCys could be detected in the exosomal fraction, and the ratio of each thiol-containing amino acid was similar to those in the corresponding native serum. Cys (94.76%) was most enriched in the exosomal fraction, followed by GSH (2.97%), γ-GluCys (1.59%) and Hcy (0.68%). These findings suggest that thiol-containing amino acids, Hcy, Cys, GSH and γ-GluCys, are included in exosomes in human serum.
  3. J Anal Toxicol. 2020 Jun 15. pii: bkaa073. [Epub ahead of print]
      As the number of prescriptions, over-the-counter medications and drugs of abuse continue to increase, forensic laboratories are faced with the challenge of developing more comprehensive screening methods in order to detect them in whole blood samples. Another challenge faced by forensic laboratories is detecting and identifying novel synthetic compounds as they emerge and change. Traditional drug screening methods include enzyme immunoassay (EIA) and either gas or liquid chromatography paired with mass spectrometry (GC-MS or LC-MS-MS, respectively). While these methods are good, they have their disadvantages. For example, EIA requires special reagents for each drug class, GC-MS requires extensive sample preparation, and LC-MS-MS only detects drugs on a known inclusion lists of compounds of interest. Described below is the development of a robust and comprehensive screening method for drugs in whole blood samples that eliminates the aforementioned disadvantages of the traditional methods. Using a Q Exactive Focus ™ liquid chromatography-high resolution-accurate mass spectrometer (LC-HRMS-MS), a method was developed that is capable of detecting approximately 200 drugs at a concentration of 2 μg/L for most analytes. This method also employs a more automated data processing feature which reduces processing time. Finally, it has the added benefit of retroactive data analysis, which allows it to be used for unknown drug analysis as well. Used as an initial screening method, the comprehensive drug screen using LC-HRMS-MS has the potential to take on two of the most important challenges faced by forensic laboratories today.
  4. J Am Soc Mass Spectrom. 2020 Jun 22.
      The analysis of 13C labelled lipids by mass spectrometry is challenging due to the complexity from labelling the large number of carbon atoms in lipids. To further add to the complexity, different adducts can be produced during electrospray ionisation and in-source fragmentation which can create complex overlapping isotope patterns that can only be resolved using high resolution mass spectrometry. Co-elution of lipids even after chromatographic separation also adds to the potential for overlapping mass spectra. Here, we describe a procedure that enables full 13C labelled patterns to be resolved in complex microalgal lipid extracts as well a procedure that provides structural labelling information. Mass resolving powers of 240,000 full width half maximum (FWHM) and fast targeted MS/MS allowed the differentiation of isotopologues, adducts and unresolved lipid species after chromatographic separation. This enabled the percentage of 13C enrichment to be calculated for each individual lipid species over a time series in the microalgal lipidome. The application of tandem mass spectrometry (MS/MS) also allowed the degree of labelling within the head-group vs acyl chains to be determined, further adding to the detail of information collected. This information is particularly useful for studying lipid synthesis and re-modelling processes and can be extended to other biological systems.
  5. Nat Protoc. 2020 Jun 24.
      Untargeted methods are typically used in the detection and discovery of small organic compounds in metabolomics research, and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) is one of the most commonly used platforms for untargeted metabolomics. Although they are non-biased and have high coverage, untargeted approaches suffer from unsatisfying repeatability and a requirement for complex data processing. Targeted metabolomics based on triple-quadrupole mass spectrometry (TQMS) could be a complementary tool because of its high sensitivity, high specificity and excellent quantification ability. However, it is usually applicable to known compounds: compounds whose identities are known and/or are expected to be present in the analyzed samples. Pseudotargeted metabolomics merges the advantages of untargeted and targeted metabolomics and can act as an alternative to the untargeted method. Here, we describe a detailed protocol of pseudotargeted metabolomics using UHPLC-TQMS. An in-depth, untargeted metabolomics experiment involving multiple UHPLC-HRMS runs with MS at different collision energies (both positive and negative) is performed using a mixture obtained using small amounts of the analyzed samples. XCMS, CAMERA and Multiple Reaction Monitoring (MRM)-Ion Pair Finder are used to find and annotate peaks and choose transitions that will be used to analyze the real samples. A set of internal standards is used to correct for variations in retention time. High coverage and high-performance quantitative analysis can be realized. The entire protocol takes ~5 d to complete and enables the simultaneously semiquantitative analysis of 800-1,300 metabolites.
  6. Anal Chem. 2020 Jun 22.
      The human faecal metabolome is complex, but rich in information and allows investigation of host metabolism as a function of diet and health. Faecal metabolome is still much less explored than the plasma and urine metabolome, and in order to generate comparable data across laboratories and cohorts standard operating procedures are required. This study evaluates 10 protocols, using different extraction solvents and sample processing methods for measuring the human faecal metabolome using proton nuclear magnetic resonance (1H-NMR) spectroscopy. Three solvents: water, methanol and dimethyl sulfoxide (DMSO) were investigated at varying concentrations for their ability to extract metabolites directly from faecal slurry or after freeze-drying. The protocols were evaluated on four different pools of human feces. The study also demonstrates a novel signature mapping (SigMa) method for rapid and unbiased processing of complex NMR spectra applied for the first time to human faecal metabolomics. The method is provided with a library containing the chemical shift ranges of 81 common faecal metabolites for future unambiguous and rapid faecal metabolite annotations. The result from the 10 faecal extraction protocols were investigated in terms of reproducibility, coverage and ability to extract low concentration metabolites. The solvent type showed to induce the highest variation in the data (45.7%) and the water based extractions allowed detection of the greatest number of metabolites and resulted in the highest reproducibility. Direct extraction of faecal slurry proved to be more reproducible than freeze drying. In addition freeze drying caused a relative loss of short chain fatty acids (SCFA). DMSO was used for the first time to extract faecal metabolites and enabled the detection of certain bile acids. Some derivatives of SCFA were only detected using methanol as solvent.
  7. J Adv Pharm Technol Res. 2020 Apr-Jun;11(2):11(2): 64-68
      The aim of the study was to develop and validate a practical assay of clinically relevant testosterone levels in human plasma and saliva. We performed ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis on Atlantis dC18 steel column using a mobile phase of 2-mM ammonium acetate and acetonitrile (20:80, v: v) that was delivered at 0.3 ml/min. After adding d3-testosterone as an internal standard (IS), we extracted plasma and salivary samples with methyl tert-butyl ether. Mass spectrometry was performed in electrospray positive-ion mode. Targeted ion transitions were examined at m/z 289.18 → 97.04 and 292.24 → 97.04 for testosterone and IS, respectively. We validated the method according to the US Food and Drug Administration guidelines. Elution times for testosterone and IS were both around 1.35 min. Testosterone level was linearly associated (r 2 = 0.9975 and 0.9958) with peak area ratio of testosterone to IS between 0.5-50 ng/ml and 10-400 pg/ml in plasma and saliva, respectively. The coefficient of variation and bias were ≤12.6% and ≤±12.1% in plasma and ≤10.2% and ≤±5.3% in saliva. The extraction recovery of testosterone was ≥92% from plasma and ≥94% from saliva. Testosterone was stable (≥91%) for 24 h at room temperature and for 8 weeks at -20°C in both plasma and salivary samples. We report a simple, validated, UPLC-MS/MS assay that can be used to determine clinically relevant levels of testosterone in human plasma and saliva.
    Keywords:  Artificial saliva; d3-testosterone; human plasma; testosterone; ultra-performance liquid chromatography-tandem mass spectrometry
  8. Anal Chem. 2020 Jun 22.
      A new class of cationic ion-interaction reagents for reversed-phase chromatography is introduced in the present work. Compounds belonging to a homologous series of linear fluoroalkyl chains including trifluoroethylamine (TFEAm), pentafluoropropylamine (PFPAm), heptafluorobutylamine (HFBAm), and nonafluoropentylamine (NFPAm) were tested and compared with ammonia and triethylamine (TEA) for the separation of selected organic acids of general interest such as the herbicides glyphosate, ethephon, and fosamine and the arsenic metabolites methylarsonic acid and dimethylarsinic acid, as well as other compounds. Depending on carbon and fluorine atom number, the fluoroalkylamines were shown to be effective cationic ion-interaction reagents, significantly enhancing the retention of organic acids on a C18 reversed-phase column. Contrary to the general behavior of ion-interaction reagents (a broader term than "ion-pairing reagent"), significant (up to 5-fold) and consistent enhancement in the electrospray ionization mass spectrometry signal (ESI-MS) was observed relative to ammonia and triethylamine. Overall, among the tested series the HFBAm was found to offer the best overall properties among the tested series as it provided a good compromise between column equilibration time (ca. 25 column volumes), and retaining behavior (up to 10-fold increase in the retention factor of acids relative to ammonia), while providing the same general advantages found for the fluoroalkylamines such as fast washout times from the ESIMS system (ca. 30 min) and 3-5 fold signal enhancement. The fluoroalkylamines are a new class of cationic ion-interaction reagents with clear advantages over the currently employed alkylamines, and may revive the general interest in ion-interaction chromatography.
  9. Redox Biol. 2020 Jun 01. pii: S2213-2317(20)30563-2. [Epub ahead of print]36 101595
      Oxysterols are critical regulators of inflammation and cholesterol metabolism in cells. They are oxidation products of cholesterol and may be differentially metabolised in subcellular compartments and in biological fluids. New analytical methods are needed to improve our understanding of oxysterol trafficking and the molecular interplay between the cellular compartments required to maintain cholesterol/oxysterol homeostasis. Here we describe a method for isolation of oxysterols using solid phase extraction and quantification by liquid chromatography-mass spectrometry, applied to tissue, cells and mitochondria. We analysed five monohydroxysterols; 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, 7 ketocholesterol and three dihydroxysterols 7α-24(S)dihydroxycholesterol, 7α-25dihydroxycholesterol, 7α-27dihydroxycholesterol by LC-MS/MS following reverse phase chromatography. Our new method, using Triton and DMSO extraction, shows improved extraction efficiency and recovery of oxysterols from cellular matrix. We validated our method by reproducibly measuring oxysterols in mouse brain tissue and showed that mice fed a high fat diet had significantly lower levels of 24S/25diOHC, 27diOHC and 7ketoOHC. We measured oxysterols in mitochondria from peripheral blood mononuclear cells and highlight the importance of rapid cell isolation to minimise effects of handling and storage conditions on oxysterol composition in clinical samples. In addition, in vitro cell culture systems, of THP-1 monocytes and neuronal-like SH-SH5Y cells, showed mitochondrial-specific oxysterol metabolism and profiles were lineage specific. In summary, we describe a robust and reproducible method validated for improved recovery, quantitative linearity and detection, reproducibility and selectivity for cellular oxysterol analysis. This method enables subcellular oxysterol metabolism to be monitored and is versatile in its application to various biological and clinical samples.
    Keywords:  Blood; Brain oxysterol; Cholesterol; Dihydroxycholesterol; Liquid chromatography-mass spectrometry; Metabolism; Mitochondria; Monocytes; Neuroblastoma; Oxysterol; Peripheral blood mononuclear cell; Subcellular; Whole cell
  10. Metabolomics. 2020 Jun 23. 16(7): 78
      INTRODUCTION: Mitochondria represent an important milieu for studying the pathogenesis of several major diseases. The need for organelle-level metabolic resolution exists, as mitochondrial/cytosolic metabolites are often diluted beyond detection limits in complex samples. Compartment-specific studies are still hindered by the lack of efficient, cost-effective fractioning methods-applicable to laboratories of all financial/analytical standing.OBJECTIVES: We established a novel mitochondrial/cytosolic purification pipeline for complimentary GC-TOF-MS and 1H-NMR metabolomics using robust, commercially available fractionation strategies.
    METHODS: Magnetic based mitochondria isolation kits (MACS) were adapted for this purpose, accompanied by cytosolic filtering. Yield was assessed through the percentage recovery of citrate synthase (CS; a mitochondrial marker), purity by immunoblotting against compartment-specific proteins and integrity interrogated through the respiratory coupling ratio (RCR). The effects of the kit-based buffers on MS/NMR analyses of pure metabolite standards were evaluated. Finally, biological applicability to mammalian disease models was shown using Ndufs4 mouse brain tissue.
    RESULTS: With minor modifications, MACS produced around 60% more mitochondria compared to a differential centrifugation method. Less than 15% of lysosomal LAMP-2 protein was found in the MACS isolates, confirming relative purity-while RCR's above 6 indicate sufficient mitochondrial integrity. The filtering approach effectively depleted mitochondria from the cytosolic fraction, as indicated by negligible Hsp60 and CS levels. Our GC-MS pilot yielded 60-70 features per fraction, while NMR analyses could quantify 6-10 of the most abundant compounds in each fraction.
    CONCLUSION: This study provides a simple and flexible solution for mitochondrial and cytosolic metabolomics in animal model tissues, towards large-scale application of such methodologies in disease research.
    Keywords:  Compartment-specific metabolomics; Cytosol; Gas chromatography time-of-flight mass spectrometry (GC-TOF–MS); MACS; Mitochondria; Proton nuclear magnetic resonance (1H-NMR)
  11. J Mass Spectrom. 2020 Sep;55(9): e4526
      A fast methodology for the assay of parabens in drug and cosmetic preparations has been presented. The procedure developed is based on paper spray tandem mass spectrometry and isotope dilution approach. For each investigated paraben, the corresponding labeled standard has been used in order to improve the accuracy and reproducibility of the analyses. The MS experiments have been performed under MRM conditions, monitoring the transitions [M-H]- → m/z 92 and [M-H]- → m/z 98, respectively, for each analyte and the corresponding labeled internal standard. The quantitative assay has been performed using a calibration curve built from 2 to 15 mg/L. The method accuracy, in all case near 100%, was evaluated using fortified samples at two concentration levels, which are representative of the lower and the higher portion of calibration curve. The good values of LOQ, LOD, and reproducibility confirm the consistency of the developed approach.
    Keywords:  cosmetics; drugs; labeled internal standards; paper spray ionization; parabens; tandem mass spectrometry
  12. Stud Health Technol Inform. 2020 Jun 23. 271 39-48
      Changes in lipid homeostasis can lead to a plethora of diseases, raising the importance of reliable identification and measurement of lipids enabled by bioinformatics tools. However, due to the enormous diversity of lipids, most contemporary tools cover only a marginal range of lipid classes. To reduce such a shortcoming, this work extends the lipid species covered by Lipid Data Analyzer (LDA) to galactolipids and oxidized lipids. Appropriate mass lists were generated for MS1 identifications and the proprietary decision rule sets were extended for MS2 identifications of the novel lipid classes. Furthermore, LDA was extended to enable identification of oxidatively modified fatty acyl chains. With these extensions, LDA can reliably identify the most important galactolipids as well as oxidatively modified versions of the 22 previously implemented lipid classes. Comparison with other up to date lipidomics tools show that LDA has a better coverage of the newly implemented lipid species. The extended version of LDA provides researchers with a powerful platform to elucidate diseases caused by perturbations in the oxidized lipidome. LDA is freely available from
    Keywords:  bioinformatics; cheminformatics; galactolipids; lipidomics; mass spectrometry; oxidized lipids
  13. Nat Commun. 2020 Jun 24. 11(1): 3186
      Mass spectrometry based metabolomics is a widely used approach in biomedical research. However, current methods coupling mass spectrometry with chromatography are time-consuming and not suitable for high-throughput analysis of thousands of samples. An alternative approach is flow-injection mass spectrometry (FI-MS) in which samples are directly injected to the ionization source. Here, we show that the sensitivity of Orbitrap FI-MS metabolomics methods is limited by ion competition effect. We describe an approach for overcoming this effect by analyzing the distribution of ion m/z values and computationally determining a series of optimal scan ranges. This enables reproducible detection of ~9,000 and ~10,000 m/z features in metabolomics and lipidomics analysis of serum samples, respectively, with a sample scan time of ~15 s and duty time of ~30 s; a ~50% increase versus current spectral-stitching FI-MS. This approach facilitates high-throughput metabolomics for a variety of applications, including biomarker discovery and functional genomics screens.
  14. Anal Chem. 2020 Jun 26.
      We introduce Goslin, a polyglot grammar for common lipid shorthand nomenclatures based on the LIPID MAPS nomenclature and the shorthand nomenclature established by Liebisch and co-authors and used by LipidHome and SwissLipids. Goslin was designed to address the following pressing issues in the lipidomics field: 1) to simplify the implementation of lipid name handling for developers of mass spectrometry-based lipidomics tools; 2) to offer a tool that unifies and normalizes the main existing lipid name dialects enabling a lipidomics analysis in a high-throughput fashion and 3) to provide a consistent mapping from lipid shorthand names to lipid building blocks and structural properties. We provide implementations of Goslin in four major programming languages, namely C++, Java, Python 3, and R to kick-start adoption and integration. Further, we set up a web service for users to work with Goslin directly. All implementations are available free of charge under a permissive open source license.