bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒09‒27
fourteen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Metabolites. 2020 Sep 21. pii: E378. [Epub ahead of print]10(9):
      The evaluation of liquid chromatography high-resolution mass spectrometry (LC-HRMS) raw data is a crucial step in untargeted metabolomics studies to minimize false positive findings. A variety of commercial or open source software solutions are available for such data processing. This study aims to compare three different data processing workflows (Compound Discoverer 3.1, XCMS Online combined with MetaboAnalyst 4.0, and a manually programmed tool using R) to investigate LC-HRMS data of an untargeted metabolomics study. Simple but highly standardized datasets for evaluation were prepared by incubating pHLM (pooled human liver microsomes) with the synthetic cannabinoid A-CHMINACA. LC-HRMS analysis was performed using normal- and reversed-phase chromatography followed by full scan MS in positive and negative mode. MS/MS spectra of significant features were subsequently recorded in a separate run. The outcome of each workflow was evaluated by its number of significant features, peak shape quality, and the results of the multivariate statistics. Compound Discoverer as an all-in-one solution is characterized by its ease of use and seems, therefore, suitable for simple and small metabolomic studies. The two open source solutions allowed extensive customization but particularly, in the case of R, made advanced programming skills necessary. Nevertheless, both provided high flexibility and may be suitable for more complex studies and questions.
    Keywords:  A-CHMINACA; LC-HRMS; data processing; feature detection; untargeted metabolomics
  2. BMC Bioinformatics. 2020 Sep 23. 21(1): 418
      BACKGROUND: Mass spectrometry (MS) uses mass-to-charge ratios of measured particles to decode the identities and quantities of molecules in a sample. Interpretation of raw MS depends upon data processing algorithms that render it human-interpretable. Quantitative MS workflows are complex experimental chains and it is crucial to know the performance and bias of each data processing method as they impact accuracy, coverage, and statistical significance of the result. Creation of the ground truth necessary for quantitatively evaluating MS1-aware algorithms is difficult and tedious task, and better software for creating such datasets would facilitate more extensive evaluation and improvement of MS data processing algorithms.RESULTS: We present JS-MS 2.0, a software suite that provides a dependency-free, browser-based, one click, cross-platform solution for creating MS1 ground truth. The software retains the first version's capacity for loading, viewing, and navigating MS1 data in 2- and 3-D, and adds tools for capturing, editing, saving, and viewing isotopic envelope and extracted isotopic chromatogram features. The software can also be used to view and explore the results of feature finding algorithms.
    CONCLUSIONS: JS-MS 2.0 enables faster creation and inspection of MS1 ground truth data. It is publicly available with an MIT license at
    Keywords:  MS1 feature finding; Mass spectrometry; Mass spectrometry viewer; Open source mass spectrometry software
  3. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Sep 06. pii: S1570-0232(20)30507-9. [Epub ahead of print]1160 122370
      Monosaccharide composition of biological samples can reflect an individual's health status. Monitoring the concentration of individual monosaccharides in human serum requires a technique for the simultaneous analysis of multiple monosaccharide molecules. Furthermore, certified reference materials (CRMs) for overall monosaccharide composition of human serum are required in order to validate the performance of clinical laboratory instruments. In the present study, we present a novel method for the simultaneous analysis of numerous monosaccharide molecules without the need for derivatization or post-column treatment. We utilized ultra-high-performance liquid chromatography (UHPLC)-quadrupole/orbitrap mass spectrometry incorporating a hydrophilic interaction chromatography (HILIC) column. We optimized the precursor ions, product ions, mobile phase composition and gradient program, flow rate, and column temperature. Seven monosaccharides (D-Ribose, L-Arabinose, D-Xylose, D-Fructose, D-Mannose, D-Galactose and D-Glucose) were able to be separated and quantified. We validated the method and the seven molecules showed favorable limits of detection and quantification, recovery rates, carry-over effects, intra- and inter-day accuracy and precision, resolution, and measurement uncertainty. We analyzed human serum samples using the method. To avoid ion suppression and D-d2-Glucose peak interference, compounds present at concentrations outside of the calibration range were analyzed from diluted samples. Quantification of serum samples corroborated some previous clinical research, in that increased D-Glucose concentration was associated with increased concentrations of D-Mannose and D-Ribose. We also validated the CRMs, and expect these to have utility as standards for serum monosaccharide profiling, thus contributing to public health.
    Keywords:  Certified reference material; High resolution mass spectrometry; Monosaccharide; Ultra high-performance liquid chromatography
  4. Anal Chem. 2020 Sep 22.
      Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma and urine samples spiked with the same metabolite mixture, were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning and rinsing procedure, etc.) were left to the discretion of the contributing labs. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs. 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the thirteen labs. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.
  5. MethodsX. 2020 ;7 101050
      Past analyses of sugar and amino acid composition of aphid honeydews have been completed using diverse instrumentation. Here we report the use of hydrophilic interaction liquid chromatography (HILIC) coupled with a triple quadrupole mass spectrometric (MS/MS) detector for the analysis of seven saccharides (xylose, fructose, glucose, sucrose, trehalose, melezitose and raffinose) and five amino acids (glutamic acid, glutamine, aspartic acid, serine, and asparagine). Limits of quantitation ranged from 0.05 mg/L (melezitose) to 1.0 mg/L (fructose) for sugars and from 0.10 mg/L (glutamic acid) to 3.66 mg/L (asparagine) for amino acids. Sample preparation was fast and simple, requiring only the washing of foils used to collect aphid honeydew with hot (80 °C) water and sonication of samples prior to HILIC/MS/MS analysis for both classes of analytes. No analyte derivatization was required and excellent chromatographic characteristics were observed. For those studying honeydew-mediated interactions in the field, this technique allows for rapid characterization of ecologically important amino acids and sugars.•Composition of seven saccharides in Aphis asclepiadis honeydew including xylose, fructose, glucose, sucrose, trehalose, melezitose,and raffinose, and five standard amino acids including glutamic acid, glutamine, aspartic acid, serine, and asparagine, were analyzed using hydrophilic interaction liquid chromatography-mass spectrometry.•All polar analytes were analyzed without derivatization using HILIC-MS with chromatographic run times of 7 min (sugars) and 10 min (amino acids).
    Keywords:  Aphis asclepiadis; HILIC; Honeydew composition; LC/MS; Melezitose; Raffinose
  6. Anal Chem. 2020 Sep 24.
      The quantitative detection methods for many microplastics (MPs) polymers in the environment are inadequate. For example, effective detection methods of nylon (polyamide, PA), a widely used plastic, in different environmental samples is still lacking. In the present study, a method based on acid depolymerization-liquid chromatography-tandem mass spectrometry (LC-MS/MS) and without the separation of MPs from samples was developed to quantify nylon MPs. After removing the background monomer compounds, PA6 and PA66 were efficiently depolymerized to 6-aminocaproic acid and adipic acid and detected by LC-MS/MS, respectively. Accordingly, the quantity of nylon MPs was accurately calculated. By using the proposed method, the recovery of spiked PA6 and PA66 MPs in the environmental samples ranged from 90.8% to 98.8%. The limits of quantification for PA6 and PA66 MPs were 0.680 and 4.62 mg/kg, respectively. PA MPs were widely detected in indoor dust, sludge, marine sediment, freshwater sediment, fishery sediment, and fish guts and gills with concentrations of 0.725-321 mg/kg. Extremely high concentrations of PA66 MPs were detected in indoor dust and fish guts and gills, indicating the unequivocal risk of human exposure through dust ingestion and dietary exposure.
  7. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Sep 11. pii: S1570-0232(20)30889-8. [Epub ahead of print]1160 122384
      3', 5' - Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in many cellular functions and biological processes. In several cell types, cholera toxin will increase the level of cAMP, which mediates toxic effects on cells. In this context, we have developed a fast and simple method based on extraction with 5% trichloroacetic acid (TCA) and quantitation with liquid chromatography-mass tandem spectrometry (LC-MS/MS) for measuring cAMP in cells. A main feature of the LC-MS method was employing a reversed phase C18 column (2.1 mm × 50 mm, 1.6 µm particles) compatible with a 100% aqueous mobile phase, providing retention of the highly polar analyte. Isocratic separations allowed for fast subsequent injections. Negative mode electrospray ionization detection was performed with a triple quadrupole (QqQ)MS. cAMP was extracted from cell samples (~106 cells per well) and spiked with a labelled internal standard, using 200 µL of 5% TCA. The extraction solvent was fully compatible for direct injection onto the reversed phase column. After 10 min incubation, the supernatant was removed, and 10 µL of the supernatant was directly analysed by LC-MS. The method was characterized by the simplicity of the extraction, and the speed (3 min retention time of cAMP), sensitivity (250 pg/mL detection limit), and selectivity (separation from interferences e.g. isomeric compounds) of the LC-MS method, and could be used for quantitation of cAMP in the range 1-500 ng/mL cell extract.
    Keywords:  Cells; Cholera toxin; Mass spectrometry; Reversed phase liquid chromatography; cAMP
  8. Metabolites. 2020 Sep 19. pii: E376. [Epub ahead of print]10(9):
      Urine is a popular biofluid for metabolomics studies due to its simple, non-invasive collection and its availability in large quantities, permitting frequent sampling, replicate analyses, and sample banking. The biggest disadvantage with using urine is that it exhibits significant variability in concentration and composition within an individual over relatively short periods of time (arising from various external factors and internal processes regulating the body's water and solute content). In treating the data from urinary metabolomics studies, one must account for the natural variability of urine concentrations to avoid erroneous data interpretation. Amongst various proposed approaches to account for broadly varying urine sample concentrations, normalization to creatinine has been widely accepted and is most commonly used. MS total useful signal (MSTUS) is another normalization method that has been recently reported for mass spectrometry (MS)-based metabolomics studies. Herein, we explored total useful peak area (TUPA), a modification of MSTUS that is applicable to GC×GC-TOFMS (and data from other separations platforms), for sample normalization in urinary metabolomics studies. Performance of TUPA was compared to the two most common normalization approaches, creatinine adjustment and Total Peak Area (TPA) normalization. Each normalized dataset was evaluated using Principal Component Analysis (PCA). The results showed that TUPA outperformed alternative normalization methods to overcome urine concentration variability. Results also conclusively demonstrate the risks in normalizing data to creatinine.
    Keywords:  GC×GC-TOFMS; creatinine; mass spectrometry; metabolomics; normalization; urine
  9. J Mass Spectrom. 2020 Jul 04. e4614
      Imaging mass spectrometry (IMS) technologies are capable of mapping a wide array of biomolecules in diverse cellular and tissue environments. IMS has emerged as an essential tool for providing spatially targeted molecular information due to its high sensitivity, wide molecular coverage, and chemical specificity. One of the major challenges for mapping the complex cellular milieu is the presence of many isomers and isobars in these samples. This challenge is traditionally addressed using orthogonal liquid chromatography (LC)-based analysis, though, common approaches such as chromatography and electrophoresis are not able to be performed at timescales that are compatible with most imaging applications. Ion mobility offers rapid, gas-phase separations that are readily integrated with IMS workflows in order to provide additional data dimensionality that can improve signal-to-noise, dynamic range, and specificity. Here, we highlight recent examples of ion mobility coupled to IMS and highlight their importance to the field.
    Keywords:  IMS; desorption electrospray ionization, DESI; drift tube ion mobility spectrometry, DTIMS; high-field asymmetric waveform ion mobility, FAIMS; imaging mass spectrometry; infrared matrix-assisted laser desorption electrospray ionization, IR-MALDESI; ion mobility; laser ablation electrospray ionization, LAESI; lipids; liquid extraction surface analysis, LESA; liquid microjunction, (LMJ); matrix-assisted laser desorption electrospray ionization, MALDI; metabolites; proteins; tissue analysis; trapped ion mobility spectrometry, TIMS; travelling wave ion mobility spectrometry, TWIMS
  10. J Pharm Biomed Anal. 2020 Sep 12. pii: S0731-7085(20)31492-8. [Epub ahead of print]191 113606
      Ventromedial hypothalamic nucleus (VMN) control of glucostasis is estradiol (E-2)-dependent. E-2 regulation of VMN reactivity to hypoglycemia may involve changes in signal volume due to altered aromatase expression. Here, high-resolution micropunch dissection tools for isolation of segmental VMN tissue were used with Design of Experiments-refined uHPLC-electrospray ionization-mass spectrometry (LC-ESI-MS) methodology to investigate the premise that effects of acute and/or recurring hypoglycemia on VMN E-2 content are sex-dimorphic. Relationships among multiple independent mass spectrometric operational variables were assessed by Central Composite Design (CCD) to amplify E-2 chromatogram area. Combinations of spectrometric temperature and gas pressure variable combinations were screened by Akaike Information Criterion correction modeling. A Fibonacci Sequence design using CCD minimum and maximal variable limits produced a small-run model that replicated maximal response from CCD. E-2 chromatographic response was further enhanced by optimization of solid phase extraction and instrument source and collision-induced dissociation voltages. In male rats, acute and chronic hypoglycemia respectively elevated or diminished E-2 concentrations relative to baseline in both rostral and caudal VMN. However, females exhibited regional variability in tissue E-2 profiles during acute (increased, rostral VMN; no change, caudal VMN) and recurring (no change, rostral VMN; increased, caudal VMN) hypoglycemia. Outcomes demonstrate requisite LC-ESI-MS sensitivity for E-2 quantification in small-volume brain tissue samples acquired with high-neuroanatomical specificity. Current methodology will facilitate efforts to investigate physiological consequences of VMN rostro-caudal segment-specific acclimation of E-2 profiles to recurring hypoglycemia, including effects on gluco-regulatory function, in each sex.adjuca.
  11. Metabolites. 2020 Sep 22. pii: E379. [Epub ahead of print]10(9):
      In the past two decades, metabolomics has proved to be a valuable tool with many potential applications in different areas of science. However, there are still some challenges that need to be addressed, particularly for multicenter studies. These challenges are mainly attributed to various sources of fluctuation and unwanted variations that can be introduced at pre-analytical, analytical, and/or post-analytical steps of any metabolomics experiment. Thus, this study aimed at using Streptomyces lividans TK24 as the model organism in a cross-laboratory experiment in Manchester and Leuven to evaluate the reproducibility of a standard sample preparation method, and determine the optimal sample format (cell extract or quenched biomass) required to preserve the metabolic profile of the cells during cross-lab sample transportation and storage. Principal component analysis (PCA) scores plot of the gas chromatography-mass spectrometry (GC-MS) data from both laboratories displayed clear growth-dependent clustering patterns which was in agreement with the Procrustes analysis findings. In addition, the data generated in Manchester displayed tight clustering of cell pellets (quenched biomass) and metabolite extracts, confirming the stability of both sample formats during the transportation and storage period.
    Keywords:  GC-MS; Streptomyces; biotechnology; metabolomics; sample preparation
  12. Sci Rep. 2020 Sep 23. 10(1): 15550
      Therapeutic drug monitoring (TDM) is necessary for the optimal administration of anti-arrhythmic drugs in the treatment of heart arrhythmia. The present study aimed to develop and validate a direct analysis in real time tandem mass spectrometry (DART-MS/MS) method for the rapid and simultaneous determination of five anti-arrhythmic drugs (metoprolol, diltiazem, amiodarone, propafenone, and verapamil) and one metabolite (5-hydroxy(OH)-propafenone) in human serum. After the addition of isotope-labeled internal standards and protein precipitation with acetonitrile, anti-arrhythmic drugs were ionized by DART in positive mode followed by multiple reaction monitoring (MRM) detection. The use of DART-MS/MS avoided the need for chromatographic separation and allowed rapid and ultrahigh throughput analysis of anti-arrhythmic drugs in a total run time of 30 s per sample. The DART-MS/MS method yielded satisfactory linearity (R2 ≥ 0.9906), accuracy (86.1-109.9%), and precision (≤ 14.3%) with minimal effect of biological matrixes. The method was successfully applied to analyzing 30 clinical TDM samples. The relative error (RE) of the concentrations obtained by DART-MS/MS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was within ± 13%. This work highlights the potential usefulness of DART for the rapid quantitative analysis of anti-arrhythmic drugs in human serum and gives rapid feedback in the clinical TDM practices.
  13. J Mass Spectrom. 2020 Sep 01. e4648
      Methotrexate (MTX), a folate antagonist, is the anchor drug used to treat several diseases. Therapeutic effects are attributed to intracellular levels of various methotrexate conjugates that are present in the cell as polyglutamates (MTX-Glu). The present study was conducted to develop a new liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based assay to separately quantitate the MTX-Glu in hair cells, red blood cells, and serum using internal standards. Sample preparation consisted of extraction with an organic solution followed by solid-phase extraction. The presented methodology was applied for the analysis of methotrexate and its polyglutamates in hair cells, red blood cells, and serum obtained from clinical patients. The developed LC-ESI-MS/MS method for the quantitative measurement of MTX-Glu was both sensitive and precise within the clinically relevant range. This method is possibly be superior with respect to sensitivity, selectivity, and speed than all previously described approaches and can be easily applied in routine clinical tests owing to the combination of a simple pretreatment process with robust LC-MS/MS.
  14. Adv Clin Chem. 2020 ;pii: S0065-2423(20)30021-4. [Epub ahead of print]99 147-191
      Today, metabolomics is becoming an indispensable tool to get a more comprehensive analysis of complex living systems, providing insights on multiple aspects of physiology. Although its application in large scale population-based studies is very challenging due to the processing of large sample sets as well as the complexity of data information, its potential to characterize human health is well recognized. Technological advances in metabolomics pave the way for the efficient biomarker discovery of disease etiology, diagnosis and prognosis. Here, different steps of the metabolomics workflow, particularly mass spectrometry-based approaches, are discussed to demonstrate the potential of metabolomics to address biological questioning in human health. First an overview of metabolomics is provided with its interest in human health studies. Analytical development and advances in mass spectrometry instrumentation and computational tools are discussed regarding their application limits. Advancing metabolomics for applicability in human health and large-scale studies is presented and discussed in conclusion.
    Keywords:  Data mining; Human health; Large-scale studies; Mass spectrometry; Metabolite identification; Metabolomics; Sample preparation