bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒03‒21
nine papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Analyst. 2021 Mar 18.
      We propose a fully automated novel workflow for lipidomics based on flow injection, followed by liquid chromatography-high-resolution mass spectrometry (FI/LC-HRMS). The workflow combined in-depth characterization of the lipidome achieved via reversed-phase LC-HRMS with absolute quantification by using a large number of lipid species-specific and/or retention time (RT)-matched/class-specific calibrants. The lipidome of 13C-labelled yeast (LILY) provided a large panel of cost-effective internal standards (ISTDs) covering triacylglycerols (TG), steryl esters (SE), free fatty acids (FA), diacylglycerols (DG), sterols (ST), ceramides (Cer), hexosyl ceramides (HexCer), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidic acids (PA), cardiolipins (CL), phosphatidylinositols (PI), phosphatidylserines (PS), phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE). The workflow in combination with the LILY lipid panel enables simultaneous quantification via (1) external multi-point calibration with internal standardization and (2) internal one-point calibration with LILY as a surrogate ISTD, increasing the coverage while keeping the accuracy and throughput high. Extensive measures on quality control allowed us to rank the calibration strategies and to automatically select the calibration strategy of the highest metrological order for the respective lipid species. Overall, the workflow enabled a streamlined analysis, with a limit of detection in the low femtomolar range, and provided validation tools together with absolute concentration values for >350 lipids in human plasma on a species level. Based on the selected standard panel, lipids from 7 classes (LPC, LPE, PC, PE, PI, DG, TG) passed stringent quality filters, which included QC accuracy, a precision and recovery bias of <30% and concentrations within the 99% confidence interval of the international laboratory comparison of SRM 1950, NIST, USA. The quantitative values are independent of common deuterated or non-endogenous ISTDs, thus offering cross-validation of different lipid methods and further standardizing lipidomics.
  2. Anal Chem. 2021 Mar 19.
      Urine is a noninvasive biofluid that is rich in polar metabolites and well suited for metabolomic epidemiology. However, because of individual variability in health and hydration status, the physiological concentration of urine can differ >15-fold, which can pose major challenges in untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics. Although numerous urine normalization methods have been implemented (e.g., creatinine, specific gravity-SG), most are manual and, therefore, not practical for population-based studies. To address this issue, we developed a method to measure SG in 96-well-plates using a refractive index detector (RID), which exhibited accuracy within 85-115% and <3.4% precision. Bland-Altman statistics showed a mean deviation of -0.0001 SG units (limits of agreement: -0.0014 to 0.0011) relative to a hand-held refractometer. Using this RID-based SG normalization, we developed an automated LC-MS workflow for untargeted urinary metabolomics in a 96-well-plate format. The workflow uses positive and negative ionization HILIC chromatography and acquires mass spectra in data-independent acquisition (DIA) mode at three collision energies. Five technical internal standards (tISs) were used to monitor data quality in each method, all of which demonstrated raw coefficients of variation (CVs) < 10% in the quality controls (QCs) and < 20% in the samples for a small cohort (n = 87 urine samples, n = 22 QCs). Application in a large cohort (n = 842 urine samples, n = 248 QCs) demonstrated CVQC < 5% and CVsamples < 16% for 4/5 tISs after signal drift correction by cubic spline regression. The workflow identified >540 urinary metabolites including endogenous and exogenous compounds. This platform is suitable for performing urinary untargeted metabolomic epidemiology and will be useful for applications in population-based molecular phenotyping.
  3. J Chromatogr A. 2021 Mar 06. pii: S0021-9673(21)00152-7. [Epub ahead of print]1642 462028
      Measurement of chiral thiol compounds such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) in human serum plays an important role in the early diagnosis and warning of cardiovascular disease, neurodegenerative disease, and cancer. We developed a novel chiral mass spectrometry derivatization reagent, (R)-(5-(3-isothiocyanatopyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium (NCS-OTPP), with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge for the diastereomeric separation of chiral thiol compounds by ultrahigh-performance liquid chromatography coupled to quadrupole-Orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). A novel method was developed for simultaneous determination of three kinds of chiral thiol compounds based on the NCS-OTPP derivatization method. Three kinds of chiral thiol compounds on a YMC Triart C18 (2.0 × 150 mm, 1.9 μm) column with Rs were 1.56-1.68. The protonated precursor to product ion transitions monitored for GSH was m/z 780.16→747.24/473.18, Cys was m/z 594.20→561.18/473.18, and Hcy was m/z 608.21→575.19/473.18. An excellent linearity for all the analytes with correlation coefficients ≥ 0.9995 and suitable precision with inter-day and intra-day coefficients of variation RSDs was 0.83-4.06% and 0.95-3.11%. Satisfactory accuracy with recoveries between 83.73 and 103.35% was observed. The limit of detection (S/N = 3) was 2.4-7.2 fmol. Furthermore, the method was successfully applied to the simultaneous determination of three kinds of free and total thiol compounds in serum from 10 healthy volunteers at normal and stress states.
    Keywords:  Chiral thiol compound; Diastereomeric separation; NCS-OTPP; Oxidative stress, UHPLC-Q-Orbitrap HRMS
  4. Anal Chim Acta. 2021 Apr 15. pii: S0003-2670(21)00085-4. [Epub ahead of print]1154 338259
      Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography - tandem mass spectrometry. The method exploits isotope-dilution mass spectrometry for absolute quantification of target metabolites. The method is applicable for semi-quantification of other sterols for which isotope labelled surrogates are not available and approximate quantification of partially identified sterols. Values are reported for non-esterified sterols in the absence of saponification and total sterols following saponification. In this way absolute quantification data is reported for 17 sterols in the NIST SRM 1950 plasma along with semi-quantitative data for 8 additional sterols and approximate quantification for one further sterol. In a pooled (CSF) sample used for internal quality control, absolute quantification was performed on 10 sterols, semi-quantification on 9 sterols and approximate quantification on a further three partially identified sterols. The value of the method is illustrated by confirming the sterol phenotype of a patient suffering from ACOX2 deficiency, a rare disorder of bile acid biosynthesis, and in a plasma sample from a patient suffering from cerebrotendinous xanthomatosis, where cholesterol 27-hydroxylase is deficient.
    Keywords:  Bile acid; Cholestenoic acid; Cholesterol; Derivatisation; Hydroxycholesterol; Isotope-labelled standard; LC-MS
  5. Rapid Commun Mass Spectrom. 2021 Mar 17. e9085
      RATIONALE: Nitrogen isotopic compositions (δ15 N) of source and trophic amino acids (AAs) are crucial tracers of N sources and trophic enrichments in diverse fields including archeology, astrobiochemistry, ecology, oceanography, and paleo-sciences. The current analytical technique using gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) requires derivatization which is not compatible with some key AAs.. Another approach using the high-performance liquid chromatography-elemental analyzer-IRMS (HPLC/EA/IRMS) may experience co-elution issues with other compounds in certain types of samples and the highly sensitive Nano-EA/IRMS instrumentations are not widely available.METHODS: We present a method for high-precision δ15 N measurements of amino acids (δ15 N-AA) optimized for canonical source AA-phenylalanine (Phe) and trophic AA-glutamic acid (Glu). This offline approach entails a purification and separation step via high-pressure ion-exchange chromatography (IC) with automated fraction collection, followed by sequential chemical conversion of AA to nitrite and then to nitrous oxide (N2 O), and final determination of δ15 N of the produced N2 O via purge-and-trap continuous-flow isotope ratio mass spectrometry (PT/CF/IRMS).
    RESULTS: The cross-plots of δ15 N of Glu and Phe standards (four different natural abundance levels) generated by this method and their accepted values have a linear regression slope of 1 and small intercepts demonstrating high accuracy. The precisions were 0.36~0.67‰ for Phe standards, and 0.27~0.35‰ for Glu standards. Our method and the GC/C/IRMS approach produced equivalent δ15 N values for two lab standards (McCarthy Lab AA mixture and cyanobacteria) within error. We further tested our method on a wide range of natural sample matrices and obtained reasonable results.
    CONCLUSIONS: Our method provides a reliable alternative to the current methods for δ15 N-AA measurement as IC or HPLC-based techniques that can collect underivatized AAs are widely available. Our chemical approach that converts AA to N2 O can be easily implemented into laboratories currently analyzing δ15 N of N2 O using PT/CF/IRMS. This method will help promote the use of δ15 N-AA in important studies of N cycling and trophic ecology in a wide range of research areas.
  6. Anal Chim Acta. 2021 Apr 08. pii: S0003-2670(21)00126-4. [Epub ahead of print]1153 338300
      Comprehensive analysis of the liver metabolome can be very useful for discovering disease biomarkers and studying diseases, especially liver-related diseases. However, the presence of a relatively large amount of blood in liver tissue may have a profound effect on liver tissue metabolome analysis. We designed a study to address this issue in order to develop a liver metabolomics workflow based on high-coverage quantitative metabolome analysis using differential chemical isotope labeling (CIL) LC-MS. In the first set of experiments, we compared the metabolomes of mouse serum, non-perfused liver, and perfused liver without and with varying amounts of blood added. We found that there was a significant metabolome difference between the perfused liver and non-perfused liver. To illustrate the effects of perfusion conditions on tissue metabolome analysis, we analyzed the mouse livers that were subjected to perfusion under two different conditions. We found that ice-cold temperature perfusion led to less change of the liver metabolome, compared to room temperature perfusion; however, there was still a significant metabolome difference between the ice-cold-perfused liver and the non-perfused liver. Finally, we applied the method to a chemical (carbon tetrachloride) exposure liver injury model to examine the effects of blood in liver on the detection of significantly changed metabolites in two comparative groups of mice. Using multivariate and univariate analyses of the serum and liver metabolomes of control and diseased mice, we detected many unique significant metabolites in serum as well as in liver. This work demonstrates that perfusion can alter the liver metabolome significantly. Therefore, we recommend the use of non-perfused liver for high-coverage liver metabolomics.
    Keywords:  Isotope labeling; LC-MS; Liver; Metabolomics; Tissue
  7. Anal Chem. 2021 Mar 16.
      LC-HRMS experiments detect thousands of compounds, with only a small fraction of them identified in most studies. Traditional data processing pipelines contain an alignment step to assemble the measurements of overlapping features across samples into a unified table. However, data sets acquired under nonidentical conditions are not amenable to this process, mostly due to significant alterations in chromatographic retention times. Alignment of features between disparately acquired LC-MS metabolomics data could aid collaborative compound identification efforts and enable meta-analyses of expanded data sets. Here, we describe metabCombiner, a new computational pipeline for matching known and unknown features in a pair of untargeted LC-MS data sets and concatenating their abundances into a combined table of intersecting feature measurements. metabCombiner groups features by mass-to-charge (m/z) values to generate a search space of possible feature pair alignments, fits a spline through a set of selected retention time ordered pairs, and ranks alignments by m/z, mapped retention time, and relative abundance similarity. We evaluated this workflow on a pair of plasma metabolomics data sets acquired with different gradient elution methods, achieving a mean absolute retention time prediction error of roughly 0.06 min and a weighted per-compound matching accuracy of approximately 90%. We further demonstrate the utility of this method by comprehensively mapping features in urine and muscle metabolomics data sets acquired from different laboratories. metabCombiner has the potential to bridge the gap between otherwise incompatible metabolomics data sets and is available as an R package at and Bioconductor.
  8. Bioanalysis. 2021 Mar 16.
      Purpose: Develop a quantitative LC-MS/MS method for FDG, FDG-monophosphate, glucose and glucose-monophosphate in mouse tumor models to assist in validating the use of [18F]FDG-positron emission tomography (PET) imaging for anticancer therapies in a clinical setting. Methodology/results: Analytes were isolated from tumors by protein precipitation and detected on a Sciex API-5500 mass spectrometer. Improved assay robustness and selectivity were achieved through chromatographic separation of FDG-monophosphate from glucose-monophosphate, selection of a unique ion transition and incorporation of stable isotope labeled internal standards. In a mouse JIMT-1 tumor model, FDG-monophosphate levels measured by LC-MS/MS correlated with [18F]FDG-PET imaging results. Conclusion: LC-MS/MS analysis of FDG-monophosphate accumulation in tumors is a cost-effective tool to gauge the translational potential of [18F]FDG-PET imaging as a noninvasive biomarker in clinical studies.
    Keywords:  FDG; FDG-PET; LC–MS/MS assay; fluorodeoxyglucose; mass spectrometry; tumor
  9. J Pharm Anal. 2021 Feb;11(1): 77-87
      5-Fluorouracil (5-FU) is an anticancer drug extensively used for different cancers. Intracellular metabolic activation leads to several nucleoside and nucleotide metabolites essential to exert its cytotoxic activity on multiple cellular targets such as enzymes, DNA and RNA. In this paper, we describe the development of a method based on liquid chromatography coupled with high resolution mass spectrometry suitable for the simultaneous determination of the ten anabolic metabolites (nucleoside, nucleotide and sugar nucleotide) of 5-FU. The chromatographic separation was optimized on a porous graphitic carbon column allowing the analysis of the metabolites of 5-FU as well as endogenous nucleotides. The detection was performed on an Orbitrap® tandem mass spectrometer. Linearity of the method was verified in intracellular content and in RNA extracts. The limit of detection was equal to 12 pg injected on column for nucleoside metabolites of 5-FU and 150 pg injected on column for mono- and tri-phosphate nucleotide metabolites. Matrix effect was evaluated in cellular contents, DNA and RNA extracts for nucleoside and nucleotides metabolites. The method was successfully applied to i) measure the proportion of each anabolic metabolite of 5-FU in cellular contents, ii) follow the consequence of inhibition of enzymes on the endogenous nucleotide pools, iii) study the incorporation of metabolites of 5-FU into RNA and DNA, and iv) to determine the incorporation rate of 5-FUrd into 18 S and 28 S sub-units of rRNA.
    Keywords:  5-Fluorouracil; DNA; Incorporation rate; LC-MS-HRMS; Nucleotide; RNA