bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒01‒24
eighteen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Anal Chem. 2021 Jan 19.
      Existing data acquisition modes such as full-scan, data-dependent (DDA), and data-independent acquisition (DIA) often present limited capabilities in capturing metabolic information in liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. In this work, we proposed a novel metabolomic data acquisition workflow that combines DDA and DIA analyses to achieve better metabolomic data quality, including enhanced metabolome coverage, tandem mass spectrometry (MS2) coverage, and MS2 quality. This workflow, named data-dependent-assisted data-independent acquisition (DaDIA), performs untargeted metabolomic analysis of individual biological samples using DIA mode and the pooled quality control (QC) samples using DDA mode. This combination takes advantage of the high-feature number and MS2 spectral coverage of the DIA data and the high MS2 spectral quality of the DDA data. To analyze the heterogeneous DDA and DIA data, we further developed a computational program, DaDIA.R, to automatically extract metabolic features and perform streamlined metabolite annotation of DaDIA data set. Using human urine samples, we demonstrated that the DaDIA workflow delivers remarkably improved data quality when compared to conventional DDA or DIA metabolomics. In particular, both the number of detected features and annotated metabolites were greatly increased. Further biological demonstration using a leukemia metabolomics study also proved that the DaDIA workflow can efficiently detect and annotate around 4 times more significant metabolites than DDA workflow with broad MS2 coverage and high MS2 spectral quality for downstream statistical analysis and biological interpretation. Overall, this work represents a critical development of data acquisition mode in untargeted metabolomics, which can greatly benefit untargeted metabolomics for a wide range of biological applications.
  2. Anal Chem. 2021 Jan 19.
      A growing number of software tools have been developed for metabolomics data processing and analysis. Many new tools are contributed by metabolomics practitioners who have limited prior experience with software development, and the tools are subsequently implemented by users with expertise that ranges from basic point-and-click data analysis to advanced coding. This Perspective is intended to introduce metabolomics software users and developers to important considerations that determine the overall impact of a publicly available tool within the scientific community. The recommendations reflect the collective experience of an NIH-sponsored Metabolomics Consortium working group that was formed with the goal of researching guidelines and best practices for metabolomics tool development. The recommendations are aimed at metabolomics researchers with little formal background in programming and are organized into three stages: (i) preparation, (ii) tool development, and (iii) distribution and maintenance.
  3. J Chromatogr Sci. 2021 Jan 22. pii: bmaa121. [Epub ahead of print]
      High throughput and high-resolution lipid analyses are important for many biological model systems and research questions. This comprises both monitoring at the individual lipid species level and broad lipid classes. Here, we present a nontarget semiquantitative lipidomics workflow based on ultrahigh performance supercritical fluid chromatography (UHPSFC)-mass spectrometry (MS). The optimized chromatographic conditions enable the base-line separation of both nonpolar and polar classes in a single 7-minute run. Ionization efficiencies of lipid classes vary 10folds in magnitude and great care must be taken in a direct interpretation of raw data. Therefore, the inclusion of internal standards or experimentally determined Response factors (RF) are highly recommended for the conversion of raw abundances into (semi) quantitative data. We have deliberately developed an algorithm for automatic semiquantification of lipid classes by RF. The workflow was tested and validated using a bovine liver extract with satisfactory results. The RF corrected data provide a more representative relative lipid class determination, but also the interpretation of individual lipid species should be performed on RF corrected data. In addition, semiquantification can be improved by using internal or also external standards when more accurate quantitative data are of interest but this requires validation for all new sample types. The workflow established greatly extends the potential of nontarget UHPSFC-MS/MS based analysis.
  4. Molecules. 2021 Jan 18. pii: E480. [Epub ahead of print]26(2):
      Oxandrolone, a synthetic testosterone analog, is used for the treatment of several diseases associated with weight loss. Unfortunately, oxandrolone is abused by many athletes and bodybuilders due to its strong anabolic effect. We have developed and validated a highly sensitive and rapid on-line SPE-UHPLC-MS/MS method for the determination of oxandrolone and simultaneous identification of its major metabolite 17-epi-oxandrolone in urine matrices. Enrichment of the analytes via an integrated solid-phase extraction was achieved using an Acquity UPLC BEH C18 Column. Subsequently, the chromatographic separation of the on-line preconcentrated sample fraction was achieved using an Acquity HSS T3 C18 Column. For the structural identification of these analytes, a high-resolution mass spectrometer Synapt-G2Si coupled to the Acquity M-class nano-LC system with ionKey source was used. A highly sensitive determination of oxandrolone was achieved using a tandem quadrupole mass spectrometer XEVO TQD. The method was successfully validated in the linear range of oxandrolone from 81.63 pg·mL-1 (limit of quantification, LOQ) to 5000 pg·mL-1 in the human urine matrix. It was applied to the analysis of real urine samples obtained from a healthy volunteer after the oral administration of one dose (10 mg) of oxandrolone. Concentration vs. time dependence was tested in the time interval of 4 h-12 days (after oral administration) to demonstrate the ability of the method to detect the renal elimination of oxandrolone from the human body. Favorable performance parameters along with successful application indicate the usefulness of the proposed method for its routine use in antidoping control labs.
    Keywords:  17-epi-oxandrolone; human urine; on-line SPE extraction; oxandrolone; tandem mass spectrometry; ultra-high performance liquid chromatography
  5. Anal Chem. 2021 Jan 22.
      The metabolome comprises a complex network of interconnecting enzyme-catalyzed reactions that involve transfers of numerous molecular subunits. Thus, the reconstruction of metabolic networks requires metabolite substructures to be tracked. Subunit tracking can be achieved by tracing stable isotopes through metabolic transformations using NMR and ultrahigh -resolution (UHR)-mass spectrometry (MS). UHR-MS1 readily resolves and counts isotopic labels in metabolites but requires tandem MS to help identify isotopic enrichment in substructures. However, it is challenging to perform chromatography-based UHR-MS1 with its long acquisition time, while acquiring MS2 data on many coeluting labeled isotopologues for each metabolite. We have developed an ion chromatography (IC)-UHR-MS1/data-independent(DI)-HR-MS2 method to trace the fate of 13C atoms from [13C6]-glucose ([13C6]-Glc) in 3D A549 spheroids in response to anticancer selenite and simultaneously 13C/15N atoms from [13C5,15N2]-glutamine ([13C5,15N2]-Gln) in 2D BEAS-2B cells in response to arsenite transformation. This method retains the complete isotopologue distributions of metabolites via UHR-MS1 while simultaneously acquiring substructure label information via DI-MS2. These details in metabolite labeling patterns greatly facilitate rigorous reconstruction of multiple, intersecting metabolic pathways of central metabolism, which are illustrated here for the purine/pyrimidine nucleotide biosynthesis. The pathways reconstructed based on subunit-level isotopologue analysis further reveal specific enzyme-catalyzed reactions that are impacted by selenite or arsenite treatments.
  6. Nat Protoc. 2021 Jan 22.
      Cognitive computing is revolutionizing the way big data are processed and integrated, with artificial intelligence (AI) natural language processing (NLP) platforms helping researchers to efficiently search and digest the vast scientific literature. Most available platforms have been developed for biomedical researchers, but new NLP tools are emerging for biologists in other fields and an important example is metabolomics. NLP provides literature-based contextualization of metabolic features that decreases the time and expert-level subject knowledge required during the prioritization, identification and interpretation steps in the metabolomics data analysis pipeline. Here, we describe and demonstrate four workflows that combine metabolomics data with NLP-based literature searches of scientific databases to aid in the analysis of metabolomics data and their biological interpretation. The four procedures can be used in isolation or consecutively, depending on the research questions. The first, used for initial metabolite annotation and prioritization, creates a list of metabolites that would be interesting for follow-up. The second workflow finds literature evidence of the activity of metabolites and metabolic pathways in governing the biological condition on a systems biology level. The third is used to identify candidate biomarkers, and the fourth looks for metabolic conditions or drug-repurposing targets that the two diseases have in common. The protocol can take 1-4 h or more to complete, depending on the processing time of the various software used.
  7. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Dec 24. pii: S1570-0232(20)31390-8. [Epub ahead of print]1164 122514
      High quality chromatographic separation underpins robustness in LC-MS, frequently the analytical method of choice for pharmaceutical drug discovery work. The potential improvements in chromatographic selectivity afforded by serial column coupling (SCC), provide a useful means to enhance the resolution of complex samples. In this work, we present a revised high-throughput form of SCC, in which just two individual mixed phase columns were coupled together and combined with a gradient-optimised, retention-directed ultra-high pressure method to achieve rapid separations, with no further method optimisation necessary. The overall performance was evaluated from an open access DMPK analytical working environment perspective; where in anticipation of bioanalytical or metabolite identification chromatography challenges, or with the knowledge that stronger resolution was required for in-vitro sample analysis, the methodology could be immediately implemented by the analyst. Retention-directed selection of a shallow SCC gradient method was successful in separating peaks throughout the chromatographic window, resulting in a runtime still congruent to high-throughput analyses (3.5 min). In-vitro assay sample interferences were resolved 44-72% of the time, and the overall resolving power for isomeric separations significantly improved against single column comparisons (1.7-fold mean RS improvement). Over a sustained period of time in our laboratory, SCC methods have been used for metabolite identification and bioanalytical samples, where both convenience and effectiveness in solving analytical challenges has been consistently demonstrated. Examples that highlight SCC chromatography, and a guided discussion of the main high-throughput considerations, are included. The technique offers wide applicability, and we would recommend it as a toolbox consideration to the laboratory analyst.
    Keywords:  Drug metabolism and pharmacokinetics (DMPK); High-throughput screening (HTS); Liquid chromatography-tandem mass spectrometry (LC-MS); Pentafluorophenyl (PFP) column; Resolution (R(S)); Serial column coupling (SCC)
  8. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Dec 28. pii: S1570-0232(20)31395-7. [Epub ahead of print]1164 122519
      Gamma-aminobutyric acid (GABA) and its precursor glutamic acid are important neurotransmitters. Both are also present in peripheral tissues and the circulation, where abnormal plasma concentrations have been linked to specific mental disorders. In addition to endogenous synthesis, GABA and glutamic acid can be obtained from dietary sources. An increasing number of studies suggest beneficial cardio-metabolic effects of GABA intake, and therefore GABA is being marketed as a food supplement. The need for further research into their health effects merits accurate and sensitive methods to analyze GABA and glutamic acid in plasma. To this end, an ultra-pressure liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of GABA and glutamic acid in human plasma. Samples were prepared by a protein precipitation step and subsequent solid phase extraction using acetonitrile. Chromatographic separation was achieved on an Acquity UPLC HSS reversed phase C18 column using gradient elution. Analytes were detected using electrospray ionization and selective reaction monitoring. Standard curve concentrations for GABA ranged from 3.4 to 2500 ng/mL and for glutamic acid from 30.9 ng/mL to 22,500 ng/mL. Within- and between-day accuracy and precision were <10% in quality control samples at low, medium and high concentrations for both GABA and glutamic acid. GABA and glutamic acid were found to be stable in plasma after freeze-thaw cycles and up to 12 months of storage. The validated method was applied to human plasma from 17 volunteers. The observed concentrations ranged between 11.5 and 20.0 ng/ml and 2269 and 7625 ng/ml for respectively GABA and glutamic acid. The reported method is well suited for the measurement of plasma GABA and glutamic acid in pre-clinical or clinical studies.
    Keywords:  GABA; Glutamic acid; Plasma; SPE; UPLC-MS/MS
  9. J Mass Spectrom. 2021 Feb;56(2): e4680
      The amino acid glutamine (Gln) is a likely source of energy in the brain during neuroglucopenia. Effects of glucose deficiency on astrocyte Gln homeostasis remain unclear, as analytical tools of requisite sensitivity for quantification of intracellular levels of this molecule are not currently available. Here, a primary hypothalamic astrocyte culture model was used in conjunction with design of experiments (DOE)-refined high-performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) methodology to investigate the hypothesis that glucoprivation alters astrocyte Gln content in a sex-specific manner. Critical mass spectrometric parameters for Gln derivative chromatographic response were identified by comparing the performance of central composite design, Box-Behnken design, and Optimal Design (OD)-A, -D, -I, -Distance, and -Modified Distance DOE models. The outcomes showed that the OD-A-generated response was superior relative to other design outcomes. Forecasted surface plot critical mass spectrometric parameters were maximized by OD-A, OD-Distance, and OD-Modified Distance designs. OD-A produced a high-performance method that yielded experimental run and forecasted surface plot maximal responses. Optimized mass spectrometric analysis of male versus female astrocyte Gln content provides novel evidence that glucoprivation significantly depletes this amino acid in female, but not in male, and that this sex-specific response may involve differential sensitivity to estrogen receptor signaling. This technological advance will facilitate efforts to ascertain how distinctive physiological and pathophysiological stimuli impact astrocyte Gln metabolism in each sex.
    Keywords:  Box-Behnken design; Optimal Design-A; Optimal Design-D; Optimal Design-Distance; Optimal Design-I; Optimal Design-Modified Distance; central composite design
  10. J Fungi (Basel). 2021 Jan 18. pii: E63. [Epub ahead of print]7(1):
      Due to the growing burden of fungal infections and a recent rise in antifungal resistance, antifungal susceptibility testing (AFST) is of increasing importance. The common methods of AFST have turnaround times of 24 to 48 h, and the available rapid methods are limited by applicability, cost-efficiency or accuracy. Given the urgency of adequate antifungal treatment in invasive mycoses, the need for the rapid and reliable detection of resistance is evident. In this systematic review and meta-analysis, we evaluated the diagnostic accuracy of AFST based on matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Twelve studies were reviewed, and data for the comparative analysis of their accuracy and methodology were systematically extracted. Compared to broth dilution as the gold standard, MALDI-TOF MS-based AFST reached a pooled sensitivity and specificity of 91% (95% Confidence Interval [CI], 84% to 96%) and 95% (95% CI, 90% to 98%), respectively. A comparative analysis showed that the sensitivity was higher for the semi-quantitative matrix-assisted laser desorption ionization Biotyper antibiotic susceptibility test rapid assay (MBT ASTRA) technique (96%) than for the correlate composite index (CCI) approach (85%), which is based on spectrum changes. Turnaround times below eight hours reached better diagnostic values than longer incubation periods, qualifying MALDI-TOF MS-based AFST as a rapid and accurate method for the detection of antifungal resistance.
    Keywords:  MALDI-TOF mass spectrometry; antifungal susceptibility testing; detection of antifungal resistance; rapid AFST
  11. Elife. 2021 Jan 20. pii: e61980. [Epub ahead of print]10
      Little is known about the metabolic regulation of rare cell populations because most metabolites are hard to detect in small numbers of cells. We previously described a method for metabolomic profiling of flow cytometrically-isolated hematopoietic stem cells (HSCs) that detects 60 metabolites in 10,000 cells (Agathocleous et al., 2017). Here we describe a new method involving hydrophilic liquid interaction chromatography and high-sensitivity orbitrap mass spectrometry that detected 160 metabolites in 10,000 HSCs, including many more glycolytic and lipid intermediates. We improved chromatographic separation, increased mass resolution, minimized ion suppression, and eliminated sample drying. Most metabolite levels did not significantly change during cell isolation. Mouse HSCs exhibited increased glycerophospholipids relative to bone marrow cells and methotrexate treatment altered purine biosynthesis. Circulating human melanoma cells were depleted for purine intermediates relative to subcutaneous tumors, suggesting decreased purine synthesis during metastasis. These methods facilitate the routine metabolomic analysis of rare cells from tissues.
    Keywords:  mouse; regenerative medicine; stem cells
  12. Analyst. 2021 Jan 22.
      Herein, a novel fluorescent method for the determination of GSH levels in aqueous solutions involving the utilization of citric acid as a derivatization reagent was developed. Therefore, the crucial parameters of the derivatization process were established from what has resulted in the development of a sensitive, reproducible, and accurate GSH assay. The method was validated, and its applicability in the characterization of the GSH concentration in dietary supplements concerning the selectivity in the determination of GSH over GSSG was both confirmed. The chemical structure of the new fluorophore 3-[(carboxymethyl)carbamoyl]-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyridine-7-carboxylic acid - CTPC was elucidated using detailed NMR: one-dimensional (1H, 13C), as well as two-dimensional NMR spectra (1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC, 1H-15N HSQC, 1H-15N HMBC) experiments. Besides, the essential optical, biological and antioxidative properties of CTPC were investigated.
  13. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Dec 24. pii: S1570-0232(20)31391-X. [Epub ahead of print]1164 122515
      As metabolic phenotyping (metabolomics, metabonomics and also lipidomics) gains in popularity and new investigators enter the field, the need to maintain and improve standards in publication is ever more pressing. In this perspective the requirements for information that should be included in manuscripts published in the Journal of Chromatography B, to ensure that the work is both credible and repeatable, are discussed. These include aspects such as study design, ethics, quality assurance (QA), quality control (QC) and data processing. In addition, aspects such as the level of confidence required for reporting metabolite identification (to a level where they could be subsequently used to develop hypotheses) are discussed.
    Keywords:  Data analysis; GC–MS; LC–MS; Mass spectrometry; Metabolite identification; Quality assurance; Quality control; Reporting standards
  14. Tunis Med. 2020 Jun;98(6): 496-500
      BACKGROUND: Paclitaxel (PTX) is an anticancer drug used in the treatment of many cancer , alone or in combination with other anti-tumors. It has a narrow therapeutic range, a large inter and intra-individual pharmacokinetic variability and haematological toxicity. The most effective pharmacokinetic parameter seems to be the time during which the plasma concentration is over 0.05 μmol/L.AIM: To develop and validate a new method for PTX quantitation in plasma using HPLC with UV/visible detection.
    METHODS: A rapid HPLC-UV method was developed for the determination of PTX level in plasma. All solvents used were HPLC grade.
    RESULTS: After liquid-liquid extraction, chromatographic separation was achieved using an RP 18 (250 mm) column. The mobile phase was composed of acetonitrile and 0.1 M potassium dihyrogenophosphate (49/51) (v/v). Clonazepam was used as internal standard. This technique was linear over the range 50 ng/mL to 1500 ng/mL (r= 0.998). The evaluation of precision showed that our method is repeatable with a within-day coefficient of variation (CV) ranging from 6.94 to 18.78 % and reproducible for three studied concentrations low, medium and high with day-to-day CV of 14.92, 10.46 and 11.8% respectively. Under these conditions, each analysis required no longer than 12.81 min.
    CONCLUSION: We have developed and validate a new assay for PTX monitoring using HPLC with UV detection which is sensible, specific, reliable and easy to carry out in clinical use for its therapeutic drug monitoring.
  15. Int J Mol Sci. 2021 Jan 16. pii: E864. [Epub ahead of print]22(2):
      Flavonoids represent an important class of secondary metabolites because of their potential health benefits and functions in plants. We propose a novel method for the comprehensive flavonoid filtering and screening based on direct infusion mass spectrometry (DIMS) analysis. The recently invented data mining procedure, the multi-step mass-remainder analysis (M-MARA) technique is applied for the effective mass spectral filtering of the peak rich spectra of natural herb extracts. In addition, our flavonoid-filtering algorithm facilitates the determination of the elemental composition. M-MARA flavonoid-filtering uses simple mathematical and logical operations and thus, it can easily be implemented in a regular spreadsheet software. A huge benefit of our method is the high speed and the low demand for computing power and memory that enables the real time application even for tandem mass spectrometric analysis. Our novel method was applied for the electrospray ionization (ESI) DIMS spectra of various herb extract, and the filtered mass spectral data were subjected to chemometrics analysis using principal component analysis (PCA).
    Keywords:  direct infusion mass spectrometry; flavonoid; mass spectral filtering; mass-remainder analysis; metabolomics; principal component analysis
  16. Methods Mol Biol. 2021 ;2251 1-17
      Phosphoinositide (PPI) lipids are a crucial class of low-abundance signaling molecules that regulate many processes within cells. Methods that enable simultaneous detection of all PPI lipid species provide a wholistic snapshot of the PPI profile of cells, which is critical for probing PPI biology. Here we describe a method for the simultaneous measurement of cellular PPI levels by metabolically labeling yeast or mammalian cells with myo-3H-inositol, extracting radiolabeled glycerophosphoinositides, and separating lipid species on an anion exchange column via HPLC.
    Keywords:  PI3,4,5P3; PI3,4P2; PI3,5P2; PI3P; PI4,5P2; PI4P; PI5P; Phosphatidylinositol; Phosphoinositide lipids; PtdIns; Radioactive metabolic labeling
  17. Anal Chim Acta. 2021 Feb 15. pii: S0003-2670(20)31215-0. [Epub ahead of print]1146 77-87
      Defects in sphingolipid metabolism have emerged as a common link across neurodegenerative disorders, and a deeper understanding of the lipid content in preclinical models and patient specimens offers opportunities for development of new therapeutic targets and biomarkers. Sphingolipid metabolic pathways include the formation of glycosphingolipid species that branch into staggeringly complex structural heterogeneity within the globoside and ganglioside sub-lipidomes. Characterization of these sub-lipidomes has typically relied on liquid chromatography-mass spectrometry-based (LC-MS) approaches, but such assays are challenging and resource intensive due to the close structural heterogeneity, the presence of isobaric and isomeric species, and broad dynamic range of endogenous glycosphingolipids. Here, we apply Structures for Lossless Ion Manipulations (SLIM)-based High Resolution Ion Mobility (HRIM)-MS to enable rapid, repeatable, quantitative assays with deep structural information sufficient to resolve endogenous brain gangliosides at the level of individual molecular species. Analyses were performed using a prototype SLIM-MS instrument equipped with a 13-m serpentine path which enabled resolution of closely related isomeric analytes such as GD1a d36:1 and GD1b d36:1 based on recorded mass-to-charge (m/z) and arrival times. To demonstrate the power of our methodology, brain extracts derived from wild-type mice hemi-brains were analyzed by HRIM-MS using flow injection analyses (FIA) without the need for additional separation by liquid chromatography. Endogenous ganglioside species were readily resolved, identified, and quantified by FIA-SLIM-MS analyses within 2 min per sample. Thus, the FIA-SLIM-MS platform enables robust quantification across a broad range of lipid species in biological specimens in a standardized assay format that is readily scalable to support studies with large sample numbers.
    Keywords:  Gangliosides; High resolution ion mobility; Isomers; Lipidomics; Mass spectrometry
  18. J Lipid Res. 2020 Dec;pii: S0022-2275(20)60017-7. [Epub ahead of print]61(12): 1539-1555
      A comprehensive and standardized system to report lipid structures analyzed by MS is essential for the communication and storage of lipidomics data. Herein, an update on both the LIPID MAPS classification system and shorthand notation of lipid structures is presented for lipid categories Fatty Acyls (FA), Glycerolipids (GL), Glycerophospholipids (GP), Sphingolipids (SP), and Sterols (ST). With its major changes, i.e., annotation of ring double bond equivalents and number of oxygens, the updated shorthand notation facilitates reporting of newly delineated oxygenated lipid species as well. For standardized reporting in lipidomics, the hierarchical architecture of shorthand notation reflects the diverse structural resolution powers provided by mass spectrometric assays. Moreover, shorthand notation is expanded beyond mammalian phyla to lipids from plant and yeast phyla. Finally, annotation of atoms is included for the use of stable isotope-labeled compounds in metabolic labeling experiments or as internal standards. This update on lipid classification, nomenclature, and shorthand annotation for lipid mass spectra is considered a standard for lipid data presentation.
    Keywords:  fatty acyls; glycerolipids; glycerophospholipids; lipidomics; mass spectrometry; sphingolipids; sterols