bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒05‒30
fourteen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Anal Chim Acta. 2021 Jun 08. pii: S0003-2670(21)00334-2. [Epub ahead of print]1163 338508
      The focus of this work was the implementation of ion mobility (IM) and a prototype quadrupole driver within data independent acquisition (DIA) using a drift tube IM-QTOFMS aiming to improve the level of confidence in identity confirmation workflows for non-targeted metabolomics. In addition to conventional all ions (IM-AI) acquisition, quadrupole resolved all ions (IM-QRAI) acquisition allows a drift time-directed precursor ion isolation in DIA using sequential isolation of precursor ions using mass windows of up to 100 Da which can be rapidly ramped across single ion mobility transients (i.e., <100 ms) according to the arrival times of precursor ions. Both IM-AI and IM-QRAI approaches were used for identity confirmation and relative quantification of metabolites in cellular extracts of the cell factory host Pichia pastoris. Samples were spiked with a uniformly 13C-labeled (U13C) internal standard and LC with low-field drift tube IM separation was used in combination with IM-AI and IM-QRAI. Combining excellent hardware performance and correlation of IM arrival times of natural (natC) and U13C metabolites enabled alignment of signals in the arrival time domain (DTCCSN2 differences ≤0.3%), and, in the case of IM-QRAI operation, maintenance of quantitative signals in comparison to IM-AI. The combination of tailored IM-QRAI methods for precursor ion isolation and IM separation also minimized the occurrence of spectral interferences in complex DIA datasets. Combined use of the software tools MS-DIAL, MS-Finder and Skyline for peak picking, feature alignment, reconciliation of natC and U13C isotopologue pairs, deconvolution of fragment spectra from DIA data, identity confirmation (including DTCCSN2) and targeted re-extraction of datafiles were employed for the data processing workflow. Overall, the combined new acquisition and data processing approaches enabled 87 metabolites to be identified between Level 1 (identified by standard compound) and Level 3.2 (accurate mass spectrum and number of carbons confirmed). The developed methods constitute promising metabolomics discovery tools and can be used to elucidate the number of carbon atoms present in unknown metabolites in stable isotope-supported metabolomics.
    Keywords:  CCS; HPLC; Ion mobility-mass spectrometry; Metabolomics; Stable isotope labeling; Yeast
  2. Mass Spectrom Rev. 2021 May 24.
      Single cell analysis has drawn increasing interest from the research community due to its capability to interrogate cellular heterogeneity, allowing refined tissue classification and facilitating novel biomarker discovery. With the advancement of relevant instruments and techniques, it is now possible to perform multiple omics including genomics, transcriptomics, metabolomics or even proteomics at single cell level. In comparison with other omics studies, single-cell metabolomics (SCM) represents a significant challenge since it involves many types of dynamically changing compounds with a wide range of concentrations. In addition, metabolites cannot be amplified. Although difficult, considerable progress has been made over the past decade in mass spectrometry (MS)-based SCM in terms of processing technologies and biochemical applications. In this review, we will summarize recent progress in the development of promising MS platforms, sample preparation methods and SCM analysis of various cell types (including plant cell, cancer cell, neuron, embryo cell, and yeast cell). Current limitations and future research directions in the field of SCM will also be discussed.
    Keywords:  mass spectrometry; metabolomics; single cell analysis; single-cell metabolomics
  3. J Chromatogr A. 2021 May 05. pii: S0021-9673(21)00307-1. [Epub ahead of print]1649 462183
      In the European Union, cosmetic products are regulated by the Cosmetics Regulation (EC) No. 1223/2009. The Analytical Working Group of the International Fragrance Association (IFRA) suggested that a new sensitive analytical method is needed to determine psoralens in finished cosmetic products. This research provides an HPLC-MS/MS method for the quality control of 20 furocoumarins, 8 coumarins and 7 polymethoxyflavones in cosmetics. Thanks to the high sensitivity of the tandem mass spectrometry detection in Multiple Reaction Monitoring mode, psoralens contained in trace have been quantified in different products. The Limits of Quantifications were in the range 0.3-74 μg L-1. A reliable identification was achieved combining the Linear Retention Index (LRI) system with the MS and MS/MS libraries. In particular, the attribution was based on the spectra similarity filtered by the LRI parameter. The robustness and reproducibility of the LRI approach was demonstrated by the comparison of the HPLC-MS/MS results here reported with those obtained in our previous study, by using an HPLC-PDA system. This method could be taken into account for quality control of furocoumarins in cosmetics, and by the main associations, such as IFRA, for the purpose of issuing new opinions.
    Keywords:  Furocoumarins; LC-MS/MS; Linear Retention Index; Method validation; Quality control; Weighting factors
  4. Anal Chim Acta. 2021 Jun 29. pii: S0003-2670(21)00399-8. [Epub ahead of print]1166 338573
      Acidic lipids are associated with the regulation of the structure and function of membrane proteins. Therefore, accurate and highly precise analysis of acidic lipids is important for elucidating their biological roles and pathological mechanisms. In this study, an enhanced analytical method for the separation and quantification of acidic lipids, including phosphatidylserine (PS), phosphatidic acid (PA), cardiolipin, and their lyso-derivatives, was developed using nanoflow ultrahigh performance liquid chromatography-electrospray ionisation-tandem mass spectrometry. The separation and mass spectrometry detection of acidic lipids were optimised in terms of peak tailing and time-based separation efficiencies, with carbamate-embedded C18 as the stationary phase, in the presence of an appropriate liquid chromatography solvent modifier. This newly developed method was applied to analyse a lipid extract from porcine brain. A significant increase in the number of acidic lipids identified (176 vs. 134), including intact monolysocardiolipin (17 vs. 4), was observed with the new method compared with conventional C18. The quantification of acidic lipids was validated with plasma standard (NIST SRM 1950) spiked with a number of LPS and PS standards, and acceptable accuracy (<15%) was obtained. The present method was found to be reliable for the acidic lipid analysis based on qualitative results from tissue extract and plasma samples.
    Keywords:  Acidic lipid; Lipidomics; Mass spectrometry; Nanoflow UHPLC; Porcine brain lipids; Validation
  5. Anal Chim Acta. 2021 Jul 04. pii: S0003-2670(21)00403-7. [Epub ahead of print]1167 338577
      LC-MS-based metabolomics offers the potential of discovering biomarkers and exploring the mechanisms of underlying diseases. However, given the enormous polarity difference between metabolites, simultaneous across-polarity quantification for broad metabolome coverage has still been challenged by limited sample preparation methods and other hurdles. Herein, we proposed a consecutive extraction strategy based on nanoconfined liquid phase nanoextraction (NLPNE) technique. By modulating the nanoconfined solvents and coupling with LC-MS/MS, this method could simultaneously quantify metabolites with different polarities assigned to three classes, including amines (high polarity), steroids (middle polarity) and lysophosphatidylcholines (LPCs, low polarity) with high selectivity and high efficiency. During the systematical optimization of the extraction workflow, response surface methodology (RSM) was used for key parameters optimization. And consecutive extraction mode and parallel extraction mode were proposed in the choice of integrated extraction strategy. Then the consecutive NLPNE method was compared with two conventional sample preparation methods in metabolomics, protein precipitation (PP) and liquid-liquid extraction (LLE). After systematical validation, the consecutive NLPNE method coupled with LC-MS/MS was successfully applied in the identification of multi-metabolites indexes for lung, colorectal, and gastric cancer plasma samples from healthy controls, and among different types of cancer with student's t-test, partial least squares discriminant analysis (PLS-DA) and logistic regression-receiver operating characteristic (ROC) curve analysis. Taken together, the developed methodology is a versatile candidate in metabolomics for high coverage detection and may be used as a powerful tool for cancer diagnosis.
    Keywords:  Cancer biomarkers; Consecutive nanoconfined liquid phase nanoextraction; Metabolomics; Sample preparation
  6. Methods Mol Biol. 2021 ;2295 441-454
      Along with the increase in knowledge on lipid metabolism during the last years, different lipid databases were established in a web-based system. This chapter presents an overview on plant lipid databases for simple and complex lipids focusing on nomenclature, structures as well as physical and chemical properties. Many databases provide information on methods and protocols for lipid isolation, fractionation, and analysis, including lipidomic procedures. References to the lipid literature are included in all databases. Additional data including mass spectra derived from GC-MS, LC-MS, and LC-MS/MS experiments are included in specialized lipid databases. An introduction is presented on how to use the most important lipid databases.
    Keywords:  Database; Library; Lipidomics; Mass spectrometry; Structures
  7. Methods Mol Biol. 2021 ;2295 117-133
      Total acyl lipid collision-induced dissociation time-of-flight (TAL-CID-TOF) mass spectrometry uses a quadrupole time-of-flight (QTOF) mass spectrometer to rapidly provide a comprehensive fatty acid composition of a biological lipid extract. Samples are infused into a QTOF instrument, operated in negative mode, and the quadrupole is used to transfer all, or a wide mass range of, precursor ions to the collision cell for fragmentation. Time-of-flight-acquired mass spectra provide mass accuracy and resolution sufficient for chemical formula determination of fatty acids in the complex mixture. Considering the limited number of reasonable CHO variants in fatty acids, one can discern acyl anions with the same nominal mass but different chemical formulas. An online application, LipidomeDB Data Calculation Environment, is employed to process the mass spectral output data and match identified fragments to target fragments at a resolution specified by the user. TAL-CID-TOF methodology is a useful discovery or screening tool to identify and compare fatty acid profiles of biological samples.
    Keywords:  Collision-induced dissociation; Fatty acid analysis; Fatty acid composition; Fatty acyl; LipidomeDB Data Calculation Environment (DCE); Lipidomics; Mass spectrometry; QTOF; TAL-CID-TOF
  8. Methods Mol Biol. 2021 ;2295 417-438
      Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has emerged as a major analytical platform for the determination and localization of lipid metabolites directly from tissue sections. Unlike analysis of lipid extracts, where lipid localizations are lost due to homogenization and/ or solvent extraction, MALDI-MSI analysis is capable of revealing spatial localization of metabolites while simultaneously collecting high chemical resolution mass spectra. Important considerations for obtaining high quality MALDI-MS images include tissue preservation, section preparation, MS data collection and data processing. Errors in any of these steps can lead to poor quality metabolite images and increases the chance for metabolite misidentification and/ or incorrect localization. Here, we present detailed methods and recommendations for specimen preparation, MALDI-MS instrument parameters, software analysis platforms for data processing, and practical considerations for each of these steps to ensure acquisition of high-quality chemical and spatial resolution data for reconstructing MALDI-MS images of plant tissues.
    Keywords:  Camelina sativa; Data processing; Lipids; MALDI mass spectrometry imaging; Phosphatidylcholine; Plant tissues; Seeds; Triacylglycerol
  9. Front Chem. 2021 ;9 659656
      Metabolomics data analysis depends on the utilization of bioinformatics tools. To meet the evolving needs of metabolomics research, several integrated platforms have been developed. Our group has developed a desktop platform IP4M (integrated Platform for Metabolomics Data Analysis) which allows users to perform a nearly complete metabolomics data analysis in one-stop. With the extensive usage of IP4M, more and more demands were raised from users worldwide for a web version and a more customized workflow. Thus, iMAP (integrated Metabolomics Analysis Platform) was developed with extended functions, improved performances, and redesigned structures. Compared with existing platforms, iMAP has more methods and usage modes. A new module was developed with an automatic pipeline for train-test set separation, feature selection, and predictive model construction and validation. A new module was incorporated with sufficient editable parameters for network construction, visualization, and analysis. Moreover, plenty of plotting tools have been upgraded for highly customized publication-ready figures. Overall, iMAP is a good alternative tool with complementary functions to existing metabolomics data analysis platforms. iMAP is freely available for academic usage at (License MPL 2.0).
    Keywords:  correlation-based network; metabolomics; statistical analysis; visualization; workflow
  10. Data Brief. 2021 Jun;36 107091
      Measuring bile acids in feces has an important role in disease prevention, diagnosis, treatment, and can be considered a measure of health status. Therefore, the primary aim was to develop a sensitive, robust, and high throughput liquid chromatography tandem mass spectrometry method with minimal sample preparation for quantitative determination of bile acids in human feces applicable to large cohorts. Due to the chemical diversity of bile acids, their wide concentration range in feces, and the complexity of feces itself, developing a sensitive and selective analytical method for bile acids is challenging. A simple extraction method using methanol suitable for subsequent quantification by liquid chromatography tandem mass spectrometry has been reported in, "Extraction and quantitative determination of bile acids in feces" [1]. The data highlight the importance of optimization of the extraction procedure and the stability of the bile acids in feces post-extraction and prior to analysis and after several freeze-thaw cycles.
    Keywords:  Bile acids; Extraction; Feces; LC-MS/MS; Stability
  11. Amino Acids. 2021 May 24.
      The quantitation and qualification of amino acids are most commonly used in clinical and epidemiological studies, and provide an excellent way of monitoring compounds in human fluids which have not been monitored previously, to prevent some diseases. Because of this, it is not surprising that scientific interest in evaluating these compounds has resurfaced in recent years and has precipitated the development of a multitude of new analytical techniques. This review considers recent developments in HPLC analytics on the basis of publications from the last few years. It helps to update and systematize knowledge in this area. Particular attention is paid to the progress of analytical methods, pointing out the advantages and drawbacks of the various techniques used for the preparation, separation and determination of amino acids. Depending on the type of sample, the preparation conditions for HPLC analysis change. For this reason, the review has focused on three types of samples, namely urine, blood and cerebrospinal fluid. Despite time-consuming sample preparation before HPLC analysis, an additional derivatization technique should be used, depending on the detection technique used. There are proposals for columns that are specially modified for amino acid separation without derivatization, but the limit of detection of the substance is less beneficial. In view of the fact that amino acid analyses have been performed for years and new solutions may generate increased costs, it may turn out that older proposals are much more advantageous.
    Keywords:  Amino acids; Blood; Cerebrospinal fluid; HPLC; Sample preparation; Urine
  12. J Sep Sci. 2021 May 27.
      γ-Glutamylpeptides have been identified as potential biomarkers for a number of diseases including cancer, diabetes, and liver disease. In this study, we developed and validated a novel quantitative analytical strategy for measuring γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline, all of which have been previously reported as potential biomarkers for prostate cancer, in HeLa cells using UHPLC-MS/MS. A BEH C18 column was used as the stationary phase. Mobile phase A was 99:1 water:formic acid, and mobile phase B was acetonitrile. Chemical isotope labeling using benzoyl chloride was used as the internal standardization strategy. Sample preparation consisted of the addition of water to a frozen cell pellet, sonication, derivatization, centrifugation, and subsequent addition of an internal standard solution. The method was validated for selectivity, accuracy, precision, linearity, and stability. The determined concentrations of γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline in HeLa cells were 1.92 ± 0.06, 10.8 ± 0.4, and 1.96 ± 0.04 pmol/ mg protein, respectively. In addition, the qualitative analysis of these analytes in human serum was achieved using a modified sample preparation strategy. To the best of our knowledge, this is the first report of the use of benzoyl chloride for chemical isotope labeling for metabolite quantitation in cells. This article is protected by copyright. All rights reserved.
    Keywords:  Chemical isotope labeling; Derivatization; Internal standard; Quantification; γ-Glutamylpeptide
  13. Curr Opin Biotechnol. 2021 May 25. pii: S0958-1669(21)00065-3. [Epub ahead of print]71 1-8
      Identifying the factors and mechanisms that regulate metabolism under normal and diseased states requires methods to quantify metabolic fluxes of live tissues within their physiological milieu. A number of recent developments have expanded the reach and depth of isotope-based in vivo flux analysis, which have in turn challenged existing dogmas in metabolism research. First, minimally invasive techniques of intravenous isotope infusion and sampling have advanced in vivo metabolic tracer studies in animal models and human subjects. Second, recent breakthroughs in analytical instrumentation have expanded the scope of isotope labeling measurements and reduced sample volume requirements. Third, innovative modeling approaches and publicly available software tools have facilitated rigorous analysis of sophisticated experimental designs involving multiple tracers and expansive metabolomics datasets. These developments have enabled comprehensive in vivo quantification of metabolic fluxes in specific tissues and have set the stage for integrated multi-tissue flux assays.
  14. J Chromatogr A. 2021 May 11. pii: S0021-9673(21)00360-5. [Epub ahead of print]1649 462236
      The widespread presence of lipid hydroperoxides in foodstuffs and biological samples has aroused great attentions in recent years, while it remains challenging for analysis of the fragility of O - O bond linkage of peroxides. In this present study, we explored the utility of electrospray ionization mass spectrometry (ESI-MS) for characterization of two fatty acid hydroperoxides from oxidation of linoleic acid and α-linolenic acid, which are the essential fatty acids abundant in many seeds and vegetable oils. The results indicated that in-source fragmentation occurred in the detection of the two fatty acid hydroperoxides in both positive and negative ion modes, which yielded characteristic fragments for ESI-MS analysis. In addition, the genotoxicity of fatty acid hydroperoxides for generation of nucleoside adducts was investigated. It was found that a variety of nucleoside adducts were formed from the reactions of fatty acid hydroperoxides and nucleosides. Furthermore, the decomposition products of the fatty acid hydroperoxides were determined, which provided evidence to elucidate the reaction mechanism for formation of nucleoside adducts.
    Keywords:  Fatty acid hydroperoxides; Mass spectrometry; Nucleoside adducts; Vegetable oil; α, β-unsaturated aldehydes