bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒02‒28
nine papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Anal Chem. 2021 Feb 23.
      Fatty acyl-coenzyme As (acyl-CoAs) are of central importance in lipid metabolism pathways. Short-chain acyl-CoAs are usually part of metabolomics, and medium- to (very) long-chain acyl-CoAs are focus of lipidomics studies. However, owing to the specific complex and amphiphilic nature contributed by fatty acyl chains and hydrophilic CoA moiety, lipidomic analysis of acyl-CoAs is still challenging, especially in terms of sample preparation and chromatographic coverage. In this work, we propose a derivatization strategy of acyl-CoAs based on phosphate methylation. After derivatization, full coverage (from free CoA to C25:0-CoA) and good peak shape in liquid chromatography were achieved. At the same time, analyte loss due to the high affinity of phosphate groups to glass and metallic surfaces was resolved, which is beneficial for routine analysis in large-scale lipidomics studies. A sample preparation method based on mixed-mode SPE was developed to optimize extraction recoveries and allow optimal integration of the derivatization process in the analytical workflow. LC-MS/MS was performed with targeted data acquisition by SRM transitions, which were constructed based on similar fragmentation rules observed for all methylated acyl-CoAs. To achieve accurate quantification, uniformly 13C-labeled metabolite extract from yeast cells was taken as internal standards. Odd-chain and stable isotope-labeled acyl-CoAs were used as surrogate calibrants in the same matrix. LOQs were between 16.9 nM (short-chain acyl-CoAs) and 4.2 nM (very-long-chain acyl-CoAs). This method was validated in cultured cells and was applied in HeLa cells and human platelets of coronary artery disease patients. It revealed distinct acyl-CoA profiles in HeLa cells and platelets. The results showed that this method can effectively detect acyl-CoAs in biological samples. Considering their central importance in many de novo lipid biosynthesis and remodeling processes, this targeted method offers a valid foundation for future lipidomics analysis of acyl-CoA profiles in biological samples, particularly those concerning metabolic syndrome.
  2. J Pharm Biomed Anal. 2021 Jan 29. pii: S0731-7085(21)00050-9. [Epub ahead of print]197 113938
      Sample pretreatment of cerebrospinal fluid (CSF) in metabolomics plays an important role in metabolic profiling study, especially for samples related to central nervous system diseases. However, there is few study about optimization of CSF metabolomics pretreatment. Therefore, it is an urgent need to optimize CSF pretreatment in order to promote the extraction efficiency of metabolites. In this study, CSF samples were separately subjected to nine different protein precipitation solvents and five different reconstitution solvents to establish the most effective pretreatment method before hydrophilic interaction (HILIC) and reverse-phase (RP) ultrahigh performance liquid chromatography mass spectrometry (UPLC/MS) analysis. The optimal conditions for different sample pretreatment methods were analyzed based on coverage (number of detected potential metabolites), stability (the relative standard deviation (RSD) distribution of metabolites) and the reproducibility of the data. Our results suggested that using EtOH or MeOH-EtOH-ACN (1:1:1, v/v/v) as the protein precipitation solvents and H2O-MeOH-ACN (2:1:1, v/v/v) as the reconstitution solvent were optimal methods for T3 column analysis. For HILIC column analysis, using EtOH to precipitate protein and H2O-MeOH-ACN (2:1:1, v/v/v) to reconstitute or MeOH to precipitate and 5 %ACN to reconstitute performed best. This developed UPLC/MS pretreatment method could provide better protein precipitation solvents and reconstitution solvents for global CSF metabolic analysis, potentially facilitating the comprehensive understanding of many central nervous system diseases.
    Keywords:  Cerebrospinal fluid; Metabolomics; Pretreatment methods; Protein precipitation solvent; Reconstitution solvent; UPLC/MS
  3. J Chromatogr A. 2021 Feb 08. pii: S0021-9673(21)00094-7. [Epub ahead of print]1641 461970
      Taking into account the growing demand for new analytical procedures that are appropriate for analysis of complex biological samples with increased sensitivity, accuracy and precision, a novel analytical method was described for the determination of underivatized amino acids in human plasma and urine samples. The presented analytical procedure involved the direct analysis of urine samples and the analysis of plasma samples followed by a simple protein precipitation protocol. Samples were analyzed using a simple and fast chromatographic method developed for the determination of 17 different amino acids by liquid chromatography - tandem mass spectrometry. The limit of detection and quantification values for amino acids were ranged between 0.03-2.26 µmol kg-1 and 0.09-7.54 µmol kg-1. Matrix effects of plasma and urine on the quantification of analytes were determined by spiking experiments. The accuracy of method was evaluated by matrix matching and quadruple isotope dilution strategies. Excellent accuracy and precision were obtained with the use isotope labeled amino acids demonstrating the high reliability and reproducibility of the proposed method. The percent recovery values were found to be between 98.70 - 101.68% with%RSD below than 1.62% for human plasma and 99.14 - 101.78% with%RSD below than 2.44% for urine samples.
    Keywords:  Free amino acids; Human plasma; Human urine; Isotope dilution; Liquid chromatography – mass spectrometry
  4. J Chromatogr A. 2021 Feb 09. pii: S0021-9673(21)00106-0. [Epub ahead of print]1641 461982
      The performance of an original CE-MS interface that allows the in-axis positioning of the electrospray with respect to the MS inlet was evaluated. The variations in the geometrical alignment of this configuration in the absence of a nebulizing gas afforded a significant reduction in the sheath-liquid flow rate from 3 µL/min to as low as 300 nL/min. The sheath liquid and BGE were respectively composed of H2O-iPrOHCH3COOH 50:50:1 (v/v/v) and 10% acetic acid (pH 2.2). A significant gain in sensitivity was obtained, and it was correlated to the effective mobility of the analytes. Compounds with low mobility values showed a greater sensitivity gain. Special attention was paid to the detection of proteinogenic amino acids. Linear response functions were obtained from 15 ng/mL to 500 ng/mL. The limits of quantification, as low as 34.3 ng/mL, were improved by a factor of up to six compared to the conventional configuration. The in-axis setup was ultimately applied to the absolute quantification of four important amino acids, alanine, tyrosine, methionine and valine, in standard reference material (NIST plasma). The accuracies ranged from 78 to 113%, thus demonstrating the potential of this configuration for metabolomics.
    Keywords:  Amino acids; CE-ESI-MS; Capillary electrophoresis; Mass spectrometry; Nanoflow interface
  5. J Mass Spectrom. 2021 Mar;56(3): e4709
      Derivatization reactions are commonly used in mass spectrometry to improve analyte signals, specifically by enhancing the ionization efficiency of those compounds. Vicinal diols are one group of biologically important compounds that have been commonly derivatized using boronic acid. In this study, a boronic acid with a tertiary amine was adapted for the derivatization of vicinal diol metabolites in B73 maize tissue cross-sections for mass spectrometry imaging analysis. Using this method, dozens of vicinal diol metabolites were derivatized, effectively improving the signal of those metabolites. Many of these metabolites were tentatively assigned using high-resolution accurate mass measurements. In addition, reaction interference and cross-reactivity with various other functional groups were systematically studied to verify data interpretation.
    Keywords:  MALDI; boronic acid; maize; mass spectrometry imaging; on-tissue derivatization; vicinal diol
  6. J Chromatogr A. 2021 Feb 01. pii: S0021-9673(21)00087-X. [Epub ahead of print]1640 461963
      Historically, retention parameters were reliably used as identification criterion in chromatographic analytical systems. However, pure analytical standards are necessary to determine the retention behaviour of a given compound. In recent decades, mass spectrometer (MS) became the detector of choice to obtain structural information on unknown peaks, thanks to the elucidation of fragments, often arisen from the loss of specific functional groups. The cost and the level of experience of the operators is surely higher compared to the use of retention data. Therefore, the aim of the present review is to describe the efforts in the introduction of the Linear Retention Index (LRI) in routine, interlaboratory applicable identification procedures. The requirements and the main challenges will be discussed, even compared to gas chromatography methods, in which LRI is stably used for identification purposes, usually in combination with MS spectral libraries. The higher number of LC-amenable molecules and the wide range of LC mobile phase compositions make the building of universal LRI database a very challenging task. The limitations encountered in the past decades are reported, together with new proposals in order to overcome such issues.
    Keywords:  Identification process; Linear retention index; Lipidomics; Oxygen heterocyclic compounds; Separation number
  7. J Chromatogr Sci. 2021 Feb 23. pii: bmab011. [Epub ahead of print]
      A simple and sensitive method was proposed for choline, carnitine, acetylcarnitine (ACa) and acetylcholine (ACh) separation in feed, blood and urine of animals by using ion chromatography (IC) and detection by tandem mass spectrometry (MS/MS). Analytes were extracted using a mixture of acetonitrile and water, purified by C18 solid-phase extraction columns, separated via IC with an IonPac SCS-1 column and detected by an MS/MS detector by using isotopic internal standards for quantification. The effects of different chromatographic parameters on the separation were also investigated. Under optimal conditions, the recovery was >90%, with the relative standard deviations of <15%. The proposed method was highly reliable for the simultaneous determination of choline, carnitine, ACa and ACh in feed, blood and urine.
  8. J Chromatogr A. 2021 Feb 12. pii: S0021-9673(21)00113-8. [Epub ahead of print]1641 461989
      Open tubular liquid chromatography (OT-LC) can provide superior chromatographic performance and more favorable mass spectrometry (MS) detection conditions. These features could provide enhanced sensitivity when coupled with electrospray ionization sources (ESI-) and lead to unprecedented detection capabilities if interfaced with a highly structural informative electron ionization (EI) source. In the past, the exploitation of OT columns in liquid chromatography evolved slowly. However, the recent instrumental developments in capillary/nanoLC-MS created new opportunities in developing and applying OT-LC-MS. Currently, the analytical advantages of OT-LC-MS are mainly exploited in the fields of proteomics and biosciences analysis. Nevertheless, under the right conditions, OT-LC-MS can also offer superior chromatographic performance and enhanced sensitivity in analyzing small molecules. This review will provide an overview of the latest developments in OT-LC-MS, focusing on the wide variety of employed separation mechanisms, innovative stationary phases, emerging column fabrication technologies, and new OT formats. In the same way, the OT-LC's opportunities and shortcomings coupled to both ESI and EI will be discussed, highlighting the complementary character of those two ionization modes to expand the LC's detection boundaries in the performance of targeted and untargeted studies.
    Keywords:  electron ionization; mass spectrometry; nano-electrospray ionization; nano-liquid chromatography; open tubular column; open tubular liquid chromatography
  9. J Pharm Biomed Anal. 2021 Feb 15. pii: S0731-7085(21)00081-9. [Epub ahead of print]197 113969
      Previous studies have reported that nucleic acid methylation is a critical element in cardiovascular disease, and most studies mainly focused on sequencing and biochemical research. Here we developed an Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) method for the quantification analysis of the dissociative epigenetic modified nucleosides (5mdC, 5mrC, m6A) in Myocardial Infarction (MI) SD rats from different periods (1 week, 4 weeks, 8 weeks) after the surgery. The samples for analysis were obtained from heart tissue and blood of the rats. All the quantification results are compared with the sham-operated group. Total RNA and DNA were isolated by enzymatic hydrolytic methods before the UPLC-MS/MS analysis. The statistical analysis demonstrates the dynamic changes of modified nucleosides in MI rats, and it showed good specificity, accuracy, stability and less samples were needed in the method. In this paper, we discovered that the concentration of 5mdC, 5mrC, m6A from heart tissue significantly increased at 8 weeks after the surgery. Furthermore, UPLC-MS/MS helps us observe the similar change of the concentration of those 3 methylated biomarkers in peripheral blood after 8 weeks. The result shows that the dynamic process of those 3 methylated biomarkers in peripheral blood is related to the content of methylated biomarkers from the heart tissue. Based on the scientific evidence available, we proved that the methylation of genetic materials in peripheral blood is similar to myocardial infarction tissue. The relation between them indicates that peripheral blood could be a promising alternative to the heart tissue which monitor the level of methylation and MI diagnosis-aided.
    Keywords:  LC–MS/MS; Myocardial infarction; Nucleic acid methylation; Peripheral blood