bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒08‒16
ten papers selected by
Sofia Costa
Cold Spring Harbor Laboratory


  1. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Jul 29. pii: S1570-0232(19)31797-0. [Epub ahead of print]1154 122288
    Darville LNF, Cline JK, Rozmeski C, Martinez YC, Rich S, Eschrich SA, Egan KM, Yaghjyan L, Koomen JM.
      In order to undertake an epidemiologic study relating levels of parent estrogens (estrone and estradiol) and estrogen metabolites (EMs) to other breast cancer risk factors, we have optimized methods for EM quantification with ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). A two-step approach was adopted; the first step comprised method development and evaluation of the method performance. The second step consisted of applying this method to quantify estrogens in postmenopausal women and determine if the observed patterns are consistent with the existing literature and prior knowledge of estrogen metabolism. First, 1-methylimidazole-2-sulfonyl chloride (MIS) was used to derivatize endogenous estrogens and estrogen metabolites in urine from study participants. Since C18 reversed phase columns have not been able to separate all the structurally related EMs, we used a C18-pentafluorophenyl (PFP) column. The parent estrogens and EMs were baseline resolved with distinct retention times on this C18-PFP column using a 30 min gradient. This method was used to quantify the parent estrogens and 13 EMs in urine samples collected in an initial pilot study involving males as well as pre- and peri-menopausal females to assess a range of EM levels in urine samples and enable comparison to the previous literature for assay evaluation. Detection limits ranged from 1 - 20 pg/mL depending on the EM. We evaluated matrix effects and interference as well as the intra- and inter-batch reproducibility including hydrolysis, extraction, derivatization and LC-MS analysis using charcoal-stripped human urine as a matrix. Methods were then applied to the measurement of estrogens in urine samples from 169 postmenopausal women enrolled in an epidemiological study to examine relationships between breast cancer risk, the intestinal microbiome, and urinary EMs. The results from our cohort are comparable to previous reports on urinary EMs in postmenopausal women and enabled thorough evaluation of the method.
    Keywords:  Derivatization; Estrogens; Liquid chromatography-high resolution mass spectrometry; Metabolites
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122288
  2. Anal Chem. 2020 Aug 11.
    Sun C, Liu W, Geng Y, Wang X.
      Carboxyl-Containing Metabolites (CCMs) play indispensable roles in cell energy metabolism and cell-cell signalling. Profiling tissue CCMs with spatial signatures is significant for the understanding of molecular histology, and may provide new clues to uncover the complex metabolic reprogramming of organisms in response to external or internal stimuli. Here, we develop a sensitive on-tissue CCMs derivatization method, coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to visualize the spatial distributions of CCMs in biological tissues. A novel reagent, N,N,N-trimethyl-2-(piperazin-1-yl)ethan-1-aminium iodide (TMPA), was synthesized and used for the on-tissue derivatization of CCMs. Meanwhile, the on-tissue derivatization efficiency was significantly improved by introducing acetonitrile gas in the incubation system. With this methodology, a total of 28 CCMs including 5 tricarboxylic acid cycle intermediates, 20 fatty acids, and 3 bile acids were successfully detected and imaged in rat kidney tissues. More importantly, the introduction of quaternary ammonium group into the chemical structure of CCMs enables simultaneous MALDI-MS imaging of tricarboxylic acid cycle intermediates, fatty acids, bile acids, and their metabolic pathway-related metabolites such as carnitines, cholines, glycerophosphocholine, phospholipids, etc. in positive ion mode. This on-tissue derivatization MALDI-MSI approach was proved to be a powerful tool for probing the distributions and spatial metabolic networks of CMMs in biological tissues.
    DOI:  https://doi.org/10.1021/acs.analchem.0c02303
  3. Biochem Med (Zagreb). 2020 Oct 15. 30(3): 030701
    Debeljak Ž, Marković I, Pavela J, Lukić I, Mandić D, Mandić S, Horvat V, Šerić V.
      Introduction: There is a growing amount of evidence showing the significant analytical bias of steroid hormone immunoassays, but large number of available immunoassays makes conduction of a single comprehensive study of this issue hardly feasible. Aim of this study was to assess the analytical bias of six heterogeneous immunoassays for serum aldosterone, cortisol, dehydroepiandrosterone sulphate (DHEAS), testosterone, 17-hydroxyprogesterone (OHP) and progesterone using the liquid chromatography coupled to the tandem mass spectrometry (LC-MS/MS).Materials and methods: This method comparison study included 49 serum samples. Testosterone, DHEAS, progesterone and cortisol immunoassays were performed on the Abbott Architect i2000SR or Alinity i analysers (Abbott Diagnostics, Chicago, USA). DiaSorin's Liaison (DiaSorin, Saluggia, Italy) and DIAsource's ETI-Max 3000 analysers (DIAsource ImmunoAssays, Louvain-La-Neuve, Belgium) were chosen for aldosterone and OHP immunoassay testing, respectively. All immunoassays were evaluated against the LC-MS/MS assay relying on the commercial kit (Chromsystems, Gräfelfing, Germany) and LCMS-8050 analyser (Shimadzu, Kyoto, Japan). Analytical biases were calculated and method comparison was conducted using weighted Deming regression analysis.
    Results: Depending on the analyte and specific immunoassay, mean relative biases ranged from -31 to + 137%. Except for the cortisol, immunoassays were positively biased. For none of the selected steroids slope and intercept 95% confidence intervals simultaneously contained 0 and 1, respectively.
    Conclusions: Evaluated immunoassays failed to satisfy requirements for methods' comparability and produced significant analytical biases in respect to the LC-MS/MS assay, especially at low concentrations.
    Keywords:  LC-MS/MS; bias; immunoassay; method comparison; steroid hormone
    DOI:  https://doi.org/10.11613/BM.2020.030701
  4. Methods Mol Biol. 2021 ;2187 27-35
    Qin C, Pan M, Han X.
      Lipid rafts are microdomains on plasma membrane that contain high levels of cholesterol and sphingolipids. Because of the detergent-resistant property of lipid rafts, lipid rafts isolated by methods that use detergents frequently yield different results. Artifacts can also be introduced through the use of detergents. These limitations could be overcome with a detergent-free method which eliminates possible artificial influences. Importantly, lipid rafts prepared with a detergent-free method is more compatible to mass spectrometric analysis since the ion suppression effect is largely reduced.This chapter describes a detergent-free two-step method for preparation of lipid rafts. Firstly, a purified plasma membrane fraction is prepared from cells by sedimentation of the postnuclear supernatant (PNS) in a Percoll gradient. Secondly, the as-prepared plasma membranes are sonicated to release lipid rafts which are further isolated by flotation in a continuous gradient of Optiprep solution. Then, we introduce a typical shotgun lipidomics workflow that can be used as a cost-effective and relatively high throughput method to determine the lipidomes of lipid rafts.The method also makes an easy start for lipidomics studies.
    Keywords:  Detergent-free preparation; Lipid rafts; Mass spectrometry; Plasma membrane; Shotgun lipidomics
    DOI:  https://doi.org/10.1007/978-1-0716-0814-2_2
  5. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Aug 01. pii: S1570-0232(20)30666-8. [Epub ahead of print]1155 122289
    Hu B, Song C, Li L, Wang M, Jia S, Li S, Du Z, Ding X, Jiang H.
      PCs and SMs are the major types of glycerophospholipids and sphingophospholipids, the two main categories of phospholipids (PLs). To study the qualitative distribution of serum phosphatidylcholine (PC) and sphingomyelin (SM) in human and three rodent species, liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap-MS/MS) was used to identify them comprehensively through the accurate mass measurement of both precursor ions and their corresponding product ions. Based on the fragmentation rules of standards, the product ions at m/z 184.0733 were filtered to maximally screen possible PC and SM molecules. For PC, the fatty acid at sn-1 and sn-2 of the glycerol backbone was identified based on the product ions in negative mode. A total of 91 PCs and 31 SMs molecular species, consisting of 166 PCs and 39 SMs regioisomers, were detected in human serum, which is the most comprehensive identification of PC and SM species in serum. The qualitative distributions of PC in rat and SM in golden hamster, respectively, were more similar with that of human from an overall perspective. Those results provided guidance regarding to the animal model selection for mimicking lipid related-syndromes or diseases in human.
    Keywords:  LC-MS/MS; Phosphatidylcholine; Profiling; Sphingomyelin
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122289
  6. Forensic Sci Int. 2020 Jul 31. pii: S0379-0738(20)30291-7. [Epub ahead of print]315 110429
    Fan SY, Zang CZ, Shih PH, Ko YC, Hsu YH, Lin MC, Tseng SH, Wang DY.
      Synthetic cathinones, which are a group of β-keto analogs of phenethylamine, have been reported as the most emerging new psychoactive substances in the past decade. The quantity and variety of synthetic cathinones have continued to increase, which poses considerable risks to public health and social security. In this study, an analytical method based on liquid chromatography-tandem mass spectrometry (LCMS/MS) was established for the simultaneous determination of 73 synthetic cathinones and related metabolites in urine. The chromatographic analysis was performed using a Kinetex® Biphenyl column (10 cm ×2.1 mm, 1.7 μm), applying a gradient mobile phase, comprising 0.1 % formic acid aqueous solution with 5 mM ammonium acetate and 0.1 % formic acid methanolic solution; the entire run time of the analysis was within 8 min. The multiple reaction monitoring (MRM) mode was employed to collect the monitoring and quantitative ion pairs. Intra-day/inter-day precision and accuracy were less than 10 % for all the studied analytes. The limits of detection and quantification for all the analytes were 0.1-0.5 ng/mL and 0.5-1.0 ng/mL, respectively. The matrix effect was satisfactory for all the analytes, with a deviation lower than 20 %. The present method was further applied to 67 authentic urine samples in which 13 different synthetic cathinones were detected from 32 positive samples. The abuse of poly-synthetic cathinones was examined that up to seven items was detected in one case from authentic samples in this study.
    Keywords:  Drug of abuse; LC–MS/MS; New psychoactive substance; Synthetic cathinones; Urine
    DOI:  https://doi.org/10.1016/j.forsciint.2020.110429
  7. Electrophoresis. 2020 Aug 09.
    Höcker O, Knierman M, Meixner J, Neusüß C.
      Capillary electrophoresis hyphenated to electrospray ionization mass spectrometry (CE-ESI-MS) is a well-established technique to analyze charged analytes in complex samples. Although various interfaces for CE-MS coupling are commercially available, the development of alternatives which combine sensitivity, simplicity and robustness remains a topic of research. In this work, a nanoflow sheath liquid CE-MS interface with two movable capillaries inside a glass emitter is described. The setup enables a separation mode and a conditioning mode to guide the separation capillary effluent either into the electrospray or to the waste, respectively. This enables to exclude parts of the analysis from MS detection and unwanted matrix components reaching the mass spectrometer, comparable to divert valves in LC-MS coupling. Also, this function improves the overall robustness of the system by reduction of particles blocking the emitter. Preconditioning with electrospray interfering substances and even the application of coating materials for every analysis is enabled, even while the separation capillary is built into the interface with running electrospray. The functionality is demonstrated by analyses of heavy matrix bioreactor samples. Overall, this innovation offers a more convenient installation of the interface, improved handling with an extended lifetime of the emitter tips and additional functions compared to previous approaches, while keeping the higher sensitivity of nanoflow CE-MS-coupling. This article is protected by copyright. All rights reserved.
    Keywords:  CE-MS; Capillary Electrophoresis; Interfacing; Mass Spectrometry
    DOI:  https://doi.org/10.1002/elps.202000169
  8. J Microbiol Methods. 2020 Aug 11. pii: S0167-7012(20)30736-3. [Epub ahead of print] 106020
    Horvath TD, Haidacher SJ, Oezguen N, Hoch KM, Auchtung JM, Haag AM.
      Recently, an opportunity to perform a broad ruggedness assessment of our liquid chromatography-tandem mass spectrometry (LC-MS/MS) system presented itself during the analytical planning phase of a large-scale human fecal microbiome study. The specific aim of this project was to study the microbial-mediated metabolism of a targeted set of bile acids/salts by mixed bacterial communities cultured from the feces of 12 healthy volunteers when grown in a custom growth medium and following exposure to different clinically-relevant antibiotics. The magnitude of this study offered a rare opportunity to significantly stress procedures and LC-MS/MS system components comprised in our bile acid/salt targeted metabolomics method. With this second specific aim in mind, we modified the sample analysis plan to include a series of figure-of-merit (FoM)-based tests that are commonly used in regulated bioanalytical labs to assess LC and MS system ruggedness for a specific assay - these FoM-based testing parameters were monitored continuously over the course of sample analysis and the results are presented in this report. In total, the assessment included 1206 sequential injections (180 calibration standards, 136 blank-internal standard samples, and 890 diluted medium samples) that took place over 8-days. Completion of the 8-days of non-stop sample analysis revealed no critical hardware or software failures, and the analysis of the FoM-based tests indicated no observable degradation of system performance over the number of samples and time tested. The FoM-based test metrics presented may be used as a template to assess the ruggedness of any LC-MS/MS-based targeted metabolomics workflow.
    Keywords:  Bile acids; Bile salts; Bioanalytical chemistry; High-performance liquid chromatography-tandem mass spectrometry; High-throughput sample analysis
    DOI:  https://doi.org/10.1016/j.mimet.2020.106020
  9. Sci Rep. 2020 Aug 07. 10(1): 13368
    Manzini S, Busnelli M, Colombo A, Kiamehr M, Chiesa G.
      Lipidomic analyses address the problem of characterizing the lipid components of given cells, tissues and organisms by means of chromatographic separations coupled to high-resolution, tandem mass spectrometry analyses. A number of software tools have been developed to help in the daunting task of mass spectrometry signal processing and cleaning, peak analysis and compound identification, and a typical finished lipidomic dataset contains hundreds to thousands of individual molecular lipid species. To provide researchers without a specific technical expertise in mass spectrometry the possibility of broadening the exploration of lipidomic datasets, we have developed liputils, a Python module that specializes in the extraction of fatty acid moieties from individual molecular lipids. There is no prerequisite data format, as liputils extracts residues from RefMet-compliant textual identifiers and from annotations of other commercially available services. We provide three examples of real-world data processing with liputils, as well as a detailed protocol on how to readily process an existing dataset that can be followed with basic informatics skills.
    DOI:  https://doi.org/10.1038/s41598-020-70259-9
  10. J Proteome Res. 2020 Aug 07.
    Tobias F, Hummon AB.
      Significant advances in mass spectrometry imaging (MSI) have pushed the boundaries in obtaining spatial information and quantification in biological samples. Quantitative MSI (qMSI) has typically been challenging to achieve because of matrix and tissue heterogeneity, inefficient analyte extraction and ion suppression effects but recent studies have demonstrated approaches to obtain highly robust methods and reproducible results. In this perspective, we share our insights into sample preparation, how the choice of matrix influences sensitivity, construction of calibration curves, signal normalization, and visualization of MSI data. We hope that by articulating these guidelines that qMSI can be routinely conducted while retaining the analytical merits of other mass spectrometry modalities.
    DOI:  https://doi.org/10.1021/acs.jproteome.0c00443