bims-metlip 2021-09-26 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2021‒09‒26
twenty-two papers selected by
Sofia Costa
Icahn School of Medicine at Mount Sinai

Multiplexed analysis of amino acids in mice brain microdialysis samples using isobaric labeling and liquid chromatography-high resolution tandem mass spectrometry.
Fast accurate quantification of salivary cortisol and cortisone in a large-scale clinical stress study by micro-UHPLC-ESI-MS/MS using a surrogate calibrant approach.
Enantioselective multiple heart cutting online two-dimensional liquid chromatography-mass spectrometry of all proteinogenic amino acids with second dimension chiral separations in one-minute time scales on a chiral tandem column.
Current applications of CE-MS for the analysis of biologically important analytes in urine (2017 - mid 2021). A review.
A simple method for the determination of glyphosate, glufosinate and their metabolites in biological specimen by liquid chromatography/tandem mass spectrometry: an application for forensic toxicology.
Ethyl chloroformate mediated gas chromatographic-mass spectrometric biomonitoring of acidic biomarkers of occupational exposure and endogenous metabolites in human urine.
Peak learning of mass spectrometry imaging data using artificial neural networks.
Absolute quantification of eleven A, D, E and K vitamers in human plasma using automated extraction and UHPLC-Orbitrap MS.
WaveICA 2.0: a novel batch effect removal method for untargeted metabolomics data without using batch information.
FT-ICR-MS-based metabolomics: A deep dive into plant metabolism.
Differential adsorption of an analyte and its D4, D5 and 13C6 labeled analogues combined with instrument-specific carry-over issues: The Achilles' heel of ibrutinib TDM.
Recent Developments Toward Integrated Metabolomics Technologies (UHPLC-MS-SPE-NMR and MicroED) for Higher-Throughput Confident Metabolite Identifications.
In-silico-library-based method enables rapid and comprehensive annotation of cardiolipins and cardiolipin oxidation products using high resolution tandem mass spectrometer.
Mass spectrometry-based metabolomics in microbiome investigations.
High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry.
Rapid analysis of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) isotopologues in stable isotope-resolved metabolomics (SIRM) using direct infusion nanoelectrospray ultra-high-resolution Fourier transform mass spectrometry (DI-nESI-UHR-FTMS).
High-performance thin-layer chromatography combined with effect-directed assays and high-resolution mass spectrometry as an emerging hyphenated technology: A tutorial review.
Quantitative analysis of 17 hypoglycemic drugs in fingerprints using UHPLC-QTRAP-MS/MS.
IonFlow: a galaxy tool for the analysis of ionomics data sets.
Overview, consequences, and strategies for overcoming matrix effects in LC-MS analysis: a critical review.
AlphaTims: Indexing trapped ion mobility spectrometry - time of flight data for fast and easy accession and visualization.
The Min-Max Test: An Objective Method for Discriminating Mass Spectra.

J Chromatogr A. 2021 Sep 07. pii: S0021-9673(21)00661-0. [Epub ahead of print]1656 462537

Multiplexed analysis of amino acids in mice brain microdialysis samples using isobaric labeling and liquid chromatography-high resolution tandem mass spectrometry.

Juho Heininen, Ulrika Julku, Timo Myöhänen, Tapio Kotiaho, Risto Kostiainen.

  We developed a new multiplexed reversed phase liquid chromatography-high resolution tandem mass spectrometric (LC-MS/MS) method. The method is based on isobaric labeling with a tandem mass tag (TMT10-plex) and stable isotope-labeled internal standards, and was used to analyze amino acids in mouse brain microdialysis samples. The TMT10-plex labeling of amino acids allowed analysis of ten samples in one LC-MS/MS run, significantly increasing the sample throughput. The method provides good chromatographic performance (peak half-width between 0.04-0.12 min), allowing separation of all TMT-labeled amino acids with acceptable resolution and high sensitivity (limits of detection typically around 10 nM). The use of stable isotope-labeled internal standards, together with TMT10-plex labeling, ensured good repeatability (relative standard deviation ≤ 12.1 %) and linearity (correlation coefficient > 0.994), indicating good quantitative performance of the multiplexed method. The method was applied to study the effect of d-amphetamine microdialysis perfusion on amino acid concentrations in the mouse brain. All amino acids were reliably detected and quantified, indicating that the method is sensitive enough to detect low concentrations of amino acids in brain microdialysis samples.

Keywords:  Amino acids; High resolution tandem mass spectrometry; Isobaric labeling; Isotope dilution; Metabolites; Multiplexing

DOI:  https://doi.org/10.1016/j.chroma.2021.462537
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Sep 11. pii: S1570-0232(21)00420-7. [Epub ahead of print]1182 122939

Fast accurate quantification of salivary cortisol and cortisone in a large-scale clinical stress study by micro-UHPLC-ESI-MS/MS using a surrogate calibrant approach.

Ece Aydin, Bernhard Drotleff, Hannes Noack, Birgit Derntl, Michael Lämmerhofer.

  Cortisol and cortisone are common markers for stress and thus preferentially analyzed in matrices that allow non-invasive sampling such as saliva. Though the major drawback of immunoassays is lack of specificity due to cross reactivities, they are still most commonly used for quantification of steroid hormones. To overcome such problems, sensitive methods based on liquid chromatography-mass spectrometry are becoming more and more accepted as the golden standard for steroid bioanalysis as they achieve accurate quantification at trace levels for multiple analytes in the same run. Along this line, the aim of this study was the development of a new microflow UHPLC-ESI-MS/MS method for the measurement of salivary cortisol and cortisone, which due to its microflow regime provides enhanced sensitivity and is more ecofriendly. The developed method implemented sample preparation by Solid-Phase Extraction (SPE) in a 96-well plate format. Data acquisitions were carried out in MRM (multiple reaction monitoring) mode. The quantitative determination of endogenous compounds in saliva remains a challenge since analyte-free matrix is lacking. Hence, a surrogate calibrant approach with cortisol-d4 andcortisone-13C3 was applied for the target compounds in the presented method. A number of factors were optimized and the method validated. The lower limit of quantitation (LLOQ) was 72 and 62 pg mL-1for cortisol and cortisone, respectively. Linear calibration was achieved in the range from 0.062 to 75.5 ng mL-1for cortisol-d4 and 0.072 to 44 ng mL-1forcortisone-13C3. The performance of the method was also evaluated via proficiency test for salivary cortisol. Finally, it was applied successfully to evaluate cortisol and cortisone concentrations in multiple batches in routine clinical stress study samples (4056 total injections with 1983 study samples). Moreover, the instrument performance (in particular retention time variability) within each batch, between different batches and lot-to-lot of 5 investigated capillary columns over time is described. The work documents that micro-UHPLC-ESI-MS/MS is suitable and robust enough to carry out a full clinical study with greater than 1000s of samples over an extended period if adequate internal standards can be used.

Keywords:  Clinical analysis; Lipid extraction; Mass spectrometry; Steroidomics; Surrogate calibration; micro-UHPLC

DOI:  https://doi.org/10.1016/j.jchromb.2021.122939
Anal Chim Acta. 2021 Oct 02. pii: S0003-2670(21)00684-X. [Epub ahead of print]1180 338858

Enantioselective multiple heart cutting online two-dimensional liquid chromatography-mass spectrometry of all proteinogenic amino acids with second dimension chiral separations in one-minute time scales on a chiral tandem column.

Ryan Karongo, Min Ge, Christian Geibel, Jeannie Horak, Michael Lämmerhofer.

  In this work, we present a unique, robust and fully automated analytical platform technology for the enantioselective amino acid analysis using a multiple heart cutting RPLC-enantio/stereoselective HPLC-ESI-QTOF-MS method. This 2D-LC method allows the full enantioselective separation of 20 proteinogenic AAs plus 5 isobaric analogues, namely allo-Threonine (aThr), homoserine (Hse), allo-isoleucine (aIle), tert-Leucine (Tle) and Norleucine (Nle), after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ). This N-terminal AA-derivatization method introduces on the one hand beneficial chromatographic properties for 1D RP-LC (stronger retention) and 2D chiral separation (better chiral recognition), and on the other hand favorable detection properties with its chromophoric, fluorophoric, and easily ionizable quinoline mass tag. The entire separation occurs within a total 2DLC run time of 45 min, which includes the 1D-RP run and the 68 s 2D chiral separations of 30 heart-cuts (from the 1D-RP-run) on a chiral quinine carbamate (core-shell QNAX/fully porous ZWIX) tandem column. This relatively short overall run time was only possible by utilizing the highly efficient "smart peak parking" algorithm for the heart cuts and the resulting optimized analysis order thereof. 1D retention time precisions of <0.21% RSD were a requirement for the time-based sampling mode and finally led to a robust, fully automated enantioselective amino acid analysis platform. This achiral-chiral 2DLC method was applied for the amino acid stereoconfiguration assignment of three peptides (aureobasidin A, a lipopeptide research sample, and octreotide) using an L-[u-13C15N] labelled internal AA standard mix spiked to each sample. The isotopically labelled L-AA standard allowed an easy and straightforward identification and configuration assignment, as well as the relative quantification of amino acids within the investigated peptides, allowing the direct determination of the number of respective amino acids and their chirality within a peptide.

Keywords:  6-Aminoquinolyl-N-Hydroxysuccinimidyl carbamate; Enantioselective amino acid separation; L-[u-(13)C(15)N] amino acid internal standard; Multiple heart-cutting; Two-dimensional liquid chromatography; UHPLC

DOI:  https://doi.org/10.1016/j.aca.2021.338858
J Sep Sci. 2021 Sep 19.

Current applications of CE-MS for the analysis of biologically important analytes in urine (2017 - mid 2021). A review.

Hrušková Helena, Voráčová Ivona, Řemínek Roman, Foret František.

  The capillary electrophoresis coupled online with mass detection (CE-MS) is a modern tool for analyzing wide ranges of compounds in complex samples, including urine. CE-MS allows the separation and identification of various analytes spanning from small ions to high molecular weight protein complexes. Similarly to the much more common liquid chromatography-mass spectrometry techniques, the capillary electrophoresis separation reduces the complexity of the mixture of analytes entering the mass spectrometer resulting in reduced ion suppression and a more straightforward interpretation of the mass spectrometry data. [2] This review summarizes the CE-MS studies published between the years 2017-2021, aiming at the determination of various compounds excreted in urine. The properties of the urine, including its diagnostical and analytical features and chemical composition, are also discussed including general protocols for the urine sample preparation. The mechanism of the electrophoretic separation and the instrumentation for the CE-MS coupling is also included. This review shows the potential of the CE-MS technique for the analyses of different kinds of analytes in a complex biological matrix. The discussed applications are divided into two main groups (the CE-MS for the determination of drugs and drugs of abuse in urine and CE-MS for the studies of urinary metabolome). This article is protected by copyright. All rights reserved.

Keywords:  capillary electrophoresis - mass spectrometry; drugs; metabolome; urine analysis

DOI:  https://doi.org/10.1002/jssc.202100621
Nagoya J Med Sci. 2021 Aug;83(3): 567-587

A simple method for the determination of glyphosate, glufosinate and their metabolites in biological specimen by liquid chromatography/tandem mass spectrometry: an application for forensic toxicology.

Tomomi Ohara, Takashi Yoshimoto, Yujin Natori, Akira Ishii.

  Glyphosate (GLYP) and glufosinate (GLUF) are phosphorus-containing amino acid type herbicides that are used worldwide. With their rising consumptions, fatal intoxication cases due to these herbicides, whether accidental or intentional, cannot be ignored. Both compounds are difficult to detect, and their pretreatment for instrumental analysis are complicated and time-consuming. Our aim was to develop a simple and rapid quantification method for the two herbicides and their metabolites with liquid chromatography/tandem mass spectrometry (LC/MS/MS). We also compared 2-amino-4-phosphonobutyric acid and DL-2-amino-5-phosphonopentanoic acid as alternative internal standards (IS) to GLYP13C2 15N. Herbicide-containing specimens were highly diluted, evaporated to dryness, and derivatized with acetate/acetic anhydride and trimethyl orthoacetate for 30 min. at 120°C. Our optimized LC conditions successfully separated the target analytes, with acceptable linearities (R 2>0.98) and matrix effects (65%-140%). Accuracy and precision ranged from 80.2 % to 111 %, and from 1.3 % to 13 % at the higher concentration, respectively.The concentration of the herbicides and their metabolites were investigated in a postmortem case of suspected herbicide poisoning cases, in which we detected GLYP and its metabolites. Using one of the three ISs, the GLYP concentrations ranged from 3.1 to 3.5 mg/mL, and 3.3 to 4.5 mg/mL in plasma and urine, respectively; GLYP metabolite concentrations in plasma and urine were 18 to 20 μg/mL and 44 to 54 μg/mL. We thus succeeded in developing a rapid method without extraction for measuring GLYP and GLUF along with their metabolites, and demonstrated its practical applicability.

Keywords:  forensic toxicology; glufosinate; glyphosate; liquid chromatography/tandem mass spectrometry; postmortem specimens

DOI:  https://doi.org/10.18999/nagjms.83.3.567
J Chromatogr A. 2021 Sep 10. pii: S0021-9673(21)00671-3. [Epub ahead of print]1656 462547

Ethyl chloroformate mediated gas chromatographic-mass spectrometric biomonitoring of acidic biomarkers of occupational exposure and endogenous metabolites in human urine.

Lucie Řimnáčová, Martin Moos, Stanislav Opekar, Petr Vodrážka, Vladimír Pejchal, Jaroslav Mráz, Petr Šimek.

  Numerous industrial organic pollutants such as aromates, alkoxyalcohols, other organic solvents and monomers are absorbed, metabolized, and finally excreted in urine mostly as carboxylic acids that are determined as biomarkers of exposure. For a number of these xenometabolites, biological limits (levels of biomarkers in biological material) have been established to prevent damage to human health. Till now, most of the analytical procedures used have been optimized for one or a few analytes. Here, we report a more comprehensive approach enabling rapid GC-MS screening of sixteen acidic biomarkers in urine that are metabolized in the human body from several important industrial chemicals; benzene, toluene, styrene, xylenes, alkoxyalcohols, carbon disulfide, furfural and N,N-dimethylformamide. The new method involves immediate in situ derivatization - liquid liquid microextraction of urine by an ethyl chloroformate-ethanol-chloroform-pyridine medium and GC-MS analysis of the derivatized analytes in the lower organic phase. The xenometabolite set represents diverse chemical structures and some of hippuric and mercapturic acids also provided unusual derivatives that were unambiguously elucidated by means of new ethyl chloroformates labeled with stable isotopes and by synthesis of the missing reference standards. In the next step, an automated routine was developed for GC-MS/MS analysis using a MetaboAuto® sample preparation workstation and the new method was validated for fourteen metabolites over the relevant concentration range of each analyte in the spiked pooled human urine. It shows good linearity (R2 ≥ 0.982), accuracy (from 85% to 120%), precision (from 0.7% to 20%) and recovery (from 89% to 120%). The method performance was further successfully proved by GC-MS/MS analysis of the certified IP45 and RM6009 reference urines. Moreover, we show that the new method opens up the possibility for biomonitoring of combined and cumulative occupational exposures as well as for urinary metabolite profiling of persons exposed to harmful industrial chemicals.

Keywords:  Biomarker of occupational exposure; Endogenous metabolites; Ethyl chloroformate mediated derivatization; Urine; Xenometabolite

DOI:  https://doi.org/10.1016/j.chroma.2021.462547
Nat Commun. 2021 Sep 20. 12(1): 5544

Peak learning of mass spectrometry imaging data using artificial neural networks.

Walid M Abdelmoula, Begona Gimenez-Cassina Lopez, Elizabeth C Randall, Tina Kapur, Jann N Sarkaria, Forest M White, Jeffrey N Agar, William M Wells, Nathalie Y R Agar.

Mass spectrometry imaging (MSI) is an emerging technology that holds potential for improving, biomarker discovery, metabolomics research, pharmaceutical applications and clinical diagnosis. Despite many solutions being developed, the large data size and high dimensional nature of MSI, especially 3D datasets, still pose computational and memory complexities that hinder accurate identification of biologically relevant molecular patterns. Moreover, the subjectivity in the selection of parameters for conventional pre-processing approaches can lead to bias. Therefore, we assess if a probabilistic generative model based on a fully connected variational autoencoder can be used for unsupervised analysis and peak learning of MSI data to uncover hidden structures. The resulting msiPL method learns and visualizes the underlying non-linear spectral manifold, revealing biologically relevant clusters of tissue anatomy in a mouse kidney and tumor heterogeneity in human prostatectomy tissue, colorectal carcinoma, and glioblastoma mouse model, with identification of underlying m/z peaks. The method is applied for the analysis of MSI datasets ranging from 3.3 to 78.9 GB, without prior pre-processing and peak picking, and acquired using different mass spectrometers at different centers.

DOI: https://doi.org/10.1038/s41467-021-25744-8
Anal Chim Acta. 2021 Oct 09. pii: S0003-2670(21)00703-0. [Epub ahead of print]1181 338877

Absolute quantification of eleven A, D, E and K vitamers in human plasma using automated extraction and UHPLC-Orbitrap MS.

G R P Arachchige, E B Thorstensen, M Coe, J M O'Sullivan, C J Pook.

  Fat-Soluble Vitamers [FSV] deficiencies and hypervitaminosis are associated with lifestyle diseases such as cardiovascular disease, diabetes, and cancer. Quantification of FSV and their metabolites in plasma has proved to be one of the most demanding analytical chemistry challenges. Current FSV quantification methods are compromises between breadth of coverage and sensitivity across the physiological range. Here, we developed and validated a sensitive, robust, semi-automated method using liquid-liquid extraction coupled with LC-ESI-MS/MS to quantify 11 FSV across their physiological concentrations in plasma. The addition of Phree® phospholipid removal plates as the last step in the extraction process reduced matrix effects, improving precision, recoveries, and the method's final sensitivity. This method can detect and quantify: retinol, retinoic acid, retinyl palmitate, 25 hydroxyvitamin D3 [25-OH-D3], 1-α-25-dihydroxy-D3, α-tocopherol, γ-tocopherol, α-tocotrienol, phylloquinone [K1], Menatetrenone [MK-4], and menaquinone-7 [MK-7].The Instrument Quantitation Limit [IQL]s for retinol (64.1 ng/mL), 25-OH-D3 (10.2 ng/mL), and α-tocopherol (3000 ng/mL) can detect clinical deficiencies. Our automated method will assist in the understanding of the complex interaction between these compounds and their possible role in health and disease.

Keywords:  Fat soluble vitamers [FSV]; Liquid-liquid extraction [LLE]; Mass spectrometry [MS]; Phree® phospholipid removal plates [Phree®]; Ultra high-performance liquid chromatography [UHPLC]

DOI:  https://doi.org/10.1016/j.aca.2021.338877
Metabolomics. 2021 Sep 20. 17(10): 87

WaveICA 2.0: a novel batch effect removal method for untargeted metabolomics data without using batch information.

Kui Deng, Falin Zhao, Zhiwei Rong, Lei Cao, Liuchao Zhang, Kang Li, Yan Hou, Zheng-Jiang Zhu.

  INTRODUCTION: Untargeted metabolomics based on liquid chromatography-mass spectrometry is inevitably affected by batch effects that are caused by non-biological systematic bias. Previously, we developed a novel method called WaveICA to remove batch effects for untargeted metabolomics data. To detect batch effect information, the method relies on a batch label. However, it cannot be used in the scenario in which there is only one batch of data or the batch label is unknown.OBJECTIVES: We aim to improve the WaveICA method to remove batch effects for untargeted metabolomics data without using batch information.
METHODS: We improved the WaveICA method by developing WaveICA 2.0 to remove batch effects for metabolomics data, and provided an R package WaveICA_2.0 to implement this method.
RESULTS: The performance of the WaveICA 2.0 method was evaluated on real metabolomics data. For metabolomics data with three batches, the performance of the WaveICA 2.0 method was similar to that of the WaveICA method in terms of gathering quality control samples (QCSs) and subject samples together in principle component analysis score plots, increasing the similarity of QCSs, increasing differential peaks, and improving classification accuracy. For metabolomics data with only one batch, the WaveICA 2.0 method had a strong ability to remove intensity drift and reveal more biological information and outperformed the QC-RLSC and QC-SVRC methods in our study using our metabolomics data.
CONCLUSION: Our results demonstrated that the WaveICA 2.0 method can be used in practice to remove batch effects for untargeted metabolomics data without batch information.

Keywords:  Batch effects; Generalized additive model; Intensity drift removal; Untargeted metabolomics; Wavelet transform

DOI:  https://doi.org/10.1007/s11306-021-01839-7
Mass Spectrom Rev. 2021 Sep 20.

FT-ICR-MS-based metabolomics: A deep dive into plant metabolism.

Marisa Maia, Andreia Figueiredo, Carlos Cordeiro, Marta Sousa Silva.

  Metabolomics involves the identification and quantification of metabolites to unravel the chemical footprints behind cellular regulatory processes and to decipher metabolic networks, opening new insights to understand the correlation between genes and metabolites. In plants, it is estimated the existence of hundreds of thousands of metabolites and the majority is still unknown. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is a powerful analytical technique to tackle such challenges. The resolving power and sensitivity of this ultrahigh mass accuracy mass analyzer is such that a complex mixture, such as plant extracts, can be analyzed and thousands of metabolite signals can be detected simultaneously and distinguished based on the naturally abundant elemental isotopes. In this review, FT-ICR-MS-based plant metabolomics studies are described, emphasizing FT-ICR-MS increasing applications in plant science through targeted and untargeted approaches, allowing for a better understanding of plant development, responses to biotic and abiotic stresses, and the discovery of new natural nutraceutical compounds. Improved metabolite extraction protocols compatible with FT-ICR-MS, metabolite analysis methods and metabolite identification platforms are also explored as well as new in silico approaches. Most recent advances in MS imaging are also discussed.

Keywords:  FT-ICR-MS; MALDI-FT-ICR-MS imaging; metabolite profiling; plant metabolism; untargeted and targeted approaches

DOI:  https://doi.org/10.1002/mas.21731
J Pharm Biomed Anal. 2021 Sep 14. pii: S0731-7085(21)00477-5. [Epub ahead of print]206 114366

Differential adsorption of an analyte and its D4, D5 and 13C6 labeled analogues combined with instrument-specific carry-over issues: The Achilles' heel of ibrutinib TDM.

Martin Mžik, Nela Váňová, Michal Kriegelstein, Miloš Hroch.

  At present, therapeutic drug monitoring is the standard in pharmacotherapy using medications with a narrow therapeutic index or showing serious adverse effects, such as in the case of ibrutinib. A technique commonly used for this purpose is liquid chromatography-tandem mass spectrometry combined with isotope dilution in sample processing. Although this method provides a high degree of reliability, its use can be complicated with some specific factors and does not guarantee trouble-free analysis. This paper is focused on investigating issues related to the differential adsorption of ibrutinib and its D4, D5 and 13C6 isotopically labeled analogues combined with instrument-specific carry-over. The results of the research point out the significantly different adsorption behavior of ibrutinib in fluidics of LC-MS compared with that of its D4, D5 and 13C6 stable isotope labeled analogues, showing preferential adsorption of non-labeled compound. The investigation also pointed to a strong affinity of ibrutinib to polymeric surfaces under specific conditions, which has to be taken into consideration during sample preparation and analysis. Our work opens a new field for the discussion of scarcely reported problem related to the use of stable isotope labeled internal standards in LC-MS/MS analysis.

Keywords:  Carry-over; Differential adsorption; Ibrutinib; Isotope dilution; Quantification; SIL-IS

DOI:  https://doi.org/10.1016/j.jpba.2021.114366
Front Mol Biosci. 2021 ;8 720955

Recent Developments Toward Integrated Metabolomics Technologies (UHPLC-MS-SPE-NMR and MicroED) for Higher-Throughput Confident Metabolite Identifications.

Rajarshi Ghosh, Guanhong Bu, Brent L Nannenga, Lloyd W Sumner.

  Metabolomics has emerged as a powerful discipline to study complex biological systems from a small molecule perspective. The success of metabolomics hinges upon reliable annotations of spectral features obtained from MS and/or NMR. In spite of tremendous progress with regards to analytical instrumentation and computational tools, < 20% of spectral features are confidently identified in most untargeted metabolomics experiments. This article explores the integration of multiple analytical instruments such as UHPLC-MS/MS-SPE-NMR and the cryo-EM method MicroED to achieve large-scale and confident metabolite identifications in a higher-throughput manner. UHPLC-MS/MS-SPE allows for the simultaneous automated purification of metabolites followed by offline structure elucidation and structure validation by NMR and MicroED. Large-scale study of complex metabolomes such as that of the model plant legume Medicago truncatula can be achieved using an integrated UHPLC-MS/MS-SPE-NMR metabolomics platform. Additionally, recent developments in MicroED to study structures of small organic molecules have enabled faster, easier and precise structure determinations of metabolites. A MicroED small molecule structure elucidation workflow (e.g., crystal screening, sample preparation, data collection and data processing/structure determination) has been described. Ongoing MicroED methods development and its future scope related to structure elucidation of specialized metabolites and metabolomics are highlighted. The incorporation of MicroED with a UHPLC-MS/MS-SPE-NMR instrumental ensemble offers the potential to accelerate and achieve higher rates of metabolite identification.

Keywords:  MicroED; NMR; UHPLC-MS-SPE; integrated metabolomics; metabolite identification

DOI:  https://doi.org/10.3389/fmolb.2021.720955
Anal Chim Acta. 2021 Oct 02. pii: S0003-2670(21)00705-4. [Epub ahead of print]1180 338879

In-silico-library-based method enables rapid and comprehensive annotation of cardiolipins and cardiolipin oxidation products using high resolution tandem mass spectrometer.

Yu He, Binghuan Yuan, Yao Lu, Xia Zhao, Cunsi Shen, Jianjian Ji, Lili Lin, Jianya Xu, Tong Xie, Jinjun Shan.

  Accumulated evidences suggest that cardiolipins (CLs) and cardiolipin oxidation products (oxCLs) are a class of essential molecules that play critical roles in many physiological functions. Diversity of four acyl chains leads to high structure complexity for cardiolipin species including CLs, monolysocardiolipins (MLCLs) and their oxCLs. The ability to rapidly identify CL species can be implemented by the match of mass spectrometry (MS)-based in-silico spectral database. In this study, after optimizing the chromatography conditions and MS detection, an in-silico library containing 377,754 simulated tandem mass spectra deducing from 31,578 CLs to 52,160 of MLCLs was successfully augmented based on LipidBlast templates. For the construction of the oxCLs' library, twenty-five fatty acyls oxidation products relating to nine oxidation types were permuted and combined. A total of 42,180 oxCL spectra were predicted based on the experimental measurements of oxCLs forming by artificially oxidation. Applying the in-silico database to murine mitochondria and cell samples enabled the sensitive and comprehensive annotation of 86 MLCLs, 307 CLs and 112 oxCLs with high annotation confidence. Compared to the conventional method, our proposed in-silico database provides a more comprehensive interpretation for CL species' characterization with high throughput and sensitivity in nontarget lipidomic study.

Keywords:  Cardiolipin; Cardiolipin oxidation products; In-silico database; Mass spectrometry

DOI:  https://doi.org/10.1016/j.aca.2021.338879
Nat Rev Microbiol. 2021 Sep 22.

Mass spectrometry-based metabolomics in microbiome investigations.

Anelize Bauermeister, Helena Mannochio-Russo, Letícia V Costa-Lotufo, Alan K Jarmusch, Pieter C Dorrestein.

Microbiotas are a malleable part of ecosystems, including the human ecosystem. Microorganisms affect not only the chemistry of their specific niche, such as the human gut, but also the chemistry of distant environments, such as other parts of the body. Mass spectrometry-based metabolomics is one of the key technologies to detect and identify the small molecules produced by the human microbiota, and to understand the functional role of these microbial metabolites. This Review provides a foundational introduction to common forms of untargeted mass spectrometry and the types of data that can be obtained in the context of microbiome analysis. Data analysis remains an obstacle; therefore, the emphasis is placed on data analysis approaches and integrative analysis, including the integration of microbiome sequencing data.

DOI: https://doi.org/10.1038/s41579-021-00621-9
Anal Chim Acta. 2021 Oct 02. pii: S0003-2670(21)00706-6. [Epub ahead of print]1180 338880

High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry.

Yan Sun, Stephanie Stransky, Jennifer Aguilan, Sanjay Koul, Scott J Garforth, Michael Brenowitz, Simone Sidoli.

  We present a direct injection mass spectrometry (DI-MS) platform that accurately, precisely, and quickly quantitates global levels of DNA cytidine methylation (5 mC) and hydroxymethylation (5hmC). Our platform combines an Advion TriVersa NanoMate coupled online to a Thermo Scientific Orbitrap Fusion Lumos. Following digestion to nucleosides, the DNA samples are analyzed at the rate of <1 min per injection with comparable detection limits of 0.63 ng/μL and 0.31 ng/μL, respectively. In contrast, the detection limits for 5 mC and 5hmC in state-of-art nano liquid chromatography (LC) coupled to online mass spectrometry (nLC-MS) are notably different (0.04 ng/μL and 2.5 ng/μL, respectively). The high sensitivity of DI-MS is achieved by optimizing sample buffer composition, the source fragmentation energy, and the radio frequency of the instrument ion funnel. DI-MS accurately reports the relative abundance of 5 mC and 5hmC over a range of 1%-7% (R2 > 0.98) and 0.13%-1.75% (R2 > 0.99), respectively. Accurate measurement of C, 5 mC and 5hmC is achieved by optimizing in-source fragmentation to obtain a population of up to 93% of just the nucleoside base. This protocol minimizes base dimer formation and partial base-deoxyribose dissociation in gas phase and greatly improves modified base quantitation. We also demonstrate that DI-MS overcomes biases in differential chromatographic retention and issues of sample degradation in the autosampler due to its high throughput. Finally, we present an application of our workflow to quantify DNA modifications on a batch of 81 samples in about 1.5 h.

Keywords:  5hmC; 5mC; DNA; Direct injection; High-throughput; Hydroxymethylation; Mass spectrometry; Methylation

DOI:  https://doi.org/10.1016/j.aca.2021.338880
Anal Chim Acta. 2021 Oct 09. pii: S0003-2670(21)00699-1. [Epub ahead of print]1181 338873

Rapid analysis of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) isotopologues in stable isotope-resolved metabolomics (SIRM) using direct infusion nanoelectrospray ultra-high-resolution Fourier transform mass spectrometry (DI-nESI-UHR-FTMS).

JoonSeon Yang, Teresa W M Fan, Jason A Brandon, Andrew N Lane, Richard M Higashi.

  S-Adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are important metabolites in the one-carbon cycle that modulates cellular methylation required for proliferation and epigenetic regulation. Their concentrations, synthesis, and turnover are difficult to determine conveniently and reliably. We have developed such a method by coupling a simple and rapid purification scheme that efficiently captures both compounds, with high sensitivity, sample throughput direct infusion nanoelectrospray ultra-high-resolution Fourier transform mass spectrometry (DI-nESI-UHR-FTMS). This method is compatible with Stable Isotope-Resolved Metabolomic (SIRM) analysis of numerous other metabolites. The limits of detection for both SAM and SAH were <1 nM, and the linearity range was up to 1000 nM. The method was first illustrated for SAM/SAH analysis of mouse livers, and lung adenocarcinoma A549 cells. We then applied the method to track 13C1-CH3-Met incorporation into SAM and 13C6-glucose transformation into SAM and SAH via de novo synthesis. We further used the method to show the distinct effects on A549 and H1299 cells with treatment of anti-cancer methylseleninic acid (MSA), selenite, and selenomethionine, notably SAM depletion and increased SAM to SAH ratio by MSA, which implicates altered epigenetic regulation.

Keywords:  DI-nESI-UHR-FTMS; Methylseleninic acid; S-adenosylhomocysteine; S-adenosylmethionine; Selenite; Stable isotope-resolved metabolomics

DOI:  https://doi.org/10.1016/j.aca.2021.338873
Anal Chim Acta. 2021 Oct 02. pii: S0003-2670(21)00470-0. [Epub ahead of print]1180 338644

High-performance thin-layer chromatography combined with effect-directed assays and high-resolution mass spectrometry as an emerging hyphenated technology: A tutorial review.

Gertrud E Morlock.

  Among the analytical advances, hyphenated HPTLC offers great potential for solving pressing questions. It provides straightforward information about effects arising from individual compounds in complex or natural samples separated in parallel. The chromatographic separation is combined with effect-directed detection using enzymatic or biological assays. This helps to select from the thousands of compounds in a sample the important ones that need to be further characterized using high-resolution mass spectrometry. Unique benefits are discussed exemplarily arising from its super-hyphenation, minimum requirements for sample preparation, detection of multi-modulating compounds or agonistic versus antagonistic effects, and miniaturized on-surface metabolization. HPTLC stands for a versatile, creative and flexible open-format technique. As miniaturized open-source LabToGo system, it shows the potential to be applied as Citizen Science.

Keywords:  Biotransformation or conversion product; Citizen science; Effect profiling; Open-source LabToGo system; Product quality and safety; nanoGIT(+active) digestive system

DOI:  https://doi.org/10.1016/j.aca.2021.338644
Rapid Commun Mass Spectrom. 2021 Sep 23. e9199

Quantitative analysis of 17 hypoglycemic drugs in fingerprints using UHPLC-QTRAP-MS/MS.

Qiuyao Du, Yunfeng Zhang, Jifen Wang, Jing Chang, Aihua Wang, Xinxin Ren, Bingjie Liu.

RATIONALE: The objective of this study was to develop, optimize, and validate a method for the determination and quantification of 17 hypoglycemic drugs in fingerprints using ultra-high-performance liquid chromatography-tandem hybrid triple quadrupole linear ion trap mass spectrometry (UHPLC-QTRAP-MS/MS). We also aimed to apply the present method to the fingerprints collected from patients with hyperglycemia.METHODS: The scheduled multiple reaction monitoring-information dependent acquisition-enhanced product ion (SMRM-IDA-EPI) scanning mode was utilized. The chromatographic system consisted of an Acquity UHPLC® BEH C18 column (3.0×100 mm, 1.7μm) and a mobile phase of 0.01% (v/v) formic acid in water and methanol. Analytes were extracted via a precipitation protein procedure. The method was validated in accordance with the US food and drug administration (FDA) guidance and applied to the analysis of fingerprint deposits from subjects who have taken the drugs.
RESULTS: The limits of detection (LODs) and the lower limits of quantification (LLOQs) of 17 hypoglycemic drugs were 0.001 to 0.020 and 0.002 to 0.050 ng/fingerprint, respectively. The correlation coefficients (r) for the calibration curves were > 0.99 in the range of 0.050-50.000 ng/fingerprint. The matrix effect and recovery of 17 hypoglycemic drugs at three concentrations ranged from 81.1 to 117.3% and 80.0 to 109.6%, respectively. The validation data (intra- and inter-day combined) for accuracy ranged from 85.5 to 117.2%, the CV (%) data were ≤ 19.7%. All analytes were found to be stable stored in the autosampler (4°C) for 24 h. This validated method was successfully applied to detect hypoglycemic drugs in fingerprints from patients with hyperglycemia.
CONCLUSIONS: A quantification method for hypoglycemic drugs in fingerprints was developed, optimized, and validated. This sensitive method could be used for drug monitoring and providing reference information in forensic investigations.

DOI: https://doi.org/10.1002/rcm.9199
Metabolomics. 2021 Sep 25. 17(10): 91

IonFlow: a galaxy tool for the analysis of ionomics data sets.

J Iacovacci, W Lin, J L Griffin, R C Glen.

  INTRODUCTION: Inductively coupled plasma mass spectrometry (ICP-MS) experiments generate complex multi-dimensional data sets that require specialist data analysis tools.OBJECTIVE: Here we describe tools to facilitate analysis of the ionome composed of high-throughput elemental profiling data.
METHODS: IonFlow is a Galaxy tool written in R for ionomics data analysis and is freely accessible at https://github.com/wanchanglin/ionflow . It is designed as a pipeline that can process raw data to enable exploration and interpretation using multivariate statistical techniques and network-based algorithms, including principal components analysis, hierarchical clustering, relevance network extraction and analysis, and gene set enrichment analysis.
RESULTS AND CONCLUSION: The pipeline is described and tested on two benchmark data sets of the haploid S. Cerevisiae ionome and of the human HeLa cell ionome.

Keywords:  Galaxy platform; Ionomics; Network biology

DOI:  https://doi.org/10.1007/s11306-021-01841-z
Analyst. 2021 Sep 21.

Overview, consequences, and strategies for overcoming matrix effects in LC-MS analysis: a critical review.

Azadeh Nasiri, Reza Jahani, Shaya Mokhtari, Hassan Yazdanpanah, Bahram Daraei, Mehrdad Faizi, Farzad Kobarfard.

The high-performance liquid chromatography-mass spectrometry (LC-MS) technique is widely applied to routine analysis in many matrices. Despite the enormous application of LC/MS, this technique is subjected to drawbacks called matrix effects (MEs) that could lead to ion suppression or ion enhancement. This phenomenon can exert a deleterious impact on the ionization efficacy of an analyte and subsequently on the important method performance parameters. LC-MS susceptibility to MEs is the main challenge of this technique in the analysis of complex matrices such as biological and food samples. Nowadays, the assessment, estimation, and overcoming of the MEs before developing a method is mandatory in any analysis. Two main approaches including the post-column infusion and post-extraction spike are proposed to determine the degree of MEs. Different strategies can be adopted to reduce or eliminate MEs depending on the complexity of the matrix. This could be done by improving extraction and clean-up methods, changing the type of ionization employed, optimization of liquid chromatography conditions, and using corrective calibration methods. This review article will provide an overview of the MEs as the Achilles heel of the LC-MS technique, the causes of ME occurrence, their consequences, and systemic approaches towards overcoming MEs during LC-MS-based multi-analyte procedures.

DOI: https://doi.org/10.1039/d1an01047f
Mol Cell Proteomics. 2021 Sep 17. pii: S1535-9476(21)00121-3. [Epub ahead of print] 100149

AlphaTims: Indexing trapped ion mobility spectrometry - time of flight data for fast and easy accession and visualization.

Sander Willems, Eugenia Voytik, Patricia Skowronek, Maximilian T Strauss, Matthias Mann.

High resolution mass spectrometry-based proteomics generates large amounts of data, even in the standard liquid chromatography (LC) - tandem mass spectrometry configuration. Adding an ion mobility dimension vastly increases the acquired data volume, challenging both analytical processing pipelines and especially data exploration by scientists. This has necessitated data aggregation, effectively discarding much of the information present in these rich data sets. Taking trapped ion mobility spectrometry (TIMS) on a quadrupole time-of-flight platform (Q-TOF) as an example, we developed an efficient indexing scheme that represents all data points as detector arrival times on scales of minutes (LC), milliseconds (TIMS) and microseconds (TOF). In our open source AlphaTims package, data are indexed, accessed and visualized by a combination of tools of the scientific Python ecosystem. We interpret unprocessed data as a sparse 4D matrix and use just-in-time compilation to machine code with Numba, accelerating our computational procedures by several orders of magnitude while keeping to familiar indexing and slicing notations. For samples with more than six billion detector events, a modern laptop can load and index raw data in about a minute. Loading is even faster when AlphaTims has already saved indexed data in a HDF5 file, a portable scientific standard used in extremely large-scale data acquisition. Subsequently, data accession along any dimension and interactive visualization happen in milliseconds. We have found AlphaTims to be a key enabling tool to explore high dimensional LC-TIMS-QTOF data and have made it freely available as an open-source Python package with a stand-alone graphical user interface at https://github.com/MannLabs/alphatims or as part of the AlphaPept 'ecosystem'.

DOI: https://doi.org/10.1016/j.mcpro.2021.100149
Anal Chem. 2021 Sep 23.

The Min-Max Test: An Objective Method for Discriminating Mass Spectra.

Arun S Moorthy, Edward Sisco.

Deciding whether the mass spectra of seized drug evidence and a reference standard are measurements of two different compounds is a central challenge in forensic chemistry. Normally, an analyst will collect mass spectra from the sample and a reference standard under identical conditions, compute a mass spectral similarity score, and make a judgment about the sample using both the similarity score and their visual interpretation of the spectra. This approach is inherently subjective and not ideal when a rapid assessment of several samples is necessary. Making decisions using only the score and a threshold value greatly improves analysis throughput and removes analyst-to-analyst subjectivity, but selecting an appropriate threshold is itself a nontrivial task. In this paper, we describe and evaluate the min-max test-a simple and objective method for classifying mass spectra that leverages replicate measurements from each sample to remove analyst subjectivity. We demonstrate that the min-max test has an intuitive interpretation for decision-making, and its performance exceeds thresholding with similarity scores even when the best performing threshold for a fixed dataset is prescribed. Determining whether the underlying framework of the min-max test can incorporate retention indices for objectively deciding whether spectra are measurements of the same compound is an ongoing work.

DOI: https://doi.org/10.1021/acs.analchem.1c03053