bims-metlip 2021-10-03 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2021–10–03
23 papers selected by
Sofia Costa, Icahn School of Medicine at Mount Sinai

Enabling High Structural Specificity to Lipidomics by Coupling Photochemical Derivatization with Tandem Mass Spectrometry.
Simultaneous Quantification of Trace and Micro Phenolic Compounds by Liquid Chromatography Tandem-Mass Spectrometry.
Rapid Automated Annotation and Analysis of N-Glycan Mass Spectrometry Imaging Data Sets Using NGlycDB in METASPACE.
Characterization and Determination of 13C-Labeled Nonessential Amino Acids in a 13C5-Glutamine Isotope Tracer Experiment with a Mass Spectrometry Strategy Combining Parallel Reaction Monitoring and Multiple Reaction Monitoring.
Development and Validation of an LC-MS/MS Based Method for the Determination of Deoxynivalenol and Its Modified Forms in Maize.
Development and validation of an ultra-high performance liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of direct oral anticoagulants in human plasma.
Powerful Steroid-Based Chiral Selector for High-Throughput Enantiomeric Separation of α-Amino Acids Utilizing Ion Mobility-Mass Spectrometry.
PROMISed: A novel web-based tool to facilitate analysis and visualization of the molecular interaction networks from co-fractionation mass spectrometry (CF-MS) experiments.
Derivatization-targeted analysis of amino compounds in plant extracts in neutral loss acquisition mode by liquid chromatography-tandem mass spectrometry.
Assessment of XCMS Optimization Methods with Machine-Learning Performance.
An Extensive Metabolomics Workflow to Discover Cardiotoxin-Induced Molecular Perturbations in Microtissues.
Retention time prediction in hydrophilic interaction liquid chromatography with graph neural network and transfer learning.
Clinical Lipidomics: Realizing the potential of lipid profiling.
A Novel Method for the Determination of Vitamin D Metabolites Assessed at the Blood-Cerebrospinal Fluid Barrier.
Circulating conjugated and unconjugated vitamin D metabolite measurements by liquid chromatography mass spectrometry.
Trace analysis of resin acids in surface waters by direct injection liquid chromatography time of flight mass spectrometry and triple quadrupole mass spectrometry.
Matrix-Free High-Resolution Atmospheric-Pressure SALDI Mass Spectrometry Imaging of Biological Samples Using Nanostructured DIUTHAME Membranes.
Unsupervised methods in LC-MS data treatment: Application for potential chemotaxonomic markers search.
Salivary Metabolomics for Diagnosis and Monitoring Diseases: Challenges and Possibilities.
Microsampling with a Solid-Phase Extraction Cartridge: Storage and Online Mass Spectrometry Analysis.
Development and Application of sequential window acquisition of all theoretical mass spectra data acquisition modes on ultra-high performance liquid chromatography triple-quadrupole/time-of-flight mass spectrometry for Metabolic profiling of amino acids in human plasma.
Development and validation of an efficient HILIC-QQQ-MS/MS method for quantitative and comparative profiling of 45 hydrophilic compounds in four types of tea (Camellia sentences).
Improving Signal to Noise Ratios in Ion Mobility Spectrometry and Structures for Lossless Ion Manipulations (SLIM) using a High Dynamic Range Analog-to-Digital Converter.

Acc Chem Res. 2021 Sep 27.

Enabling High Structural Specificity to Lipidomics by Coupling Photochemical Derivatization with Tandem Mass Spectrometry.

Xiaoxiao Ma, Wenpeng Zhang, Zishuai Li, Yu Xia, Zheng Ouyang.

ConspectusLipids have pivotal roles in many biological processes, including energy storage, signal transduction, and plasma membrane formation. A disruption of lipid homeostasis is found to be associated with a range of diseases, such as cardiovascular diseases, diabetes, and cancer. Fundamental lipid biology and disease diagnostics can benefit from monitoring lipid changes in cells, tissues, organs, or the whole biological system. Therefore, it is important to develop lipid analysis tools to achieve comprehensive lipid characterization and quantitation. Over the past two decades, mass spectrometry (MS) has become the method of choice for qualitative and quantitative analyses of lipids, owing to its high sensitivity, multiplexed analysis, and soft ionization features. With the rapid development and adoption of ultrahigh-resolution MS, isobaric lipids can now be routinely resolved. By contrast, the structural characterization and quantitation of isomeric lipids remain an analytical challenge. Although some lipid C═C location or sn-isomers can be resolved by chromatography, ion mobility, or selective ionization approaches, a detailed structural characterization on the lipidome-wide level needs to be achieved.Over the past six years, we have successfully combined the Paternò-Büchi (PB) reaction, which is a UV-promoted photocycloaddition reaction specific to the C═C, with tandem MS (MS/MS) to locate the C═C in lipids and quantify lipid C═C location isomers. The PB reactions have analytical advantages such as a simple experimental setup, rapid lipid C═C derivatization, and highly specific C═C cleavage during PB-MS/MS to produce abundant diagnostic ions. More importantly, without a need of isomer separation or a comparison to authentic standards, PB-MS/MS can be directly applied to identify and quantify a mixture of lipid C═C location isomers, often coexisting with molar ratios sensitive to the biological state of the system. The PB-MS/MS method is compatible with conventional shotgun lipidomics employing a nanoelectrospray ionization or a large-sale lipid structural analysis via liquid chromatography (LC) coupled to any mass spectrometer with tandem MS capability. The PB-MS/MS method is highly versatile, as a variety of PB reagents can be tailored to a broad range of applications. Besides UV-promoted PB reactions, visible-light PB reactions have also been developed to offer more flexibility for a lipid analysis. By using selected PB reagents, the sn-positions of fatty acyls can be resolved together with C═C locations in phospholipids. This method has been used in lipidomic analyses of tissue, blood, and plasma from animal models and clinical samples, demonstrating the potential of using lipid C═C or sn-location isomer ratios for phenotyping and disease diagnostics. Lipid isomer-resolving MS imagings of tissues and single-cell lipid analysis have also been demonstrated by a proper implementation of PB-MS/MS.

DOI: https://doi.org/10.1021/acs.accounts.1c00419
Metabolites. 2021 Aug 31. pii: 589. [Epub ahead of print]11(9):

Simultaneous Quantification of Trace and Micro Phenolic Compounds by Liquid Chromatography Tandem-Mass Spectrometry.

Dandan Wang, Liangxiao Zhang, Li Yu, Fei Ma, Peiwu Li.

  The simultaneous quantification of trace and micro metabolites is a bottleneck in food and biological analysis. Phenolic compounds are the most widely distributed and have various physiological functions. In this study, the strategy for the simultaneous liquid chromatography tandem-mass spectrometry (LC-MS/MS) quantification of 13 trace and micro phenolic compounds was proposed by taking product ions and isotopic ions as quantitative ions. The method validation results showed that the limits of detection (LODs) were from 0.01 to 9.84 μg/kg, and the limits of quantification (LOQs) were from 0.03 to 32.8 μg/kg. The intra-day precision and inter-day precision were below 8.4% and 14.4%, respectively. The recoveries ranged from 81.9% to 117.2%, and the matrix effects ranged from -11.5% to 13.7%, which indicated that the method has high sensitivity and suitable stability. The developed analytical method was applied to determine trace and micro constituents in rapeseed samples. The analysis results indicated that the contents of sinapine have significantly different between high and low total phenolic content rapeseeds. This method provides a reference strategy for the simultaneous quantitative analysis of other micro- and trace antioxidants.

Keywords:  LC-MS/MS; isotopic ion; simultaneous quantification; trace and micro-metabolites

DOI:  https://doi.org/10.3390/metabo11090589
Anal Chem. 2021 Sep 28.

Rapid Automated Annotation and Analysis of N-Glycan Mass Spectrometry Imaging Data Sets Using NGlycDB in METASPACE.

Dušan Veličković, Tamara Bečejac, Sergii Mamedov, Kumar Sharma, Namasivayam Ambalavanan, Theodore Alexandrov, Christopher R Anderton.

Imaging N-glycan spatial distribution in tissues using mass spectrometry imaging (MSI) is emerging as a promising tool in biological and clinical applications. However, there is currently no high-throughput tool for visualization and molecular annotation of N-glycans in MSI data, which significantly slows down data processing and hampers the applicability of this approach. Here, we present how METASPACE, an open-source cloud engine for molecular annotation of MSI data, can be used to automatically annotate, visualize, analyze, and interpret high-resolution mass spectrometry-based spatial N-glycomics data. METASPACE is an emerging tool in spatial metabolomics, but the lack of compatible glycan databases has limited its application for comprehensive N-glycan annotations from MSI data sets. We created NGlycDB, a public database of N-glycans, by adapting available glycan databases. We demonstrate the applicability of NGlycDB in METASPACE by analyzing MALDI-MSI data from formalin-fixed paraffin-embedded (FFPE) human kidney and mouse lung tissue sections. We added NGlycDB to METASPACE for public use, thus, facilitating applications of MSI in glycobiology.

DOI: https://doi.org/10.1021/acs.analchem.1c02347
Anal Chem. 2021 Sep 27.

Characterization and Determination of 13C-Labeled Nonessential Amino Acids in a 13C5-Glutamine Isotope Tracer Experiment with a Mass Spectrometry Strategy Combining Parallel Reaction Monitoring and Multiple Reaction Monitoring.

Yuanyuan Zheng, Hongzhi Zhao, Man Tong, Lin Zhu, Stephanie Ma, Zongwei Cai.

Isotopic tracer, a powerful technique for metabolic pathway analysis, is currently widely applied in metabolic flux analysis. However, the qualitative and quantitative analyses of 13C-labeled metabolites pose great challenges, especially in complex biological sample matrices. Here, we present an integrated method for the qualitative and quantitative analyses of various isotopologues and isotopomers of 13C-labeled nonessential amino acids (NEAAs) in HepG2 cells incubated with 13C5-glutamine (Gln) based on ultra-high-performance liquid chromatography (UHPLC) coupled with tandem mass spectrometry (MS). First, accurate mass-to-charge (m/z) values of protonated isotopologues and elution time of standards were simultaneously analyzed to characterize 13C-labeled NEAAs by high-resolution Orbitrap MS in the parallel reaction monitoring (PRM) mode. Second, isotopologues and isotopomers of 13C-labeled NEAAs were investigated in HepG2 cells incubated with 13C5-Gln at different time points. Ultimately, a total of 66 multiple reaction monitoring (MRM) transitions were performed by UHPLC coupled with triple quadrupole MS. Among them, 29 MRM transitions were monitored for pure metabolites (unambiguously identified). The other 37 MRM transitions were monitored for mixtures with exactly identical MRM transitions and retention time. The application of targeted profiling of 13C-labeled NEAAs in the dynamic 13C-labeling experiment indicated that the concentration-time profiles of NEAAs were different from each other. The concentrations of most 13C-labeled Gln, Glu, Pro, and Asp altered after 13C5-Gln incubation, indicating that Gln plays a fundamental role in the biosynthesis of Glu, Pro, and Asp. The proposed PRM-MRM combination mode LC-MS approach is expected to provide valuable insights into analyses of isotope-labeled metabolites in isotope tracer experiments.

DOI: https://doi.org/10.1021/acs.analchem.1c02554
Toxins (Basel). 2021 Aug 27. pii: 600. [Epub ahead of print]13(9):

Development and Validation of an LC-MS/MS Based Method for the Determination of Deoxynivalenol and Its Modified Forms in Maize.

Iris Fiby, Marta Magdalena Sopel, Herbert Michlmayr, Gerhard Adam, Franz Berthiller.

  The Fusarium mycotoxin deoxynivalenol (DON) is a common contaminant of cereals and is often co-occurring with its modified forms DON-3-glucoside (D3G), 3-acetyl-DON (3ADON) or 15-acetyl-DON (15ADON). A stable-isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) based method for their determination in cereals was developed and validated for maize. Therefore, 13C-labelled D3G was enzymatically produced using 13C-DON and [13C6Glc]-sucrose and used as an internal standard (IS) for D3G, while uniformly 13C labelled IS was used for the other mycotoxins. Baseline separation was achieved for the critical peak pair DON/D3G, while 3ADON/15ADON could not be fully baseline separated after testing various reversed phase, fluorinated phase and chiral LC columns. After grinding, weighing and extracting the cereal samples, the raw extract was centrifuged and a mixture of the four 13C-labelled ISs was added directly in a microinsert vial. The subsequent analytical run took 7 min, followed by negative electrospray ionization and selected reaction monitoring on a triple quadrupole MS. Maize was used as a complex cereal model matrix for validation. The use of the IS corrected the occurring matrix effects efficiently from 76 to 98% for D3G, from 86 to 103% for DON, from 68 to 100% for 15ADON and from 63 to 96% for 3ADON.

Keywords:  masked mycotoxins; mass spectrometry; modified mycotoxins; mycotoxins; stable-isotope dilution assay; trichothecenes

DOI:  https://doi.org/10.3390/toxins13090600
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Sep 21. pii: S1570-0232(21)00433-5. [Epub ahead of print]1182 122952

Development and validation of an ultra-high performance liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of direct oral anticoagulants in human plasma.

Yu-Fei Zhang, Xiao-Qin Liu, Yang Wang, Xin Xu, Ming-Kang Zhong, Pu Zhang, Chun-Lai Ma.

  Direct oral anticoagulants are widely used to treat and prevent thromboembolic disorders. With rising clinical application, monitoring concentrations of direct oral anticoagulants are necessary in certain clinical conditions. A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of dabigatran etexilate, dabigatran, rivaroxaban, edoxaban, and apixaban, in human plasma. Protein precipitation with methanol was performed for sample preparation. The direct oral anticoagulants and internal standards were separated under gradient conditions using a C18 column, at an analytical run time of 8 min. The mobile phase was composed of 0.1% (v/v) formic acid in water (solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B) at a flow rate of 0.3 mL/min. Mass detection was performed in multiple reaction monitoring using positive ionization mode. The method was validated over a range of 1.0-500 ng/mL for dabigatran etexilate, 0.1-500 ng/mL for dabigatran, and 0.5-500 ng/mL for edoxaban, rivaroxaban, and apixaban. The method detection limits of five analytes were in the range of 0.05-0.5 ng/mL. The lower limits of quantification of five analytes ranged from 0.1 to 1 ng/mL. The linearity (r2 values) was higher than 0.997. The accuracy of the low, medium, and high quality control samples were between 85.9 and 114%, and intra- and inter-day precision were below 9.47%. This validated method was successfully used to determine the plasma concentrations of rivaroxaban in 32 patients, and of dabigatran etexilate and dabigatran in 1 patient.

Keywords:  DOAC; Human plasma; Simultaneous determination; Therapeutic drug monitoring; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2021.122952
Anal Chem. 2021 Oct 01.

Powerful Steroid-Based Chiral Selector for High-Throughput Enantiomeric Separation of α-Amino Acids Utilizing Ion Mobility-Mass Spectrometry.

Yuling Li, Bowen Zhou, Keke Wang, Jing Zhang, Wenjian Sun, Li Zhang, Yinlong Guo.

Stereospecific recognition of amino acids (AAs) plays a crucial role in chiral biomarker-based diagnosis and prognosis. Separation of AA enantiomers is a long and tedious task due to the requirement of AA derivatization prior to the chromatographic or electrophoretic steps which are also time-consuming. Here, a mass-tagged chiral selector named [d0]/[d5]-estradiol-3-benzoate-17β-chloroformate ([d0]/[d5]-17β-EBC) with high reactivity and good enantiomeric resolution in regard to AAs was developed. After a quick and easy chemical derivatization step of AAs using 17β-EBC as the single chiral selector before ion mobility-mass spectrometry analysis, good enantiomer separation was achieved for 19 chiral proteinogenic AAs in a single analytical run (∼2 s). A linear calibration curve of enantiomeric excess was also established using [d0]/[d5]-17β-EBC. It was demonstrated to be capable of determining enantiomeric ratios down to 0.5% in the nanomolar range. 17β-EBC was successfully applied to investigate the absolute configuration of AAs among peptide drugs and detect trace levels of d-AAs in complex biological samples. These results indicated that [d0]/[d5]-17β-EBC may contribute to entail a valuable step forward in peptide drug quality control and discovering chiral disease biomarkers.

DOI: https://doi.org/10.1021/acs.analchem.1c02691
Comput Struct Biotechnol J. 2021 ;19 5117-5125

PROMISed: A novel web-based tool to facilitate analysis and visualization of the molecular interaction networks from co-fractionation mass spectrometry (CF-MS) experiments.

Dennis Schlossarek, Marcin Luzarowski, Ewelina Sokołowska, Michał Górka, Lothar Willmitzer, Aleksandra Skirycz.

  Co-fractionation mass spectrometry (CF-MS)-based approaches enable cell-wide identification of protein-protein and protein-metabolite complexes present in the cellular lysate. CF-MS combines biochemical separation of molecular complexes with an untargeted mass-spectrometry-based proteomics and/or metabolomics analysis of the obtained fractions, and is used to delineate putative interactors. CF-MS data are a treasure trove for biological discovery. To facilitate analysis and visualization of original or publically available CF-MS datasets, we designed PROMISed, a user-friendly tool available online via https://myshiny.mpimp-golm.mpg.de/PDP1/ or as a repository via https://github.com/DennisSchlossarek/PROMISed. Specifically, starting with raw fractionation profiles, PROMISed (i) contains activities for data pre-processing and normalization, (ii) deconvolutes complex fractionation profiles into single, distinct peaks, (iii) identifies co-eluting protein-protein or protein-metabolite pairs using user-defined correlation methods, and (iv) performs co-fractionation network analysis. Given multiple CF-MS datasets, for instance representing different environmental condition, PROMISed allows to select for proteins and metabolites that differ in their elution profile, which may indicate change in the interaction status. But it also enables the identification of protein-protein and protein-metabolite pairs that co-elute together across multiple datasets. PROMISed enables users to (i) easily adjust parameters at each step of the analysis, (ii) download partial and final results, and (iii) select among different data-visualization options. PROMISed renders CF-MS data accessible to a broad scientific audience, allowing users with no computational or statistical background to look for novel protein-protein and protein-metabolite complexes for further experimental validation.

Keywords:  CF-MS; Co-fractionation mass spectrometry; Protein-protein complexes; Proteinmetabolite complexes; User-friendly app

DOI:  https://doi.org/10.1016/j.csbj.2021.08.042
J Chromatogr A. 2021 Sep 15. pii: S0021-9673(21)00679-8. [Epub ahead of print]1656 462555

Derivatization-targeted analysis of amino compounds in plant extracts in neutral loss acquisition mode by liquid chromatography-tandem mass spectrometry.

Larissa Silva Maciel, Arianna Marengo, Patrizia Rubiolo, Ivo Leito, Koit Herodes.

  Amino compounds, such as amino acids and biogenic amines, are important metabolites that can be found in diverse natural matrices. The most common method for amino compound analysis nowadays is reversed-phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS). However, due to the polar and the basic nature of amines, their RPLC retention is often insufficient or peaks are tailing. Derivatization is a way to overcome the issue and in the present work amino compounds are derivatized with diethyl ethoxymethylenemalonate (DEEMM) and analyzed by a RPLC triple quadrupole MS system in neutral loss scan (NLS) mode (loss of 46). This allows to target all compounds in the sample that undergo derivatization with DEEMM, so that the amino compound profile of the sample is obtained. To the best of our knowledge, the NLS acquisition mode has never been employed to target amino compounds after DEEMM derivatization. In the first part of the study, eight amino acids (arginine, aspartic acid, threonine, proline, tyrosine, tryptophan, phenylalanine and isoleucine) were employed as model compounds for method optimization, with good results in terms of DEEMM derivatives detection and repeatability. The developed method was successfully applied to a complex extract from the plant species Carduus nutans subsp. macrocephalus (Desf.) Nyman, with 18 amino acids and 3 other amines being identified. The proposed approach could be employed for straightforward identification of known and unknown amino compounds in different types of matrices.

Keywords:  Amino compounds; Carduus spp; Diethyl ethoxymethylenemalonate; Neutral loss scan; Pre-column derivatization

DOI:  https://doi.org/10.1016/j.chroma.2021.462555
Anal Chem. 2021 Sep 29.

Assessment of XCMS Optimization Methods with Machine-Learning Performance.

Johan Lassen, Kirstine Lykke Nielsen, Mogens Johannsen, Palle Villesen.

The metabolomics field is under rapid development. In particular, biomarker identification and pathway analysis are growing, as untargeted metabolomics is usable for discovery research. Frequently, new processing and statistical strategies are proposed to accommodate the increasing demand for robust and standardized data. One such algorithm is XCMS, which processes raw data into integrated peaks. Multiple studies have tried to assess the effect of optimizing XCMS parameters, but it is challenging to quantify the quality of the XCMS output. In this study, we investigate the effect of two automated optimization tools (Autotuner and isotopologue parameter optimization (IPO)) using the prediction power of machine learning as a proxy for the quality of the data set. We show that optimized parameters outperform default XCMS settings and that manually chosen parameters by liquid chromatography-mass spectrometry (LC-MS) experts remain the best. Finally, the machine-learning approach of quality assessment is proposed for future evaluations of newly developed optimization methods because its performance directly measures the retained signal upon preprocessing.

DOI: https://doi.org/10.1021/acs.analchem.1c02000
Metabolites. 2021 Sep 21. pii: 644. [Epub ahead of print]11(9):

An Extensive Metabolomics Workflow to Discover Cardiotoxin-Induced Molecular Perturbations in Microtissues.

Tara J Bowen, Andrew R Hall, Gavin R Lloyd, Ralf J M Weber, Amanda Wilson, Amy Pointon, Mark R Viant.

  Discovering modes of action and predictive biomarkers of drug-induced structural cardiotoxicity offers the potential to improve cardiac safety assessment of lead compounds and enhance preclinical to clinical translation during drug development. Cardiac microtissues are a promising, physiologically relevant, in vitro model, each composed of ca. 500 cells. While untargeted metabolomics is capable of generating hypotheses on toxicological modes of action and discovering metabolic biomarkers, applying this technology to low-biomass microtissues in suspension is experimentally challenging. Thus, we first evaluated a filtration-based approach for harvesting microtissues and assessed the sensitivity and reproducibility of nanoelectrospray direct infusion mass spectrometry (nESI-DIMS) measurements of intracellular extracts, revealing samples consisting of 28 pooled microtissues, harvested by filtration, are suitable for profiling the intracellular metabolome and lipidome. Subsequently, an extensive workflow combining nESI-DIMS untargeted metabolomics and lipidomics of intracellular extracts with ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) analysis of spent culture medium, to profile the metabolic footprint and quantify drug exposure concentrations, was implemented. Using the synthetic drug and model cardiotoxin sunitinib, time-resolved metabolic and lipid perturbations in cardiac microtissues were investigated, providing valuable data for generating hypotheses on toxicological modes of action and identifying putative biomarkers such as disruption of purine metabolism and perturbation of polyunsaturated fatty acid levels.

Keywords:  biomarkers; cardiac microtissues; cardiotoxicity; in vitro metabolomics; mode of action; sample harvesting; sensitivity; untargeted toxicokinetics

DOI:  https://doi.org/10.3390/metabo11090644
J Chromatogr A. 2021 Sep 07. pii: S0021-9673(21)00660-9. [Epub ahead of print]1656 462536

Retention time prediction in hydrophilic interaction liquid chromatography with graph neural network and transfer learning.

Qiong Yang, Hongchao Ji, Xiaqiong Fan, Zhimin Zhang, Hongmei Lu.

  The combination of retention time (RT), accurate mass and tandem mass spectra can improve the structural annotation in untargeted metabolomics. However, the incorporation of RT for metabolite identification has received less attention because of the limitation of available RT data, especially for hydrophilic interaction liquid chromatography (HILIC). Here, the Graph Neural Network-based Transfer Learning (GNN-TL) is proposed to train a model for HILIC RTs prediction. The graph neural network was pre-trained using an in silico HILIC RT dataset (pseudo-labeling dataset) with ∼306 K molecules. Then, the weights of dense layers in the pre-trained GNN (pre-GNN) model were fine-tuned by transfer learning using a small number of experimental HILIC RTs from the target chromatographic system. The GNN-TL outperformed the methods in Retip, including the Random Forest (RF), Bayesian-regularized neural network (BRNN), XGBoost, light gradient-boosting machine (LightGBM), and Keras. It achieved the lowest mean absolute error (MAE) of 38.6 s on the test set and 33.4 s on an additional test set. It has the best ability to generalize with a small performance difference between training, test, and additional test sets. Furthermore, the predicted RTs can filter out nearly 60% false positive candidates on average, which is valuable for the identification of compounds complementary to mass spectrometry.

Keywords:  Graph neural network; HILIC RT prediction; Pseudo-labeling; Transfer learning

DOI:  https://doi.org/10.1016/j.chroma.2021.462536
J Lipid Res. 2021 Sep 25. pii: S0022-2275(21)00109-7. [Epub ahead of print] 100127

Clinical Lipidomics: Realizing the potential of lipid profiling.

Thomas G Meikle, Kevin Huynh, Corey Giles, Peter J Meikle.

Dysregulation of lipid metabolism plays a major role in the aetiology and sequelae of inflammatory disorders, cardiometabolic and neurological diseases, and several forms of cancer. Recent advances in lipidomic methodology allow comprehensive lipidomic profiling of clinically relevant biological samples, enabling researchers to associate lipid species and metabolic pathways with disease onset and progression. The resulting data serve not only to advance our fundamental knowledge of the underlying disease process, but also to develop risk assessment models to assist in the diagnosis and management of disease. Currently, clinical applications of in-depth lipidomic profiling are largely limited to the use of research-based protocols in the analysis of clinical trial sample sets. However, we foresee the development of purpose-built clinical platforms designed for continuous operation and clinical integration - assisting healthcare providers with disease risk assessment, diagnosis, and monitoring. Herein, we review the current state of clinical lipidomics and the techniques employed in lipidomic profiling, with a primary focus on mass spectrometry-based analysis. We discuss the prospective design of clinically integrated platforms, including practical considerations for sample collection, storage, and preparation; automation solutions for high-throughput analysis; available software packages, and statistical development of risk assessment models.

DOI: https://doi.org/10.1016/j.jlr.2021.100127
Biomolecules. 2021 Aug 29. pii: 1288. [Epub ahead of print]11(9):

A Novel Method for the Determination of Vitamin D Metabolites Assessed at the Blood-Cerebrospinal Fluid Barrier.

Sieglinde Zelzer, Andreas Meinitzer, Markus Herrmann, Walter Goessler, Dietmar Enko.

  The brain's supply with vitamin D is poorly understood. Therefore, the present study aimed to determine 25-hydroxy vitamin D3 (25(OH)D) and 24,25-dihydroxy vitamin D (24,25(OH)2D3) in serum and cerebrospinal fluid (CSF) from individuals with intact and disturbed brain-CSF-barrier (BCB) function. In 292 pairs of serum and CSF samples the vitamin D metabolites were measured with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). CSF/serum ratios (QALB, Q25(OH)D, Q24,25(OH)2D3) were calculated. Median (IQR) serum concentrations of 25(OH)D and 24,25(OH)2D3 were 63.8 (43.4-83.9) nmol/L and 4.2 (2.2-6.2) nmol/L. The CSF concentrations of both metabolites accounted for 3.7 and 3.3% of the respective serum concentrations. Serum 25(OH)D correlated inversely with Q25(OH)D and Q24,25(OH)2D3 implying a more efficient transport of both metabolites across the BCB when the serum concentration of 25(OH)D is low. In patients with BCB dysfunction, the CSF concentrations and the CSF/serum ratios of both vitamin D metabolites were higher than in individuals with intact BCB. The CSF concentrations of 25(OH)D and 24,25(OH)2D3 depend on BCB function and the respective serum concentrations of both metabolites. Higher vitamin D metabolite concentrations in CSF of patients with impaired BCB function may be due to passive diffusion across the BCB.

Keywords:  biomarkers; blood-cerebrospinal fluid barrier; cerebrospinal fluid; liquid chromatography-tandem mass spectrometry; vitamin D metabolites

DOI:  https://doi.org/10.3390/biom11091288
J Clin Endocrinol Metab. 2021 Sep 27. pii: dgab708. [Epub ahead of print]

Circulating conjugated and unconjugated vitamin D metabolite measurements by liquid chromatography mass spectrometry.

Carl Jenkinson, Reena Desai, Malcolm D McLeod, Jonathan Wolf Mueller, Martin Hewison, David J Handelsman.

   CONTEXT: Vitamin D status is conventionally defined by measurement of unconjugated circulating 25-hydroxyvitamin D (25OHD), but it remains uncertain whether this isolated analysis gives sufficient weight to vitamin D's diverse metabolic pathways and bioactivity. Emerging evidence has shown that phase II endocrine metabolites are important excretory or storage forms, however the clinical significance of circulating phase II vitamin D metabolites remains uncertain. In this study we analysed the contribution of sulfate and glucuronide vitamin D metabolites relative to unconjugated levels in human serum.
METHODS: An optimized enzyme hydrolysis method using recombinant arylsulfatase (Pseudomonas aeruginosa) and beta-glucuronidase (Escherichia coli) was combined with liquid chromatography mass spectrometry (LC-MS/MS) analysis to measure conjugated and unconjugated vitamin D metabolites 25OHD3, 25OHD2, 3-epi-25OHD3 and 24,25(OH)2D3. The method was applied to the analysis of 170 human serum samples from community-dwelling men aged over 70 years, categorised by vitamin D supplementation status, to evaluate the proportions of each conjugated and unconjugated fraction.
RESULTS: As a proportion of total circulating vitamin D metabolites, sulfate conjugates (ranging between 18-53%) were a higher proportion than glucuronide conjugates (ranging between 2.7-11%). The proportion of conjugated 25OHD3 (48±9%) was higher compared to 25OHD2 conjugates (29.1±10%) across all supplementation groups. Conjugated metabolites correlated with their unconjugated forms for all four vitamin D metabolites (r=0.85 to 0.97).
CONCLUSIONS: Sulfated conjugates form a high proportion of circulating vitamin D metabolites, whereas glucuronide conjugates constitute a smaller fraction. Our findings principally in older men highlight the differences in abundance between metabolites and suggests a combination of both conjugated and unconjugated measurements may provide a more accurate assessment of vitamin D status.

Keywords:  Conjugate; Hydrolysis; LC-MS/MS; Vitamin D

DOI:  https://doi.org/10.1210/clinem/dgab708
J Chromatogr A. 2021 Sep 15. pii: S0021-9673(21)00682-8. [Epub ahead of print]1656 462558

Trace analysis of resin acids in surface waters by direct injection liquid chromatography time of flight mass spectrometry and triple quadrupole mass spectrometry.

Pamela Brunswick, Oxana Blajkevitch, Liane Chow, Ceara MacInnis, Graham van Aggelen, Marcus Kim, Dayue Shang.

  A rapid and sensitive liquid chromatography (LC) quadrupole time of flight (QTOF) method has been developed for the determination of resin acid concentrations in aqueous pulp and paper effluent related samples. Calibration R2 of ≥0.995 for twelve resin acids, namely dehydroabietic, 8(14)-abietenic, dihydroisopimaric, levopimaric, neoabietic, pimaric, sandaracopimaric, abietic, isopimaric, palustric, chlorodehydroabietic, and dichlorodehydroabietic acids, was demonstrated in the range 1 µgL-1 to 40 µgL-1. An improved lower limit of quantitation was achieved without use of complex sample extraction and clean-up procedures undertaken by other published methods. Excellent precision and accuracy results were achieved for dehydroabietic, chlorodehydroabietic, dichlorodehydroabietic, isopimaric (integrated inclusive of all C20H30O2 resin acids), dihydroisopimaric and 8(14)-abietenic resin acids, with t-99 percentile detection limits spanning the range 0.05 to 0.07 µgL-1. While measurement for the C20H30O2 resin acids by isopimaric equivalence is considered semi-quantitative and could be an under estimate for the abietic acid component, the developed method demonstrated clear advantage over time consuming, hazardous, and unstable derivatization procedures used for gas chromatography and capillary electrophoresis. The developed LC/QToF method was successfully transferred to an LC triple quadrupole mass spectrometer for routine high throughput trace level analysis. Real world samples, including sea water and estuary water, demonstrated excellent spike recoveries by this procedure, indicating that the method is well suited to the monitoring of industrially derived resin acids in environmental surface waters. While no interferences were observed during routine sample analysis using myristic-1-13C acid and palmitic-1-13C acid internal standards, these were later substituted by myristic-d27 and palmitic-d31 acid in order to improve method robustness for environmental samples where endogenous parent fatty acids could be present.

Keywords:  Environmental; LC; Pulp & paper; QTOF; Resin acid

DOI:  https://doi.org/10.1016/j.chroma.2021.462558
Metabolites. 2021 Sep 15. pii: 624. [Epub ahead of print]11(9):

Matrix-Free High-Resolution Atmospheric-Pressure SALDI Mass Spectrometry Imaging of Biological Samples Using Nanostructured DIUTHAME Membranes.

Max A Müller, Dhaka R Bhandari, Bernhard Spengler.

  Applications of mass spectrometry imaging (MSI), especially matrix-assisted laser desorption/ionization (MALDI) in the life sciences are becoming increasingly focused on single cell analysis. With the latest instrumental developments, pixel sizes in the micrometer range can be obtained, leading to challenges in matrix application, where imperfections or inhomogeneities in the matrix layer can lead to misinterpretation of MS images. Thereby, the application of premanufactured, homogeneous ionization-assisting devices is a promising approach. Tissue sections were investigated using a matrix-free imaging technique (Desorption Ionization Using Through-Hole Alumina Membrane, DIUTHAME) based on premanufactured nanostructured membranes to be deposited on top of a tissue section, in comparison to the spray-coating of an organic matrix in a MALDI MSI approach. Atmospheric pressure MALDI MSI ion sources were coupled to orbital trapping mass spectrometers. MS signals obtained by the different ionization techniques were annotated using accurate-mass-based database research. Compared to MALDI MSI, DIUTHAME MS images captivated with higher signal homogeneities, higher contrast and reduced background signals, while signal intensities were reduced by about one order of magnitude, independent of analyte class. DIUTHAME membranes, being applicable only on tissue sections thicker than 50 µm, were successfully used for mammal, insect and plant tissue with a high lateral resolution down to 5 µm.

Keywords:  DIUTHAME; LDI; MALDI; SALDI; atmospheric pressure; high resolution; mass spectrometry imaging

DOI:  https://doi.org/10.3390/metabo11090624
J Pharm Biomed Anal. 2021 Sep 21. pii: S0731-7085(21)00493-3. [Epub ahead of print]206 114382

Unsupervised methods in LC-MS data treatment: Application for potential chemotaxonomic markers search.

Polina Turova, Iain Styles, Vladimir Timashev, Konstantin Kravets, Alexander Grechnikov, Dmitry Lyskov, Tahir Samigullin, Ilya Podolskiy, Oleg Shpigun, Andrey Stavrianidi.

  The combination of Liquid Chromatography and Mass Spectrometry (LC-MS) is commonly used to determine and characterize biologically active compounds because of its high resolution and sensitivity. In this work we explore the interpretation of LC-MS data using multivariate statistical analysis algorithms to extract useful chemical information and identify clusters of similar samples. Samples of leaves from 19 plants belonging to the Apiaceae family were analyzed in unified LC conditions by high- and low-resolution mass spectrometry in a wide range scan mode. LC-MS data preprocessing was performed followed by statistical analysis using tensor decomposition in the form of Parallel Factor Analysis (PARAFAC); matrix factorization following tensor unfolding with principal component analysis (PCA), independent component analysis (ICA), non-negative matrix factorization (NMF); or unsupervised feature selection (UFS). The optimal number of components for each of these methods were found and results were compared using four different metrics: silhouette score, Davies-Bouldin index, computational time, number of noisy components. It was found that PCA, ICA and UFS give the best results across the majority of the criteria for both low- and high-resolution data. An algorithm for biomarker signal selection is suggested and 23 potential chemotaxonomic markers were tentatively identified using MS2 data. Dendrograms constructed by the methods were compared to the molecular phylogenic tree by calculating pixel-wise mean square error (MSE). Therefore, the suggested approach can support chemotaxonomic studies and yield valuable chemical information for biomarker discovery.

Keywords:  Apiaceae; Liquid chromatography; Machine learning; Mass spectrometry; Multi-way data

DOI:  https://doi.org/10.1016/j.jpba.2021.114382
Metabolites. 2021 Aug 31. pii: 587. [Epub ahead of print]11(9):

Salivary Metabolomics for Diagnosis and Monitoring Diseases: Challenges and Possibilities.

Eelis Hyvärinen, Minttu Savolainen, Jopi J W Mikkonen, Arja M Kullaa.

  Saliva is a useful biological fluid and a valuable source of biological information. Saliva contains many of the same components that can be found in blood or serum, but the components of interest tend to be at a lower concentration in saliva, and their analysis demands more sensitive techniques. Metabolomics is starting to emerge as a viable method for assessing the salivary metabolites which are generated by the biochemical processes in elucidating the pathways underlying different oral and systemic diseases. In oral diseases, salivary metabolomics has concentrated on periodontitis and oral cancer. Salivary metabolites of systemic diseases have been investigated mostly in the early diagnosis of different cancer, but also neurodegenerative diseases. This mini-review article aims to highlight the challenges and possibilities of salivary metabolomics from a clinical viewpoint. Furthermore, applications of the salivary metabolic profile in diagnosis and prognosis, monitoring the treatment success, and planning of personalized treatment of oral and systemic diseases are discussed.

Keywords:  NMR spectrometry; mass spectrometry; metabolomics; oral disease; saliva; systemic disease

DOI:  https://doi.org/10.3390/metabo11090587
Anal Chem. 2021 Sep 30.

Microsampling with a Solid-Phase Extraction Cartridge: Storage and Online Mass Spectrometry Analysis.

Dmytro S Kulyk, Taghi Sahraeian, Suji Lee, Abraham K Badu-Tawiah.

This study aims to introduce the concept of utilizing a solid-phase extraction (SPE) cartridge for remote biofluid collection, followed by direct sample analysis at a later time. For this, a dried matrix spot was prepared in a syringe, in the form of SPE cartridge for the first time to enable small biofluid collection (microsampling), storage, shipment, and online electrospray ionization (ESI) mass spectrometry (MS) analysis of the stored dried samples. The SPE sorbents were packed into an ESI syringe and the resultant cartridge was used for sampling small volumes (<20 μL) of different complex biological fluids including blood, plasma, serum, and urine. The collected sample was stored in the dry state within the confinement of the SPE sorbent at room temperature, and analyte stability (e.g., diazepam) was maintained for more than a year. Direct coupling of the SPE cartridge to MS provides excellent accuracy, precision, and sensitivity for analyzing illicit drugs present in the biofluid. The corresponding mechanism of wrong-way positive ion generation from highly basic elution solvents was explored. Without chromatography, our direct SPE-ESI-MS analysis technique afforded detection limits as low as 26 and 140 pg/mL for raw urine and untreated plasma, respectively. These promising results proved that the new syringe-based SPE cartridge can serve as a good alternative to conventional microsampling techniques in terms of analyte stability, ease of operation and versatility, and analytical sensitivity and speed.

DOI: https://doi.org/10.1021/acs.analchem.1c02960
J Sep Sci. 2021 Sep 30.

Development and Application of sequential window acquisition of all theoretical mass spectra data acquisition modes on ultra-high performance liquid chromatography triple-quadrupole/time-of-flight mass spectrometry for Metabolic profiling of amino acids in human plasma.

Wenbo Ren, Tingting Wang, Xiuhong Hu, Yanyan Li, Zhengchao Ji, Haiyang Guo, Haiwei Cao, Jing Huang.

  Accumulating evidence suggests that amino acids are important indicators of nutritional and metabolic status. A high-resolution MS method based on sequential window acquisition of all theoretical mass spectra acquisition was developed for the simultaneous determination of 16 amino acids in human plasma. Sample preparation by protein precipitation using a mixture of acetonitrile and formic acid was followed by a BEH Amide column. The superiority of this method was investigated by comparing to TOF scan and multiple reaction monitoring modes. The limit of detection in SWATH mode for threonine, methionine, histidine, citrulline and tryptophan is 0.1 ng on column; for lysine and asparagine is 0.2 ng; for alanine, pyroglutamic acid, leucine, ornithine and aspartate is 0.5 ng, for arginine is 1.0 ng; for glutamate and serine is 2.0 ng; for glutamine is 10.0 ng. This method was linear in the range 0.8-40 μg/mL for arginine, citrulline, glutamate, histidine, leucine, methionine, pyroglutamic acid, threonine, tryptophan; 2-100 μg/mL for asparagine, aspartate, lysine, ornithine, serine; and 4-200 μg/mL for alanine, glutamine with good accuracy and precision. Significantly different levels in 11 amino acids were observed between childhood and adulthood, representing the growth and development of individuals relating to the level of amino acids. This article is protected by copyright. All rights reserved.

Keywords:  amino acids; childhood; high-resolution mass spectrometry; reference interval

DOI:  https://doi.org/10.1002/jssc.202100573
Food Chem. 2021 Sep 22. pii: S0308-8146(21)02207-X. [Epub ahead of print]371 131201

Development and validation of an efficient HILIC-QQQ-MS/MS method for quantitative and comparative profiling of 45 hydrophilic compounds in four types of tea (Camellia sentences).

Dan Wang, Lijuan Shi, Xiaowei Fan, Huaqiao Lou, Wenting Li, Yonglin Li, Dabing Ren, Lunzhao Yi.

  Hydrophilic constituents are significant for the taste and nutrition of tea, but their simultaneous quantification remains challenging due to the lack of efficient methods. Based on the hydrophilic interaction chromatography coupled with triple quadrupole-tandem mass spectrometry, this work developed and validated an efficient (8.5 min per run), sensitive (LOQ: 0.002-0.493 μg/mL) and accurate method. This method was successfully used to determine the contents of 45 hydrophilic constituents in Yunnan large-leaf tea. Umami amino acids and umami-enhanced nucleotides generally exhibited higher content in green tea and Pu-erh raw tea. By contrast, a few number of amino acids (e.g., proline and γ-aminobutyric acid) and most alkaloids and nucleosides showed significantly higher contents in black tea or Pu-erh ripen tea. By performing the orthogonal partial least squares discriminant analysis, classification models for distinguishing four types of tea, and green tea from Pu-erh raw tea were established.

Keywords:  Hydrophilic constituents; Hydrophilic interaction chromatography; Quantification; Tandem mass spectrometry; Yunnan large-leaf tea

DOI:  https://doi.org/10.1016/j.foodchem.2021.131201
J Am Soc Mass Spectrom. 2021 Sep 30.

Improving Signal to Noise Ratios in Ion Mobility Spectrometry and Structures for Lossless Ion Manipulations (SLIM) using a High Dynamic Range Analog-to-Digital Converter.

Adam L Hollerbach, Cameron M Giberson, Joon-Yong Lee, Adam P Huntley, Richard D Smith, Yehia M Ibrahim.

  Signal digitization is a commonly overlooked part of ion mobility-mass spectrometry (IMS-MS) workflows, yet it greatly affects signal-to-noise ratio and MS resolution measurements. Here, we report on the integration of a 2 GS/s, 14-bit ADC with structures for lossless ion manipulations (SLIM-IMS-MS) and compare the performance to a commonly used 8-bit ADC. The 14-bit ADC provided a reduction in the digitized noise by a factor of ∼6, owing largely to the use of smaller bit sizes. The low baseline allowed threshold voltage levels to be set very close to the MCP baseline voltage, allowing for as much signal to be acquired as possible without overloading or excessive digitization of MCP baseline noise. Analyses of Agilent tuning mixture ions and a mixture of heavy labeled phosphopeptides showed that the 14-bit ADC provided a ∼1.5-2× signal-to-noise (S/N) increase for high intensity ions, such as the Agilent tuning mixture ions and the 2+ and 3+ charge states of many phosphopeptide constituents. However, signal enhancements were as much as 10-fold for low intensity ions, and the 14-bit ADC enabled discernible signal intensities otherwise lost using an 8-bit digitizer. Additionally, the 14-bit ADC required ∼14-fold fewer mass spectra to be averaged to produce a mass spectrum with a similar S/N as the 8-bit ADC, demonstrating ∼10× higher measurement throughput. The high resolution, low baseline, and fast speed of the new 14-bit ADC enables high performance digitization of MS, IMS-MS, and SLIM-IMS-MS spectra and provides a much better picture of analyte profiles in complex mixtures.

Keywords:  data acquisition; digitization; ion mobility spectrometry (IMS); mass spectrometry (MS); signal-to-noise (S/N); structures for lossless ion manipulations (SLIM); ultrahigh resolution

DOI:  https://doi.org/10.1021/jasms.1c00226