bims-mascan 2021-06-06 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2021–06–06
thirty-two papers selected by
Giovanny Rodriguez Blanco, University of Edinburgh

A Protocol for Untargeted Metabolomic Analysis: From Sample Preparation to Data Processing.
Untargeted Lipidomic Profiling of Dry Blood Spots Using SFC-HRMS.
Successive High-Resolution (H2O)n-GCIB and C60-SIMS Imaging Integrates Multi-Omics in Different Cell Types in Breast Cancer Tissue.
Deep Structural Annotation of Glycerolipids by the Charge-Tagging Paterno-Büchi Reaction and Supercritical Fluid Chromatography-Ion Mobility Mass Spectrometry.
Anthracyclins Increase PUFAs: Potential Implications in ER Stress and Cell Death.
NADomics: Measuring NAD+ and Related Metabolites Using Liquid Chromatography Mass Spectrometry.
KRAS, A Prime Mediator in Pancreatic Lipid Synthesis through Extra Mitochondrial Glutamine and Citrate Metabolism.
Assessment of Mitochondrial Cell Metabolism by Respiratory Chain Electron Flow Assays.
A Novel Solid Phase Extraction Sample Preparation Method for Lipidomic Analysis of Human Plasma Using Liquid Chromatography/Mass Spectrometry.
The Isotopic Ac-IP Tag Enables Multiplexed Proteome Quantification in Data-Independent Acquisition Mode.
Quantitative Mass Spectrometry-Based Proteomics for Biomarker Development in Ovarian Cancer.
Assessment of TMT Labeling Efficiency in Large-Scale Quantitative Proteomics: The Critical Effect of Sample pH.
The Impact of Simvastatin on Lipidomic Markers of Cardiovascular Risk in Human Liver Cells Is Secondary to the Modulation of Intracellular Cholesterol.
Cancer cell metabolic plasticity in migration and metastasis.
Simultaneous Quantification of the Concentration and Carbon Isotopologue Distribution of Polar Metabolites in a Single Analysis by Gas Chromatography and Mass Spectrometry.
Comprehensive Plasma Metabolomic Profile of Patients with Advanced Neuroendocrine Tumors (NETs). Diagnostic and Biological Relevance.
Quantification of Modified Nucleosides in the Context of NAIL-MS.
Interactive effects of aging and aerobic capacity on energy metabolism-related metabolites of serum, skeletal muscle, and white adipose tissue.
Identification of prognostic lipid droplet-associated genes in pancreatic cancer patients via bioinformatics analysis.
Cancer Cell Metabolism in Hypoxia: Role of HIF-1 as Key Regulator and Therapeutic Target.
Breast Cancer: Targeting of Steroid Hormones in Cancerogenesis and Diagnostics.
PPARgamma: A Potential Intrinsic and Extrinsic Molecular Target for Breast Cancer Therapy.
Metabolic Response of Pancreatic Carcinoma Cells under Treatment with Dichloroacetate.
Choline kinase alpha 2 acts as a protein kinase to promote lipolysis of lipid droplets.
Epigenomic and Metabolomic Integration Reveals Dynamic Metabolic Regulation in Bladder Cancer.
EMT-derived alterations in glutamine metabolism sensitize mesenchymal breast cells to mTOR inhibition.
Selected ion flow tube mass spectrometry for targeted analysis of volatile organic compounds in human breath.
Quantification of mRNA cap-modifications by means of LC-QqQ-MS.
Lactate Reprograms Energy and Lipid Metabolism in Glucose-Deprived Oxidative Glioma Stem Cells.
Tick-Tock Consider the Clock: The Influence of Circadian and External Cycles on Time of Day Variation in the Human Metabolome-A Review.
Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS.
Impact of chemically defined culture media formulations on extracellular vesicle production by amniotic epithelial cells.

Methods Mol Biol. 2021 ;2276 357-382

A Protocol for Untargeted Metabolomic Analysis: From Sample Preparation to Data Processing.

Amanda L Souza, Gary J Patti.

  Untargeted metabolomics has rapidly become a profiling method of choice in many areas of research, including mitochondrial biology. Most commonly, untargeted metabolomics is performed with liquid chromatography/mass spectrometry because it enables measurement of a relatively wide range of physiochemically diverse molecules. Specifically, to assess energy pathways that are associated with mitochondrial metabolism, hydrophilic interaction liquid chromatography (HILIC) is often applied before analysis with a high-resolution accurate mass instrument. The workflow produces large, complex data files that are impractical to analyze manually. Here, we present a protocol to perform untargeted metabolomics on biofluids such as plasma, urine, and cerebral spinal fluid with a HILIC separation and an Orbitrap mass spectrometer. Our protocol describes each step of the analysis in detail, from preparation of solvents for chromatography to selecting parameters during data processing.

Keywords:  Accurate mass; Data-dependent acquisition; HILIC; High-resolution; Liquid chromatography; Mass spectrometry; Metabolites; Metabolomics; Profiling; Quality assurance; Quality control

DOI:  https://doi.org/10.1007/978-1-0716-1266-8_27
Metabolites. 2021 May 11. pii: 305. [Epub ahead of print]11(5):

Untargeted Lipidomic Profiling of Dry Blood Spots Using SFC-HRMS.

Pauline Le Faouder, Julia Soullier, Marie Tremblay-Franco, Anthony Tournadre, Jean-François Martin, Yann Guitton, Caroline Carlé, Sylvie Caspar-Bauguil, Pierre-Damien Denechaud, Justine Bertrand-Michel.

  Lipids are essential cellular constituents that have many critical roles in physiological functions. They are notably involved in energy storage and cell signaling as second messengers, and they are major constituents of cell membranes, including lipid rafts. As a consequence, they are implicated in a large number of heterogeneous diseases, such as cancer, diabetes, neurological disorders, and inherited metabolic diseases. Due to the high structural diversity and complexity of lipid species, the presence of isomeric and isobaric lipid species, and their occurrence at a large concentration scale, a complete lipidomic profiling of biological matrices remains challenging, especially in clinical contexts. Using supercritical fluid chromatography coupled with high-resolution mass spectrometry, we have developed and validated an untargeted lipidomic approach to the profiling of plasma and blood. Moreover, we have tested the technique using the Dry Blood Spot (DBS) method and found that it allows for the easy collection of blood for analysis. To develop the method, we performed the optimization of the separation and detection of lipid species on pure standards, reference human plasma (SRM1950), whole blood, and DBS. These analyses allowed an in-house lipid data bank to be built. Using the MS-Dial software, we developed an automatic process for the relative quantification of around 500 lipids species belonging to the 6 main classes of lipids (including phospholipids, sphingolipids, free fatty acids, sterols, and fatty acyl-carnitines). Then, we compared the method using the published data for SRM 1950 and a mouse blood sample, along with another sample of the same blood collected using the DBS method. In this study, we provided a method for blood lipidomic profiling that can be used for the easy sampling of dry blood spots.

Keywords:  Dry Blood Spot 4; Lipidomic 1; Plasma 3; Supercritical Fluids Chromatography 3

DOI:  https://doi.org/10.3390/metabo11050305
Anal Chem. 2021 Jun 02.

Successive High-Resolution (H2O)n-GCIB and C60-SIMS Imaging Integrates Multi-Omics in Different Cell Types in Breast Cancer Tissue.

Hua Tian, Louis J Sparvero, Tamil Selvan Anthonymuthu, Wan-Yang Sun, Andrew A Amoscato, Rong-Rong He, Hülya Bayır, Valerian E Kagan, Nicholas Winograd.

The temporo-spatial organization of different cells in the tumor microenvironment (TME) is the key to understanding their complex communication networks and the immune landscape that exists within compromised tissues. Multi-omics profiling of single-interacting cells in the native TME is critical for providing further information regarding the reprograming mechanisms leading to immunosuppression and tumor progression. This requires new technologies for biomolecular profiling of phenotypically heterogeneous cells on the same tissue sample. Here, we developed a new methodology for comprehensive lipidomic and metabolomic profiling of individual cells on frozen-hydrated tissue sections using water gas cluster ion beam secondary ion mass spectrometry ((H2O)n-GCIB-SIMS) (at 1.6 μm beam spot size), followed by profiling cell-type specific lanthanide antibodies on the same tissue section using C60-SIMS (at 1.1 μm beam spot size). We revealed distinct variations of distribution and intensities of >150 key ions (e.g., lipids and important metabolites) in different types of the TME individual cells, such as actively proliferating tumor cells as well as infiltrating immune cells. The demonstrated feasibility of SIMS imaging to integrate the multi-omics profiling in the same tissue section at the single-cell level will lead to new insights into the role of lipid reprogramming and metabolic response in normal regulation or pathogenic discoordination of cell-cell interactions in a variety of tissue microenvironments.

DOI: https://doi.org/10.1021/acs.analchem.0c05311
Anal Chem. 2021 May 31.

Deep Structural Annotation of Glycerolipids by the Charge-Tagging Paterno-Büchi Reaction and Supercritical Fluid Chromatography-Ion Mobility Mass Spectrometry.

Tian Xia, Ming Yuan, Yongwei Xu, Feng Zhou, Kate Yu, Yu Xia.

Glycerolipids (GLs) are essential for cellular lipid homeostasis, while dysregulation in GL metabolism is often associated with the onset or progression of human-related metabolic diseases. The profile of GLs is thus frequently used as a molecular readout for disease phenotyping. Although mass spectrometry (MS) is the method of choice for GL profiling, the current MS methods are unable to differentiate two major types of structural isomers due to the fact that fatty acyls can be linked to different positions on the glycerol backbone (sn-positions) and the site(s) of unsaturation in acyl chains. Herein, by utilizing charge-tagging Paterno-Büchi (PB) derivatization of carbon-carbon double bond (C═C), supercritical fluid chromatography (SFC), and mobility aligned tandem mass spectrometry (MS/MS), a workflow has been developed for the sensitive and structurally informative analysis of GLs. SFC allows fast separation (within 25 min) of sn-isomers of diacylglycerols (DGs) and separation of triacylglycerols (TGs) of different chain lengths and degrees of unsaturation. Time-aligned parallel fragmentation enables multiple-stage MS/MS of the PB-derivatized lipids in a high-throughput fashion and allows pinpointing C═C location to a specific fatty acyl chain. This workflow reveals the presence of more than 500 molecular structures of neutral lipids from pooled human plasma. A comparison of human plasma samples between type 2 diabetes (N = 7) and control (N = 7) shows significant changes in isomer compositions (C18:1 Δ9 vs Δ11) from nine groups of TG and DG. These findings suggest that the developed workflow can be potentially applied to lipid marker discovery for disease monitoring or diagnosis.

DOI: https://doi.org/10.1021/acs.analchem.1c01379
Cells. 2021 May 11. pii: 1163. [Epub ahead of print]10(5):

Anthracyclins Increase PUFAs: Potential Implications in ER Stress and Cell Death.

David Balgoma, Fredrik Kullenberg, Carlemi Calitz, Maria Kopsida, Femke Heindryckx, Hans Lennernäs, Mikael Hedeland.

  Metabolic and personalized interventions in cancer treatment require a better understanding of the relationship between the induction of cell death and metabolism. Consequently, we treated three primary liver cancer cell lines with two anthracyclins (doxorubicin and idarubin) and studied the changes in the lipidome. We found that both anthracyclins in the three cell lines increased the levels of polyunsaturated fatty acids (PUFAs) and alkylacylglycerophosphoethanolamines (etherPEs) with PUFAs. As PUFAs and alkylacylglycerophospholipids with PUFAs are fundamental in lipid peroxidation during ferroptotic cell death, our results suggest supplementation with PUFAs and/or etherPEs with PUFAs as a potential general adjuvant of anthracyclins. In contrast, neither the markers of de novo lipogenesis nor cholesterol lipids presented the same trend in all cell lines and treatments. In agreement with previous research, this suggests that modulation of the metabolism of cholesterol could be considered a specific adjuvant of anthracyclins depending on the type of tumor and the individual. Finally, in agreement with previous research, we found a relationship across the different cell types between: (i) the change in endoplasmic reticulum (ER) stress, and (ii) the imbalance between PUFAs and cholesterol and saturated lipids. In the light of previous research, this imbalance partially explains the sensitivity to anthracyclins of the different cells. In conclusion, our results suggest that the modulation of different lipid metabolic pathways may be considered for generalized and personalized metabochemotherapies.

Keywords:  ferroptosis; hepatocellular carcinoma; lipidomics; plasmalogen; plasmanyl; plasmenyl

DOI:  https://doi.org/10.3390/cells10051163
Life (Basel). 2021 May 31. pii: 512. [Epub ahead of print]11(6):

NADomics: Measuring NAD+ and Related Metabolites Using Liquid Chromatography Mass Spectrometry.

Nady Braidy, Maria D Villalva, Ross Grant.

  Nicotinamide adenine dinucleotide (NAD+) and its metabolome (NADome) play important roles in preserving cellular homeostasis. Altered levels of the NADome may represent a likely indicator of poor metabolic function. Accurate measurement of the NADome is crucial for biochemical research and developing interventions for ageing and neurodegenerative diseases. In this mini review, traditional methods used to quantify various metabolites in the NADome are discussed. Owing to the auto-oxidation properties of most pyridine nucleotides and their differential chemical stability in various biological matrices, accurate assessment of the concentrations of the NADome is an analytical challenge. Recent liquid chromatography mass spectrometry (LC-MS) techniques which overcome some of these technical challenges for quantitative assessment of the NADome in the blood, CSF, and urine are described. Specialised HPLC-UV, NMR, capillary zone electrophoresis, or colorimetric enzymatic assays are inexpensive and readily available in most laboratories but lack the required specificity and sensitivity for quantification of human biological samples. LC-MS represents an alternative means of quantifying the concentrations of the NADome in clinically relevant biological specimens after careful consideration of analyte extraction procedures, selection of internal standards, analyte stability, and LC assays. LC-MS represents a rapid, robust, simple, and reliable assay for the measurement of the NADome between control and test samples, and for identifying biological correlations between the NADome and various biochemical processes and testing the efficacy of strategies aimed at raising NAD+ levels during physiological ageing and disease states.

Keywords:  NAD+; ageing; biomarker; nicotinamide; plasma

DOI:  https://doi.org/10.3390/life11060512
Int J Mol Sci. 2021 May 11. pii: 5070. [Epub ahead of print]22(10):

KRAS, A Prime Mediator in Pancreatic Lipid Synthesis through Extra Mitochondrial Glutamine and Citrate Metabolism.

Isaac James Muyinda, Jae-Gwang Park, Eun-Jung Jang, Byong-Chul Yoo.

  Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven pancreatic cancer is very lethal, with a five-year survival rate of <9%, irrespective of therapeutic advances. Different treatment modalities including chemotherapy, radiotherapy, and immunotherapy demonstrated only marginal efficacies because of pancreatic tumor specificities. Surgery at the early stage of the disease remains the only curative option, although only in 20% of patients with early stage disease. Clinical trials targeting the main oncogenic driver, KRAS, have largely been unsuccessful. Recently, global metabolic reprogramming has been identified in patients with pancreatic cancer and oncogenic KRAS mouse models. The newly reprogrammed metabolic pathways and oncometabolites affect the tumorigenic environment. The development of methods modulating metabolic reprogramming in pancreatic cancer cells might constitute a new approach to its therapy. In this review, we describe the major metabolic pathways providing acetyl-CoA and NADPH essential to sustain lipid synthesis and cell proliferation in pancreatic cancer cells.

Keywords:  glutaminolysis; lipidomics; metabolomics; pancreatic cancer

DOI:  https://doi.org/10.3390/ijms22105070
Methods Mol Biol. 2021 ;2276 129-141

Assessment of Mitochondrial Cell Metabolism by Respiratory Chain Electron Flow Assays.

Flavia Radogna, Déborah Gérard, Mario Dicato, Marc Diederich.

  Cellular energy metabolism is regulated by complex metabolic pathways. Although anaerobic glycolysis was reported as a primary source of energy in cancer leading to a high rate of lactate production, current evidence shows that the main energy source supporting cancer cell metabolism relies on mitochondrial metabolism. Mitochondria are the key organelle maintaining optimal cellular energy levels. MitoPlate™ S-1 provides a highly reproducible bioenergetics tool to analyze the electron flow rate in live cells. Measuring the rates of electron flow into and through the electron transport chain using different NADH and FADH2-producing metabolic substrates enables the assessment of mitochondrial functionality. MitoPlate™ S-1 are 96-well microplates pre-coated with different substrates used as probes to examine the activity of mitochondrial metabolic pathways based on a colorimetric assay. A comparative metabolic analysis between cell lines or primary cells allows to establish a specific metabolic profile and to detect possible alterations of the mitochondrial function of a tumor cell. Moreover, the direct measurements of electron flux triggered by metabolic pathway activation could highlight targets for potential drug candidates.

Keywords:  Bioinformatics; Cancer metabolism; Electron transport chain; Mitochondrial respiration; Tricarboxylic acid cycle

DOI:  https://doi.org/10.1007/978-1-0716-1266-8_9
Metabolites. 2021 May 04. pii: 294. [Epub ahead of print]11(5):

A Novel Solid Phase Extraction Sample Preparation Method for Lipidomic Analysis of Human Plasma Using Liquid Chromatography/Mass Spectrometry.

Alex Apffel, Limian Zhao, Mark J Sartain.

  Lipidomic approaches are widely used to investigate the relationship between lipids, human health, and disease. Conventional sample preparation techniques for the extraction of lipids from biological matrices like human plasma are based on liquid-liquid extraction (LLE). However, these methods are labor-intensive, time-consuming, and can show poor reproducibility and selectivity on lipid extraction. A novel, solid-phase extraction (SPE) approach was demonstrated to extract lipids from human plasma using a lipid extraction SPE in both cartridge and 96-well-plate formats, followed by analysis using a combination of targeted and untargeted liquid chromatography/mass spectrometry. The Lipid Extraction SPE method was compared to traditional LLE methods for lipid class recovery, lipidome coverage, and reproducibility. The novel SPE method used a simplified protocol with significant time and labor savings and provided equivalent or better qualitative and quantitative results than traditional LLE methods with respect to several critical performance metrics; recovery, reproducibility, and lipidome coverage.

Keywords:  SPE; lipidomics; lipids; liquid chromatography; mass spectrometry; sample preparation

DOI:  https://doi.org/10.3390/metabo11050294
Anal Chem. 2021 May 31.

The Isotopic Ac-IP Tag Enables Multiplexed Proteome Quantification in Data-Independent Acquisition Mode.

Xiaobo Tian, Marcel P de Vries, Hjalmar P Permentier, Rainer Bischoff.

Data-independent acquisition (DIA) is an increasingly used approach for quantitative proteomics. However, most current isotope labeling strategies are not suitable for DIA as they lead to more complex MS2 spectra or severe ratio distortion. As a result, DIA suffers from a lower throughput than data-dependent acquisition (DDA) due to a lower level of multiplexing. Herein, we synthesized an isotopically labeled acetyl-isoleucine-proline (Ac-IP) tag for multiplexed quantification in DIA. Differentially labeled peptides have distinct precursor ions carrying the quantitative information but identical MS2 spectra since the isotopically labeled Ac-Ile part leaves as a neutral loss upon collision-induced dissociation, while fragmentation of the peptide backbone generates regular fragment ions for identification. The Ac-IP-labeled samples can be analyzed using general DIA liquid chromatography-mass spectrometry settings, and the data obtained can be processed with established approaches. Relative quantification requires deconvolution of the isotope envelope of the respective precursor ions. Suitability of the Ac-IP tag is demonstrated with a triplex-labeled yeast proteome spiked with bovine serum albumin that was mixed at 10:5:1 ratios, resulting in measured ratios of 9.7:5.3:1.1.

DOI: https://doi.org/10.1021/acs.analchem.1c00453
Molecules. 2021 May 03. pii: 2674. [Epub ahead of print]26(9):

Quantitative Mass Spectrometry-Based Proteomics for Biomarker Development in Ovarian Cancer.

Joohyun Ryu, Stefani N Thomas.

  Ovarian cancer is the most lethal gynecologic malignancy among women. Approximately 70-80% of patients with advanced ovarian cancer experience relapse within five years and develop platinum-resistance. The short life expectancy of patients with platinum-resistant or platinum-refractory disease underscores the need to develop new and more effective treatment strategies. Early detection is a critical step in mitigating the risk of disease progression from early to an advanced stage disease, and protein biomarkers have an integral role in this process. The best biological diagnostic tool for ovarian cancer will likely be a combination of biomarkers. Targeted proteomics methods, including mass spectrometry-based approaches, have emerged as robust methods that can address the chasm between initial biomarker discovery and the successful verification and validation of these biomarkers enabling their clinical translation due to the robust sensitivity, specificity, and reproducibility of these versatile methods. In this review, we provide background information on the fundamental principles of biomarkers and the need for improved treatment strategies in ovarian cancer. We also provide insight into the ways in which mass spectrometry-based targeted proteomics approaches can provide greatly needed solutions to many of the challenges related to ovarian cancer biomarker development.

Keywords:  biomarker; mass spectrometry; ovarian cancer; proteomics

DOI:  https://doi.org/10.3390/molecules26092674
ACS Omega. 2021 May 18. 6(19): 12660-12666

Assessment of TMT Labeling Efficiency in Large-Scale Quantitative Proteomics: The Critical Effect of Sample pH.

Chelsea Hutchinson-Bunch, James A Sanford, Joshua R Hansen, Marina A Gritsenko, Karin D Rodland, Paul D Piehowski, Wei-Jun Qian, Joshua N Adkins.

Isobaric labeling via tandem mass tag (TMT) reagents enables sample multiplexing prior to LC-MS/MS, facilitating high-throughput large-scale quantitative proteomics. Consistent and efficient labeling reactions are essential to achieve robust quantification; therefore, embedded in our clinical proteomic protocol is a quality control (QC) sample that contains a small aliquot from each sample within a TMT set, referred to as "Mixing QC." This Mixing QC enables the detection of TMT labeling issues by LC-MS/MS before combining the full samples to allow for salvaging of poor TMT labeling reactions. While TMT labeling is a valuable tool, factors leading to poor reactions are not fully studied. We observed that relabeling does not necessarily rescue TMT reactions and that peptide samples sometimes remained acidic after resuspending in 50 mM HEPES buffer (pH 8.5), which coincided with low labeling efficiency (LE) and relatively low median reporter ion intensities (MRIIs). To obtain a more resilient TMT labeling procedure, we investigated LE, reporter ion missingness, the ratio of mean TMT set MRII to individual channel MRII, and the distribution of log 2 reporter ion ratios of Mixing QC samples. We discovered that sample pH is a critical factor in LE, and increasing the buffer concentration in poorly labeled samples before relabeling resulted in the successful rescue of TMT labeling reactions. Moreover, resuspending peptides in 500 mM HEPES buffer for TMT labeling resulted in consistently higher LE and lower missing data. By better controlling the sample pH for labeling and implementing multiple methods for assessing labeling quality before combining samples, we demonstrate that robust TMT labeling for large-scale quantitative studies is achievable.

DOI: https://doi.org/10.1021/acsomega.1c00776
Metabolites. 2021 May 25. pii: 340. [Epub ahead of print]11(6):

The Impact of Simvastatin on Lipidomic Markers of Cardiovascular Risk in Human Liver Cells Is Secondary to the Modulation of Intracellular Cholesterol.

Yvette L Schooneveldt, Corey Giles, Michael F Keating, Natalie A Mellett, Aaron W Jurrjens, Sudip Paul, Anna C Calkin, Peter J Meikle.

  Statins are the first-line lipid-lowering therapy for reducing cardiovascular disease (CVD) risk. A plasma lipid ratio of two phospholipids, PI(36:2) and PC(18:0_20:4), was previously identified to explain 58% of the relative CVD risk reduction associated with pravastatin, independent of a change in low-density lipoprotein-cholesterol. This ratio may be a potential biomarker for the treatment effect of statins; however, the underlying mechanisms linking this ratio to CVD risk remain unclear. In this study, we investigated the effect of altered cholesterol conditions on the lipidome of cultured human liver cells (Hep3B). Hep3B cells were treated with simvastatin (5 μM), cyclodextrin (20 mg/mL) or cholesterol-loaded cyclodextrin (20 mg/mL) for 48 hours and their lipidomes were examined. Induction of a low-cholesterol environment via simvastatin or cyclodextrin was associated with elevated levels of lipids containing arachidonic acid and decreases in phosphatidylinositol species and the PI(36:2)/PC(18:0_20:4) ratio. Conversely, increasing cholesterol levels via cholesterol-loaded cyclodextrin resulted in reciprocal regulation of these lipid parameters. Expression of genes involved in cholesterol and fatty acid synthesis supported the lipidomics data. These findings demonstrate that the PI(36:2)/PC(18:0_20:4) ratio responds to changes in intracellular cholesterol abundance per se, likely through a flux of the n-6 fatty acid pathway and altered phosphatidylinositol synthesis. These findings support this ratio as a potential marker for CVD risk reduction and may be useful in monitoring treatment response.

Keywords:  cardiovascular disease; cholesterol; lipid metabolism; low-density lipoprotein cholesterol; statins; targeted lipidomics

DOI:  https://doi.org/10.3390/metabo11060340
Clin Exp Metastasis. 2021 Jun 02.

Cancer cell metabolic plasticity in migration and metastasis.

Jenna A Mosier, Samantha C Schwager, David A Boyajian, Cynthia A Reinhart-King.

  Metabolic reprogramming is a hallmark of cancer metastasis in which cancer cells manipulate their metabolic profile to meet the dynamic energetic requirements of the tumor microenvironment. Though cancer cell proliferation and migration through the extracellular matrix are key steps of cancer progression, they are not necessarily fueled by the same metabolites and energy production pathways. The two main metabolic pathways cancer cells use to derive energy from glucose, glycolysis and oxidative phosphorylation, are preferentially and plastically utilized by cancer cells depending on both their intrinsic metabolic properties and their surrounding environment. Mechanical factors in the microenvironment, such as collagen density, pore size, and alignment, and biochemical factors, such as oxygen and glucose availability, have been shown to influence both cell migration and glucose metabolism. As cancer cells have been identified as preferentially utilizing glycolysis or oxidative phosphorylation based on heterogeneous intrinsic or extrinsic factors, the relationship between cancer cell metabolism and metastatic potential is of recent interest. Here, we review current in vitro and in vivo findings in the context of cancer cell metabolism during migration and metastasis and extrapolate potential clinical applications of this work that could aid in diagnosing and tracking cancer progression in vivo by monitoring metabolism. We also review current progress in the development of a variety of metabolically targeted anti-metastatic drugs, both in clinical trials and approved for distribution, and highlight potential routes for incorporating our recent understanding of metabolic plasticity into therapeutic directions. By further understanding cancer cell energy production pathways and metabolic plasticity, more effective and successful clinical imaging and therapeutics can be developed to diagnose, target, and inhibit metastasis.

Keywords:  ATP; Collective migration; Extracellular matrix; Glycolysis; Oxidative phosphorylation

DOI:  https://doi.org/10.1007/s10585-021-10102-1
Anal Chem. 2021 Jun 01.

Simultaneous Quantification of the Concentration and Carbon Isotopologue Distribution of Polar Metabolites in a Single Analysis by Gas Chromatography and Mass Spectrometry.

Bernard Evers, Albert Gerding, Theo Boer, M Rebecca Heiner-Fokkema, Mathilde Jalving, S Aljoscha Wahl, Dirk-Jan Reijngoud, Barbara M Bakker.

13C-isotope tracing is a frequently employed approach to study metabolic pathway activity. When combined with the subsequent quantification of absolute metabolite concentrations, this enables detailed characterization of the metabolome in biological specimens and facilitates computational time-resolved flux quantification. Classically, a 13C-isotopically labeled sample is required to quantify 13C-isotope enrichments and a second unlabeled sample for the quantification of metabolite concentrations. The rationale for a second unlabeled sample is that the current methods for metabolite quantification rely mostly on isotope dilution mass spectrometry (IDMS) and thus isotopically labeled internal standards are added to the unlabeled sample. This excludes the absolute quantification of metabolite concentrations in 13C-isotopically labeled samples. To address this issue, we have developed and validated a new strategy using an unlabeled internal standard to simultaneously quantify metabolite concentrations and 13C-isotope enrichments in a single 13C-labeled sample based on gas chromatography-mass spectrometry (GC/MS). The method was optimized for amino acids and citric acid cycle intermediates and was shown to have high analytical precision and accuracy. Metabolite concentrations could be quantified in small tissue samples (≥20 mg). Also, we applied the method on 13C-isotopically labeled mammalian cells treated with and without a metabolic inhibitor. We proved that we can quantify absolute metabolite concentrations and 13C-isotope enrichments in a single 13C-isotopically labeled sample.

DOI: https://doi.org/10.1021/acs.analchem.1c01040
Cancers (Basel). 2021 May 27. pii: 2634. [Epub ahead of print]13(11):

Comprehensive Plasma Metabolomic Profile of Patients with Advanced Neuroendocrine Tumors (NETs). Diagnostic and Biological Relevance.

Beatriz Soldevilla, Angeles López-López, Alberto Lens-Pardo, Carlos Carretero-Puche, Angeles Lopez-Gonzalvez, Anna La Salvia, Beatriz Gil-Calderon, Maria C Riesco-Martinez, Paula Espinosa-Olarte, Jacinto Sarmentero, Beatriz Rubio-Cuesta, Raúl Rincón, Coral Barbas, Rocio Garcia-Carbonero.

   PURPOSE: High-throughput "-omic" technologies have enabled the detailed analysis of metabolic networks in several cancers, but NETs have not been explored to date. We aim to assess the metabolomic profile of NET patients to understand metabolic deregulation in these tumors and identify novel biomarkers with clinical potential.
METHODS: Plasma samples from 77 NETs and 68 controls were profiled by GC-MS, CE-MS and LC-MS untargeted metabolomics. OPLS-DA was performed to evaluate metabolomic differences. Related pathways were explored using Metaboanalyst 4.0. Finally, ROC and OPLS-DA analyses were performed to select metabolites with biomarker potential.
RESULTS: We identified 155 differential compounds between NETs and controls. We have detected an increase of bile acids, sugars, oxidized lipids and oxidized products from arachidonic acid and a decrease of carnitine levels in NETs. MPA/MSEA identified 32 enriched metabolic pathways in NETs related with the TCA cycle and amino acid metabolism. Finally, OPLS-DA and ROC analysis revealed 48 metabolites with diagnostic potential.
CONCLUSIONS: This study provides, for the first time, a comprehensive metabolic profile of NET patients and identifies a distinctive metabolic signature in plasma of potential clinical use. A reduced set of metabolites of high diagnostic accuracy has been identified. Additionally, new enriched metabolic pathways annotated may open innovative avenues of clinical research.

Keywords:  NETs; diagnostic biomarkers; disease modelling; machine learning; metabolic signaling; molecular pathways; plasma metabolites

DOI:  https://doi.org/10.3390/cancers13112634
Methods Mol Biol. 2021 ;2298 279-306

Quantification of Modified Nucleosides in the Context of NAIL-MS.

Matthias Heiss, Kayla Borland, Yasemin Yoluç, Stefanie Kellner.

  Recent progress in epitranscriptome research shows an interplay of enzymes modifying RNAs and enzymes dedicated for RNA modification removal. One of the main techniques to study RNA modifications is liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) as it allows sensitive detection of modified nucleosides. Although RNA modifications have been found to be highly dynamic, state-of-the-art LC-MS/MS analysis only gives a static view on modifications and does not allow the investigation of temporal modification placement. Here, we present the principles of nucleic acid isotope labeling coupled with mass spectrometry, termed NAIL-MS, which overcomes these limitations by stable isotope labeling in human cell culture and gives detailed instructions on how to label cells and process samples in order to get reliable results. For absolute quantification in the context of NAIL-MS, we explain the production of internal standards in detail. Furthermore, we outline the requirements for stable isotope labeling in cell culture and all subsequent steps to receive nucleoside mixtures of native RNA for NAIL-MS analysis. In the final section of this chapter, we describe the distinctive features of NAIL-MS data analysis with a special focus toward absolute quantification of modified nucleosides.

Keywords:  Epitranscriptome; LC-MS/MS; Mass spectrometry; RNA modification; Stable isotope labeling; tRNA

DOI:  https://doi.org/10.1007/978-1-0716-1374-0_18
Geroscience. 2021 Jun 05.

Interactive effects of aging and aerobic capacity on energy metabolism-related metabolites of serum, skeletal muscle, and white adipose tissue.

Haihui Zhuang, Sira Karvinen, Timo Törmäkangas, Xiaobo Zhang, Xiaowei Ojanen, Vidya Velagapudi, Markku Alen, Steven L Britton, Lauren G Koch, Heikki Kainulainen, Sulin Cheng, Petri Wiklund.

  Aerobic capacity is a strong predictor of longevity. With aging, aerobic capacity decreases concomitantly with changes in whole body metabolism leading to increased disease risk. To address the role of aerobic capacity, aging, and their interaction on metabolism, we utilized rat models selectively bred for low and high intrinsic aerobic capacity (LCRs/HCRs) and compared the metabolomics of serum, muscle, and white adipose tissue (WAT) at two time points: Young rats were sacrificed at 9 months of age, and old rats were sacrificed at 21 months of age. Targeted and semi-quantitative metabolomics analysis was performed on the ultra-pressure liquid chromatography tandem mass spectrometry (UPLC-MS) platform. The effects of aerobic capacity, aging, and their interaction were studied via regression analysis. Our results showed that high aerobic capacity is associated with an accumulation of isovalerylcarnitine in muscle and serum at rest, which is likely due to more efficient leucine catabolism in muscle. With aging, several amino acids were downregulated in muscle, indicating more efficient amino acid metabolism, whereas in WAT less efficient amino acid metabolism and decreased mitochondrial β-oxidation were observed. Our results further revealed that high aerobic capacity and aging interactively affect lipid metabolism in muscle and WAT, possibly combating unfavorable aging-related changes in whole body metabolism. Our results highlight the significant role of WAT metabolism for healthy aging.

Keywords:  Aerobic capacity; Aging; Metabolites; Metabolomics

DOI:  https://doi.org/10.1007/s11357-021-00387-1
Lipids Health Dis. 2021 Jun 02. 20(1): 58

Identification of prognostic lipid droplet-associated genes in pancreatic cancer patients via bioinformatics analysis.

Rubing Bai, Artur Rebelo, Jörg Kleeff, Yoshiaki Sunami.

   BACKGROUND: Pancreatic cancer is the fourth leading cause of cancer deaths in the United States both in females and in males, and is projected to become the second deadliest cancer by 2030. The overall 5-year survival rate remains at around 10%. Cancer metabolism and specifically lipid metabolism plays an important role in pancreatic cancer progression and metastasis. Lipid droplets can not only store and transfer lipids, but also act as molecular messengers, and signaling factors. As lipid droplets are implicated in reprogramming tumor cell metabolism and in invasion and migration of pancreatic cancer cells, we aimed to identify lipid droplet-associated genes as prognostic markers in pancreatic cancer.
METHODS: We performed a literature search on review articles related to lipid droplet-associated proteins. To select relevant lipid droplet-associated factors, bioinformatics analysis on the GEPIA platform (data are publicly available) was carried out for selected genes to identify differential expression in pancreatic cancer versus healthy pancreatic tissues. Differentially expressed genes were further analyzed regarding overall survival of pancreatic cancer patients.
RESULTS: 65 factors were identified as lipid droplet-associated factors. Bioinformatics analysis of 179 pancreatic cancer samples and 171 normal pancreatic tissue samples on the GEPIA platform identified 39 deferentially expressed genes in pancreatic cancer with 36 up-regulated genes (ACSL3, ACSL4, AGPAT2, BSCL2, CAV1, CAV2, CAVIN1, CES1, CIDEC, DGAT1, DGAT2, FAF2, G0S2, HILPDA, HSD17B11, ICE2, LDAH, LIPE, LPCAT1, LPCAT2, LPIN1, MGLL, NAPA, NCEH1, PCYT1A, PLIN2, PLIN3, RAB5A, RAB7A, RAB8A, RAB18, SNAP23, SQLE, VAPA, VCP, VMP1) and 3 down-regulated genes (FITM1, PLIN4, PLIN5). Among 39 differentially expressed factors, seven up-regulated genes (CAV2, CIDEC, HILPDA, HSD17B11, NCEH1, RAB5A, and SQLE) and two down-regulation genes (BSCL2 and FITM1) were significantly associated with overall survival of pancreatic cancer patients. Multivariate Cox regression analysis identified CAV2 as the only independent prognostic factor.
CONCLUSIONS: Through bioinformatics analysis, we identified nine prognostic relevant differentially expressed genes highlighting the role of lipid droplet-associated factors in pancreatic cancer.

Keywords:  Bioinformatics; GEPIA; Lipid droplet-associated genes; Lipid metabolism; Pancreatic cancer

DOI:  https://doi.org/10.1186/s12944-021-01476-y
Int J Mol Sci. 2021 May 27. pii: 5703. [Epub ahead of print]22(11):

Cancer Cell Metabolism in Hypoxia: Role of HIF-1 as Key Regulator and Therapeutic Target.

Vittoria Infantino, Anna Santarsiero, Paolo Convertini, Simona Todisco, Vito Iacobazzi.

  In order to meet the high energy demand, a metabolic reprogramming occurs in cancer cells. Its role is crucial in promoting tumor survival. Among the substrates in demand, oxygen is fundamental for bioenergetics. Nevertheless, tumor microenvironment is frequently characterized by low-oxygen conditions. Hypoxia-inducible factor 1 (HIF-1) is a pivotal modulator of the metabolic reprogramming which takes place in hypoxic cancer cells. In the hub of cellular bioenergetics, mitochondria are key players in regulating cellular energy. Therefore, a close crosstalk between mitochondria and HIF-1 underlies the metabolic and functional changes of cancer cells. Noteworthy, HIF-1 represents a promising target for novel cancer therapeutics. In this review, we summarize the molecular mechanisms underlying the interplay between HIF-1 and energetic metabolism, with a focus on mitochondria, of hypoxic cancer cells.

Keywords:  HIF-1; TCA cycle; cancer metabolism; hypoxia; mitochondria

DOI:  https://doi.org/10.3390/ijms22115703
Int J Mol Sci. 2021 May 30. pii: 5878. [Epub ahead of print]22(11):

Breast Cancer: Targeting of Steroid Hormones in Cancerogenesis and Diagnostics.

Marcela Valko-Rokytovská, Peter Očenáš, Aneta Salayová, Zuzana Kostecká.

  Breast cancer is the most common malignancy in women with high mortality. Sensitive and specific methods for the detection, characterization and quantification of endogenous steroids in body fluids or tissues are needed for the diagnosis, treatment and prognosis of breast cancer and many other diseases. At present, non-invasive diagnostic methods are gaining more and more prominence, which enable a relatively fast and painless way of detecting many diseases. Metabolomics is a promising analytical method, the principle of which is the study and analysis of metabolites in biological material. It represents a comprehensive non-invasive diagnosis, which has a high potential for use in the diagnosis and prognosis of cancers, including breast cancer. This short review focuses on the targeted metabolomics of steroid hormones, which play an important role in the development and classification of breast cancer. The most commonly used diagnostic tool is the chromatographic method with mass spectrometry detection, which can simultaneously determine several steroid hormones and metabolites in one sample. This analytical procedure has a high potential in effective diagnosis of steroidogenesis disorders. Due to the association between steroidogenesis and breast cancer progression, steroid profiling is an important tool, as well as in monitoring disease progression, improving prognosis, and minimizing recurrence.

Keywords:  breast cancer; metabolomics; steroid hormones

DOI:  https://doi.org/10.3390/ijms22115878
Biomedicines. 2021 May 13. pii: 543. [Epub ahead of print]9(5):

PPARgamma: A Potential Intrinsic and Extrinsic Molecular Target for Breast Cancer Therapy.

Giuseppina Augimeri, Daniela Bonofiglio.

  Over the last decades, the breast tumor microenvironment (TME) has been increasingly recognized as a key player in tumor development and progression and as a promising prognostic and therapeutic target for breast cancer patients. The breast TME, representing a complex network of cellular signaling-deriving from different stromal cell types as well as extracellular matrix components, extracellular vesicles, and soluble growth factors-establishes a crosstalk with cancer cells sustaining tumor progression. A significant emphasis derives from the tumor surrounding inflammation responsible for the failure of the immune system to effectively restrain breast cancer growth. Thus, effective therapeutic strategies require a deeper understanding of the interplay between tumor and stroma, aimed at targeting both the intrinsic neoplastic cells and the extrinsic surrounding stroma. In this scenario, peroxisome proliferator-activated receptor (PPAR) γ, primarily known as a metabolic regulator, emerged as a potential target for breast cancer treatment since it functions in breast cancer cells and several components of the breast TME. In particular, the activation of PPARγ by natural and synthetic ligands inhibits breast cancer cell growth, motility, and invasiveness. Moreover, activated PPARγ may educate altered stromal cells, counteracting the pro-inflammatory milieu that drive breast cancer progression. Interestingly, using Kaplan-Meier survival curves, PPARγ also emerges as a prognostically favorable factor in breast cancer patients. In this perspective, we briefly discuss the mechanisms by which PPARγ is implicated in tumor biology as well as in the complex regulatory networks within the breast TME. This may help to profile approaches that provide a simultaneous inhibition of epithelial cells and TME components, offering a more efficient way to treat breast cancer.

Keywords:  PPARγ ligands; breast cancer; breast tumor microenvironment; peroxisome proliferator-activated receptor gamma; polyunsaturated fatty acids; thiazolidinediones

DOI:  https://doi.org/10.3390/biomedicines9050543
Metabolites. 2021 May 30. pii: 350. [Epub ahead of print]11(6):

Metabolic Response of Pancreatic Carcinoma Cells under Treatment with Dichloroacetate.

Benedikt Feuerecker, Philipp Biechl, Christian Veltkamp, Dieter Saur, Wolfgang Eisenreich.

  In modern oncology, the analysis and evaluation of treatment response are still challenging. Hence, we used a 13C-guided approach to study the impacts of the small molecule dichloroacetate (DCA) upon the metabolic response of pancreatic cancer cells. Two different oncogenic PI3K-driven pancreatic cancer cell lines, 9580 and 10,158, respectively, were treated with 75 mM DCA for 18 h. In the presence of [U-13C6]glucose, the effects of DCA treatment in the core carbon metabolism were analyzed in these cells using gas chromatography-mass spectrometry (GC/MS). 13C-enrichments and isotopologue profiles of key amino acids revealed considerable effects of the DCA treatment upon glucose metabolism. The DCA treatment of the two pancreatic cell lines resulted in a significantly decreased incorporation of [U-13C6]glucose into the amino acids alanine, aspartate, glutamate, glycine, proline and serine in treated, but not in untreated, cancer cells. For both cell lines, the data indicated some activation of pyruvate dehydrogenase with increased carbon flux via the TCA cycle, but also massive inhibition of glycolytic flux and amino acid biosynthesis presumably by inhibition of the PI3K/Akt/mTORC axis. Together, it appears worthwhile to study the early treatment response in DCA-guided or accompanied cancer therapy in more detail, since it could open new avenues for improved diagnosis and therapeutic protocols of cancer.

Keywords:  [U-13C6]glucose; dichloroacetate (DCA); isotopologue profiling; pancreatic cancer

DOI:  https://doi.org/10.3390/metabo11060350
Mol Cell. 2021 May 25. pii: S1097-2765(21)00362-2. [Epub ahead of print]

Choline kinase alpha 2 acts as a protein kinase to promote lipolysis of lipid droplets.

Rui Liu, Jong-Ho Lee, Jingyi Li, Rilei Yu, Lin Tan, Yan Xia, Yanhua Zheng, Xue-Li Bian, Philip L Lorenzi, Qianming Chen, Zhimin Lu.

  Lipid droplets are important for cancer cell growth and survival. However, the mechanism underlying the initiation of lipid droplet lipolysis is not well understood. We demonstrate here that glucose deprivation induces the binding of choline kinase (CHK) α2 to lipid droplets, which is sequentially mediated by AMPK-dependent CHKα2 S279 phosphorylation and KAT5-dependent CHKα2 K247 acetylation. Importantly, CHKα2 with altered catalytic domain conformation functions as a protein kinase and phosphorylates PLIN2 at Y232 and PLIN3 at Y251. The phosphorylated PLIN2/3 dissociate from lipid droplets and are degraded by Hsc70-mediated autophagy, thereby promoting lipid droplet lipolysis, fatty acid oxidation, and brain tumor growth. In addition, levels of CHKα2 S279 phosphorylation, CHKα2 K247 acetylation, and PLIN2/3 phosphorylation are positively correlated with one another in human glioblastoma specimens and are associated with poor prognosis in glioblastoma patients. These findings underscore the role of CHKα2 as a protein kinase in lipolysis and glioblastoma development.

Keywords:  AMPK; KAT5; PLIN2/3; acetylation; autophagy; choline kinase; lipid droplet; lipolysis; phosphorylation; tumorigenesis

DOI:  https://doi.org/10.1016/j.molcel.2021.05.005
Cancers (Basel). 2021 May 31. pii: 2719. [Epub ahead of print]13(11):

Epigenomic and Metabolomic Integration Reveals Dynamic Metabolic Regulation in Bladder Cancer.

Alba Loras, Cristina Segovia, José Luis Ruiz-Cerdá.

  Bladder cancer (BC) represents a clinical, social, and economic challenge due to tumor-intrinsic characteristics, limitations of diagnostic techniques and a lack of personalized treatments. In the last decade, the use of liquid biopsy has grown as a non-invasive approach to characterize tumors. Moreover, the emergence of omics has increased our knowledge of cancer biology and identified critical BC biomarkers. The rewiring between epigenetics and metabolism has been closely linked to tumor phenotype. Chromatin remodelers interact with each other to control gene silencing in BC, but also with stress-inducible factors or oncogenic signaling cascades to regulate metabolic reprogramming towards glycolysis, the pentose phosphate pathway, and lipogenesis. Concurrently, one-carbon metabolism supplies methyl groups to histone and DNA methyltransferases, leading to the hypermethylation and silencing of suppressor genes in BC. Conversely, α-KG and acetyl-CoA enhance the activity of histone demethylases and acetyl transferases, increasing gene expression, while succinate and fumarate have an inhibitory role. This review is the first to analyze the interplay between epigenome, metabolome and cell signaling pathways in BC, and shows how their regulation contributes to tumor development and progression. Moreover, it summarizes non-invasive biomarkers that could be applied in clinical practice to improve diagnosis, monitoring, prognosis and the therapeutic options in BC.

Keywords:  biomarkers; bladder cancer; epigenetics; metabolic pathways; metabolism; metabolomics; miRNAs

DOI:  https://doi.org/10.3390/cancers13112719
Mol Cancer Res. 2021 Jun 04. pii: molcanres.0962.2020. [Epub ahead of print]

EMT-derived alterations in glutamine metabolism sensitize mesenchymal breast cells to mTOR inhibition.

Sigurdur Trausti Karvelsson, Arnar Sigurdsson, Kotryna Seip, Maria Tunset Grinde, Qiong Wang, Freyr Johannsson, Gunhild Marie Maelandsmo, Siver Andreas Moestue, Ottar Rolfsson, Skarphedinn Halldorsson.

Epithelial-to-mesenchymal transition (EMT) is a fundamental developmental process with strong implications in cancer progression. Understanding the metabolic alterations associated with EMT may open new avenues of treatment and prevention. Here, we used 13C carbon analogs of glucose and glutamine to examine differences in their utilization within central carbon and lipid metabolism following EMT in breast epithelial cell lines. We found that there are inherent differences in metabolic profiles before and after EMT. We observed EMT-dependent re-routing of the TCA-cycle, characterized by increased mitochondrial IDH2 -mediated reductive carboxylation of glutamine to lipid biosynthesis with a concomitant lowering of glycolytic rates and glutamine-dependent glutathione (GSH) generation. Using weighted correlation network analysis, we identified cancer drugs whose efficacy against the NCI-60 Human Tumor Cell Line panel is significantly associated with GSH abundance and confirmed these in vitro. We report that EMT-linked alterations in GSH synthesis modulate the sensitivity of breast epithelial cells to mTOR inhibitors. Implications: EMT in breast cells causes an increased demand for glutamine for fatty acid biosynthesis, altering its contribution to glutathione biosynthesis which sensitizes the cells to mTOR inhibitors.

DOI: https://doi.org/10.1158/1541-7786.MCR-20-0962
Nat Protoc. 2021 Jun 04.

Selected ion flow tube mass spectrometry for targeted analysis of volatile organic compounds in human breath.

Ilaria Belluomo, Piers R Boshier, Antonis Myridakis, Bhamini Vadhwana, Sheraz R Markar, Patrik Spanel, George B Hanna.

The analysis of volatile organic compounds (VOCs) within breath for noninvasive disease detection and monitoring is an emergent research field that has the potential to reshape current clinical practice. However, adoption of breath testing has been limited by a lack of standardization. This protocol provides a comprehensive workflow for online and offline breath analysis using selected ion flow tube mass spectrometry (SIFT-MS). Following the suggested protocol, 50 human breath samples can be analyzed and interpreted in <3 h. Key advantages of SIFT-MS are exploited, including the acquisition of real-time results and direct compound quantification without need for calibration curves. The protocol includes details of methods developed for targeted analysis of disease-specific VOCs, specifically short-chain fatty acids, aldehydes, phenols, alcohols and alkanes. A procedure to make custom breath collection bags is also described. This standardized protocol for VOC analysis using SIFT-MS is intended to provide a basis for wider application and the use of breath analysis in clinical studies.

DOI: https://doi.org/10.1038/s41596-021-00542-0
Methods. 2021 May 28. pii: S1046-2023(21)00150-X. [Epub ahead of print]

Quantification of mRNA cap-modifications by means of LC-QqQ-MS.

Nils Muthmann, Petr Špaček, Dennis Reichert, Melissa van Dülmen, Andrea Rentmeister.

  Enzymatic modification of the 5'-cap is a versatile approach to modulate the properties of mRNAs. Transfer of methyl groups from S-adenosyl-L-methionine (AdoMet) or functional moieties from non-natural analogs by methyltransferases (MTases) allows for site-specific modifications at the cap. These modifications have been used to tune translation or control it in a temporal manner and even influence immunogenicity of mRNA. For quantification of the MTase-mediated cap modification, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) provides the required sensitivity and accuracy. Here, we describe the complete workflow starting from in vitro transcription to produce mRNAs, via their enzymatic modification at the cap with natural or non-natural moieties to the quantification of these cap-modifications by LC-QqQ-MS.

Keywords:  5' cap; Chemo-enzymatic; LS-MS; Methyltransferase; SAM

DOI:  https://doi.org/10.1016/j.ymeth.2021.05.018
Metabolites. 2021 May 18. pii: 325. [Epub ahead of print]11(5):

Lactate Reprograms Energy and Lipid Metabolism in Glucose-Deprived Oxidative Glioma Stem Cells.

Noriaki Minami, Kazuhiro Tanaka, Takashi Sasayama, Eiji Kohmura, Hideyuki Saya, Oltea Sampetrean.

  Fast-growing tumors satisfy their bioenergetic needs by supplementing glucose with alternative carbon sources. Cancer stem cells are the most versatile and robust cells within malignant tumors. They avoid potentially lethal metabolic and other types of stress through flexible reprogramming of relevant pathways, but it has remained unclear whether alternative carbon sources are important for the maintenance of their tumor-propagating ability. Here we assessed the ability of glycolytic and oxidative murine glioma stem cells (GSCs) to grow in an ultralow glucose medium. Sphere formation assays revealed that exogenous lactate and acetate reversed the growth impairment of oxidative GSCs in such medium. Extracellular flux analysis showed that lactate supported oxygen consumption in these cells, whereas metabolomics analysis revealed that it increased the intracellular levels of tricarboxylic acid cycle intermediates, ATP, and GTP as well as increased adenylate and guanylate charge. Lactate also reversed the depletion of choline apparent in the glucose-deprived cells as well as reprogrammed phospholipid and fatty acid biosynthesis. This metabolic reprogramming was associated with a more aggressive phenotype of intracranial tumors formed by lactate-treated GSCs. Our results thus suggest that lactate is an important alternative energetic and biosynthetic substrate for oxidative GSCs, and that it sustains their growth under conditions of glucose deprivation.

Keywords:  cancer stem cell; glioma; glioma stem cell; glucose deprivation; lactate; lipid metabolism; metabolic cooperation; metabolic reprogramming; metabolic symbiosis; metabolism

DOI:  https://doi.org/10.3390/metabo11050325
Metabolites. 2021 May 19. pii: 328. [Epub ahead of print]11(5):

Tick-Tock Consider the Clock: The Influence of Circadian and External Cycles on Time of Day Variation in the Human Metabolome-A Review.

Thomas P M Hancox, Debra J Skene, Robert Dallmann, Warwick B Dunn.

  The past decade has seen a large influx of work investigating time of day variation in different human biofluid and tissue metabolomes. The driver of this daily variation can be endogenous circadian rhythms driven by the central and/or peripheral clocks, or exogenous diurnal rhythms driven by behavioural and environmental cycles, which manifest as regular 24 h cycles of metabolite concentrations. This review, of all published studies to date, establishes the extent of daily variation with regard to the number and identity of 'rhythmic' metabolites observed in blood, saliva, urine, breath, and skeletal muscle. The probable sources driving such variation, in addition to what metabolite classes are most susceptible in adhering to or uncoupling from such cycles is described in addition to a compiled list of common rhythmic metabolites. The reviewed studies show that the metabolome undergoes significant time of day variation, primarily observed for amino acids and multiple lipid classes. Such 24 h rhythms, driven by various factors discussed herein, are an additional source of intra/inter-individual variation and are thus highly pertinent to all studies applying untargeted and targeted metabolomics platforms, particularly for the construction of biomarker panels. The potential implications are discussed alongside proposed minimum reporting criteria suggested to acknowledge time of day variation as a potential influence of results and to facilitate improved reproducibility.

Keywords:  blood; breath; circadian rhythms; diurnal rhythms; metabolite rhythms; metabolomics; saliva; skeletal muscle; urine

DOI:  https://doi.org/10.3390/metabo11050328
Metabolites. 2021 May 13. pii: 313. [Epub ahead of print]11(5):

Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS.

Boryana Petrova, Anna Warren, Nuria Yulia Vital, Andrew J Culhane, Adam G Maynard, Alan Wong, Naama Kanarek.

  Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell's metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH, and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here, we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields.

Keywords:  HILIC chromatography; NADH; NADPH; glutathione; mass-spectrometry method; redox metabolite detection in mammalian cells; redox metabolites

DOI:  https://doi.org/10.3390/metabo11050313
Proteomics. 2021 Jun 03. e2000080

Impact of chemically defined culture media formulations on extracellular vesicle production by amniotic epithelial cells.

Dandan Zhu, Haoyun Fang, Gina D Kusuma, Renate Schwab, Mehri Barabadi, Siow Teng Chan, Hannah McDonald, Cheng Mee Leong, Euan M Wallace, David W Greening, Rebecca Lim.

  The therapeutic properties of cell derived extracellular vesicles (EVs) make them promising cell-free alternative to regenerative medicine. However, clinical translation of this technology relies on the ability to manufacture EVs in a scalable, reproducible, and cGMP-compliant manner. To generate EVs in sufficient quantity, a critical step is the selection and development of culture media, where differences in formulation may influence the EV manufacturing process. In this study, we used human amniotic epithelial cells (hAECs) as a model system to explore the effect of different formulations of chemically defined, commercially sourced media on EV production. Here, we determined that cell viability and proliferation rate are not reliable quality indicators for EV manufacturing. The levels of tetraspanins and epitope makers of EVs were significantly impacted by culture media formulations. Mass spectrometry-based proteomic profiling revealed proteome composition of hAEC-EVs and the influence of media formulations on composition of EV proteome. This study has revealed critical aspects including cell viability and proliferation rate, EV yield, and tetraspanins, surface epitopes and proteome composition of EVs influenced by media formulations, and further insight into standardised EV production culture media that should be considered in clinical-grade scalable EV manufacture for generation of therapeutic EVs. This article is protected by copyright. All rights reserved.

Keywords:  culture media formulation; extracellular vesicle generation; extracellular vesicles; human amniotic epithelial cells; proteomics

DOI:  https://doi.org/10.1002/pmic.202000080