bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2020‒04‒12
thirty papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research

  1. Science. 2020 Apr 10. pii: eaaw5473. [Epub ahead of print]368(6487):
      Metabolic reprogramming is a hallmark of malignancy. As our understanding of the complexity of tumor biology increases, so does our appreciation of the complexity of tumor metabolism. Metabolic heterogeneity among human tumors poses a challenge to developing therapies that exploit metabolic vulnerabilities. Recent work also demonstrates that the metabolic properties and preferences of a tumor change during cancer progression. This produces distinct sets of vulnerabilities between primary tumors and metastatic cancer, even in the same patient or experimental model. We review emerging concepts about metabolic reprogramming in cancer, with particular attention on why metabolic properties evolve during cancer progression and how this information might be used to develop better therapeutic strategies.
  2. Biomolecules. 2020 Apr 08. pii: E568. [Epub ahead of print]10(4):
      Tumorigenesis is accompanied by the reprogramming of cellular metabolism. The shift from oxidative phosphorylation to predominantly glycolytic pathways to support rapid growth is well known and is often referred to as the Warburg effect. However, other metabolic changes and acquired needs that distinguish cancer cells from normal cells have also been discovered. The dependence of cancer cells on exogenous methionine is one of them and is known as methionine dependence or the Hoffman effect. This phenomenon describes the inability of cancer cells to proliferate when methionine is replaced with its metabolic precursor, homocysteine, while proliferation of non-tumor cells is unaffected by these conditions. Surprisingly, cancer cells can readily synthesize methionine from homocysteine, so their dependency on exogenous methionine reflects a general need for altered metabolic flux through pathways linked to methionine. In this review, an overview of the field will be provided and recent discoveries will be discussed.
    Keywords:  S-adenosylmethionine; SAM-checkpoint; cancer; cell cycle; methionine
  3. Front Pharmacol. 2020 ;11 239
      Ferroptosis is a novel regulated cell death pattern discovered when studying the mechanism of erastin-killing RAS mutant tumor cells in 2012. It is an iron-dependent programmed cell death pathway mainly caused by an increased redox imbalance but with distinct biological and morphology characteristics when compared to other known cell death patterns. Ferroptosis is associated with various diseases including acute kidney injury, cancer, and cardiovascular, neurodegenerative, and hepatic diseases. Moreover, activation or inhibition of ferroptosis using a variety of ferroptosis initiators and inhibitors can modulate disease progression in animal models. In this review, we provide a comprehensive analysis of the characteristics of ferroptosis, its initiators and inhibitors, and the potential role of its main metabolic pathways in the treatment and prevention of various diseased states. We end the review with the current knowledge gaps in this area to provide direction for future research on ferroptosis.
    Keywords:  degenerative diseases; ferroptosis; pharmacology design; reactive oxygen species; signaling pathways
  4. ACS Omega. 2020 Mar 31. 5(12): 6754-6762
      Despite its growing popularity and use, bottom-up proteomics remains a complex analytical methodology. Its general workflow consists of three main steps: sample preparation, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and computational data analysis. Quality assessment of the different steps and components of this workflow is instrumental to identify technical flaws and avoid loss of precious measurement time and sample material. However, assessment of the extent of sample losses along with the sample preparation protocol, in particular, after proteolytic digestion, is not yet routinely implemented because of the lack of an accurate and straightforward method to quantify peptides. Here, we report on the use of a microfluidic UV/visible spectrophotometer to quantify MS-ready peptides directly in the MS-loading solvent, consuming only 2 μL of sample. We compared the performance of the microfluidic spectrophotometer with a standard device and determined the optimal sample amount for LC-MS/MS analysis on a Q Exactive HF mass spectrometer using a dilution series of a commercial K562 cell digest. A careful evaluation of selected LC and MS parameters allowed us to define 3 μg as an optimal peptide amount to be injected into this particular LC-MS/MS system. Finally, using tryptic digests from human HEK293T cells and showing that injecting equal peptide amounts, rather than approximate ones, result in less variable LC-MS/MS and protein quantification data. The obtained quality improvement together with easy implementation of the approach makes it possible to routinely quantify MS-ready peptides as a next step in daily proteomics quality control.
  5. Anal Chem. 2020 Apr 07.
      Quantitative mass spectrometry imaging (MSI) is an effective technique for determining the spatial distribution of molecules in a variety of sample types; however, the quality of the ion signals is related to the chemical and morphological properties of both the same matrix and targeted analyte(s). Issues may arise with the incorporation of standards into the sample at repeatable, well-defined concentrations, as well as with the extraction and incorporation of endogenous analytes versus standards from tissue into the matrix. To address these concerns, we combine imprint MSI (iMSI) with kinetic calibration, and use it to quantify lipids in rat brain tissue samples. Briefly, tissues were imprinted on slides coated with a dopamine-modified TiO2 monolith pretreated with analyte standards, resulting in the adsorption of endogenous analytes onto the coating and desorption of standards into the tissue. The incorporation of standards into the tissue enabled quantification of the measured analytes using kinetic calibration. Moreover, matrix effects were reduced, and the intensities of analyte standard signals became more uniform. The symmetry of the adsorption of endogenous ceramides and the desorption of ceramide standards suggests that the content of adsorbed endogenous ceramide can be determined by measuring the content of desorbed ceramide standard. Using kinetic calibration, endogenous ceramide concentrations were calculated for a range of dry and wet tissue imprinting conditions and compared with quantitative MSI using a standard spiking approach. We validated quantitative iMSI using liquid chromatography tandem mass spectrometry (LC-MS/MS) and found that the concentrations determined using iMSI compared with LC-MS/MS were in the range of 70 to 200% over the concentration range of endogenous ceramides; the correlation coefficient between iMSI and LC-MS/MS was over 0.9 (Pearson's r), while the recoveries via traditional standard spiking were between 200% and 5000% depending on the brain region and sample preparation conditions. .
  6. J Lipid Res. 2020 Apr 07. pii: jlr.RA120000671. [Epub ahead of print]
      Ceramides are the predominant lipids in the stratum corneum (SC) and are crucial components for normal skin barrier function. Although the composition of various ceramide classes in the human SC has been reported, that in mice is still unknown, despite mice being widely used as animal models of skin barrier function. Here, we performed LC-MS/MS analyses using recently available ceramide class standards to measure 25 classes of free ceramides and 5 classes of protein-bound ceramides from the human and mouse SC. Phytosphingosine-type ceramides (P-ceramides) and 6-hydroxy sphingosine-type ceramides (H-ceramides), which both contain an additional hydroxyl group, were abundant in human SC (35% and 45% of total ceramides, respectively). In contrast, in mice, P-ceramides and H-ceramides were present at ~1% and undetectable levels, respectively, and sphingosine-type ceramides accounted for ~90%. In humans, ceramides containing α-hydroxy FA were abundant, whereas ceramides containing β-hydroxy FA (B-ceramides) or ω-hydroxy FA were abundant in mice. The hydroxylated β-carbon in B-ceramides was in the (R)-configuration. Genetic knockout of β-hydroxy acyl-CoA dehydratases in HAP1 cells increased B-ceramide levels, suggesting that β-hydroxy acyl-CoA, an FA-elongation cycle intermediate in the endoplasmic reticulum, is a substrate for B-ceramide synthesis. We anticipate that our methods and findings will help to elucidate the role of each ceramide class in skin barrier formation and in the pathogenesis of skin disorders.
    Keywords:  Ceramides; Epidermis; Fatty acid; Lipidomics; Mass spectrometry; Skin barrier; Sphingolipids; Stratum corneum
  7. J Am Chem Soc. 2020 Apr 10.
      Untargeted metabolomics aims to quantify the complete set of metabolites within a biological system, most commonly by liquid chromatography/mass spectrometry (LC/MS). Since nearly the inception of the field, compound identification has been widely recognized as the rate-limiting step of the experimental workflow. In spite of exponential increases in the size of metabolomic databases, which now contain experimental MS/MS spectra for over a half million reference compounds, chemical structures still cannot be confidently assigned to many signals in a typical LC/MS dataset. The purpose of this Perspective is to consider why identification rates continue to be low in untargeted metabolomics. One rationalization is that many naturally occurring metabolites detected by LC/MS are true "novel" compounds that have yet to be incorporated into metabolomic databases. An alternative possibility, however, is that research data do not provide database matches because of informatic artifacts, chemical contaminants, and signal redundancies. Increasing evidence suggests that, for at least some sample types, many unidentifiable signals in untargeted metabolomics result from the latter rather than new compounds originating from the specimen being measured. The implications of these observations on chemical discovery in untargeted metabolomics is discussed.
  8. Front Oncol. 2020 ;10 231
      To sustain their high proliferation rates, most cancer cells rely on glycolytic metabolism, with production of lactic acid. For many years, lactate was seen as a metabolic waste of glycolytic metabolism; however, recent evidence has revealed new roles of lactate in the tumor microenvironment, either as metabolic fuel or as a signaling molecule. Lactate plays a key role in the different models of metabolic crosstalk proposed in malignant tumors: among cancer cells displaying complementary metabolic phenotypes and between cancer cells and other tumor microenvironment associated cells, including endothelial cells, fibroblasts, and diverse immune cells. This cell metabolic symbiosis/slavery supports several cancer aggressiveness features, including increased angiogenesis, immunological escape, invasion, metastasis, and resistance to therapy. Lactate transport is mediated by the monocarboxylate transporter (MCT) family, while another large family of G protein-coupled receptors (GPCRs), not yet fully characterized in the cancer context, is involved in lactate/acidosis signaling. In this mini-review, we will focus on the role of lactate in the tumor microenvironment, from metabolic affairs to signaling, including the function of lactate in the cancer-cancer and cancer-stromal shuttles, as well as a signaling oncometabolite. We will also review the prognostic value of lactate metabolism and therapeutic approaches designed to target lactate production and transport.
    Keywords:  GPR81; lactate; lactate shuttles; metabolic fuel; monocarboxylate transporters; signaling molecule; warburg effect
  9. Cell Chem Biol. 2020 Apr 07. pii: S2451-9456(20)30111-2. [Epub ahead of print]
      Redox balance is essential for normal brain, hence dis-coordinated oxidative reactions leading to neuronal death, including programs of regulated death, are commonly viewed as an inevitable pathogenic penalty for acute neuro-injury and neurodegenerative diseases. Ferroptosis is one of these programs triggered by dyshomeostasis of three metabolic pillars: iron, thiols, and polyunsaturated phospholipids. This review focuses on: (1) lipid peroxidation (LPO) as the major instrument of cell demise, (2) iron as its catalytic mechanism, and (3) thiols as regulators of pro-ferroptotic signals, hydroperoxy lipids. Given the central role of LPO, we discuss the engagement of selective and specific enzymatic pathways versus random free radical chemical reactions in the context of the phospholipid substrates, their biosynthesis, intracellular location, and related oxygenating machinery as participants in ferroptotic cascades. These concepts are discussed in the light of emerging neuro-therapeutic approaches controlling intracellular production of pro-ferroptotic phospholipid signals and their non-cell-autonomous spreading, leading to ferroptosis-associated necroinflammation.
    Keywords:  cerebral hemorrhage; cerebral ischemia; glutathione peroxidase 4; lipoxygenase; neurodegeneration; phospholipid; redox lipidomics; regulated cell death; traumatic brain injury
  10. Genes Dev. 2020 Apr 09.
      Human cancers with activating RAS mutations are typically highly aggressive and treatment-refractory, yet RAS mutation itself is insufficient for tumorigenesis, due in part to profound metabolic stress induced by RAS activation. Here we show that loss of REDD1, a stress-induced metabolic regulator, is sufficient to reprogram lipid metabolism and drive progression of RAS mutant cancers. Redd1 deletion in genetically engineered mouse models (GEMMs) of KRAS-dependent pancreatic and lung adenocarcinomas converts preneoplastic lesions into invasive and metastatic carcinomas. Metabolic profiling reveals that REDD1-deficient/RAS mutant cells exhibit enhanced uptake of lysophospholipids and lipid storage, coupled to augmented fatty acid oxidation that sustains both ATP levels and ROS-detoxifying NADPH. Mechanistically, REDD1 loss triggers HIF-dependent activation of a lipid storage pathway involving PPARγ and the prometastatic factor CD36. Correspondingly, decreased REDD1 expression and a signature of REDD1 loss predict poor outcomes selectively in RAS mutant but not RAS wild-type human lung and pancreas carcinomas. Collectively, our findings reveal the REDD1-mediated stress response as a novel tumor suppressor whose loss defines a RAS mutant tumor subset characterized by reprogramming of lipid metabolism, invasive and metastatic progression, and poor prognosis. This work thus provides new mechanistic and clinically relevant insights into the phenotypic heterogeneity and metabolic rewiring that underlies these common cancers.
    Keywords:  RAS; REDD1; energy stress; fatty acid oxidation; glycolysis; lipid metabolism; lysophospholipids; metastasis; oxidative stress
  11. Metabolites. 2020 Apr 04. pii: E143. [Epub ahead of print]10(4):
      The development of improved mass spectrometers and supporting computational tools is expected to enable the rapid annotation of whole metabolomes. Essential for the progress is the identification of strengths and weaknesses of novel instrumentation in direct comparison to previous instruments. Orbitrap liquid chromatography (LC)-mass spectrometry (MS) technology is now widely in use, while Orbitrap gas chromatography (GC)-MS introduced in 2015 has remained fairly unexplored in its potential for metabolomics research. This study aims to evaluate the additional knowledge gained in a metabolomics experiment when using the high-resolution Orbitrap GC-MS in comparison to a commonly used unit-mass resolution single-quadrupole GC-MS. Samples from an osmotic stress treatment of a non-model organism, the microalga Skeletonema costatum, were investigated using comparative metabolomics with low- and high-resolution methods. Resulting datasets were compared on a statistical level and on the level of individual compound annotation. Both MS approaches resulted in successful classification of stressed vs. non-stressed microalgae but did so using different sets of significantly dysregulated metabolites. High-resolution data only slightly improved conventional library matching but enabled the correct annotation of an unknown. While computational support that utilizes high-resolution GC-MS data is still underdeveloped, clear benefits in terms of sensitivity, metabolic coverage, and support in structure elucidation of the Orbitrap GC-MS technology for metabolomics studies are shown here.
    Keywords:  Orbitrap Gas Chromatography–Mass Spectrometry (Orbitrap GC–MS), high-resolution mass spectrometry (HRMS); comparative metabolomics; diatom; instrument comparison; metabolite identification; osmotic stress
  12. Cancer Res. 2020 Apr 09. pii: canres.3580.2019. [Epub ahead of print]
      Epigenetic regulation of gene transcription has been shown to coordinate with nutrient availability, yet the mechanisms underlying this coordination remain incompletely understood. Here we show that glucose starvation suppresses histone 2A K119 monoubiquitination (H2Aub), a histone modification that correlates with gene repression. Glucose starvation suppressed H2Aub levels independently of energy stress-mediated AMPK activation and possibly through NADPH depletion and subsequent inhibition of BMI1, an integral component of polycomb repressive complex 1 (PRC1) that catalyzes H2Aub on chromatin. Integrated transcriptomic and epigenomic analyses linked glucose starvation-mediated H2Aub repression to the activation of genes involved in the endoplasmic reticulum (ER) stress response. We further showed that this epigenetic mechanism has a role in glucose starvation-induced cell death and that pharmacologic inhibition of glucose transporter 1 (GLUT1) and PRC1 synergistically promoted ER stress and suppressed tumor growth in vivo. Together, these results reveal a hitherto unrecognized epigenetic mechanism coupling glucose availability to the ER stress response.
  13. Cancers (Basel). 2020 Apr 07. pii: E900. [Epub ahead of print]12(4):
      A new pathway leading to the n-10 fatty acid series has been recently evidenced, starting from sapienic acid, a monounsaturated fatty acid (MUFA) resulting from the transformation of palmitic acid by delta-6 desaturase. Sapienic acid has attracted attention as a novel marker of cancer cell plasticity. Here, we analyzed fatty acids, including the n-10 fatty acid contents, and for the first time, compared cell membranes and the corresponding extracellular vesicles (EV) of two human prostatic adenocarcinoma cell lines of different aggressiveness (PC3 and LNCaP). The n-10 components were 9-13% of the total fatty acids in both cancer cell lines and EVs, with total MUFA levels significantly higher in EVs of the most aggressive cell type (PC3). High sapienic/palmitoleic ratios indicated the preference for delta-6 versus delta-9 desaturase enzymatic activity in these cell lines. The expressions analysis of enzymes involved in desaturation and elongation by qRT-PCR showed a higher desaturase activity in PC3 and a higher elongase activity toward polyunsaturated fatty acids than toward saturated fatty acids, compared to LNCaP cells. Our results improve the present knowledge in cancer fatty acid metabolism and lipid phenotypes, highlighting EV lipidomics to monitor positional fatty acid isomer profiles and MUFA levels in cancer.
    Keywords:  desaturase enzyme; elongase enzyme; extracellular vesicle lipidome; lipidomics; positional fatty acid isomer; sapienic acid; sebaleic acid; trans geometrical isomer
  14. J Am Soc Mass Spectrom. 2020 Apr 08.
      We developed a normalization method utilizing the expression levels of a panel of endogenous proteins as normalization standards (EPNS herein). We tested the validity of the method using two sets of TMT-labeled data and found that this normalization method effectively reduced global intensity bias at the protein level. The coefficient of variation (CV) of overall median was reduced by 55% and 82% on average, comparing to the reduction by 72% and 86% after normalization using upper quartile. Furthermore, we used differential protein expression analysis and statistical learning to identify biomarkers for colorectal cancer from a CPTAC data set. The expression changes of a panel of proteins, including NUP205, GTPBP4, CNN2, GNL3, and S100A11, all of which highly correlate with colorectal cancer. Applying these five proteins as model features, random forest modeling obtained prediction results with the maximum AUC of 0.9998 using EPNS-normalized data, comparing favorably to the AUC of 0.9739 using the raw data. Thus, the normalization method based on EPNS reduced the global intensity bias and is applicable for quantitative proteomic analysis.
  15. Anal Chem. 2020 Apr 09.
      Mapping the complete molecular composition of a lipidome is considered as an important goal of lipidomics for unraveling pathways and mechanisms behind lipid homeostasis. Conventional dissociation methods of mass spectrometry (MS) usually cannot give detailed structural information of lipids such as locations of carbon-carbon double bond (C=C) in acyl chains. Double bond derivatization via the Paternò-Büchi (PB) reaction has been demonstrated as a simple and highly efficient method for identification of C=C locations of different classes of lipids when paired with tandem mass spectrometry (MS/MS). In this work, reverse-phase lipid chromatography (RPLC)-MS was coupled with online PB reaction to achieve enhanced analysis of isomers and isobars of phospholipids. A new acetone-containing mobile phase was developed, which showed good elution performance for the separation of phospholipids by C18 columns. An improved flow microreactor was developed, enabling online derivatization of phospholipid C=C in 20 s. The workflow of RPLC-PB-MS/MS was developed and optimized for identification of C=C locations in isobaric ether-linked and diacyl phospholipids, 13C isobars, and acyl chain isomers in biological lipid extracts. Separation and identification of C=C locations of cis/trans phospholipid isomers were achieved for lipid standards. The incorporation of the PB reaction into RPLC-MS workflow enabled analysis of phospholipid isomers and isobars with high confidence, demonstrating its potential for high-throughput phospholipid identification from complex mixtures.
  16. Cancers (Basel). 2020 Apr 07. pii: E898. [Epub ahead of print]12(4):
      Cancer cells and tissues have an aberrant regulation of hydrogen ion dynamics driven by a combination of poor vascular perfusion, regional hypoxia, and increased the flux of carbons through fermentative glycolysis. This leads to extracellular acidosis and intracellular alkalinization. Dysregulated pH dynamics influence cancer cell biology, from cell transformation and tumorigenesis to proliferation, local growth, invasion, and metastasis. Moreover, this dysregulated intracellular pH (pHi) drives a metabolic shift to increased aerobic glycolysis and reduced mitochondrial oxidative phosphorylation, referred to as the Warburg effect, or Warburg metabolism, which is a selective feature of cancer. This metabolic reprogramming confers a thermodynamic advantage on cancer cells and tissues by protecting them against oxidative stress, enhancing their resistance to hypoxia, and allowing a rapid conversion of nutrients into biomass to enable cell proliferation. Indeed, most cancers have increased glucose uptake and lactic acid production. Furthermore, cancer cells have very dysregulated electrolyte balances, and in the interaction of the pH dynamics with electrolyte, dynamics is less well known. In this review, we highlight the interconnected roles of dysregulated pH dynamics and electrolytes imbalance in cancer initiation, progression, adaptation, and in determining the programming and reprogramming of tumor cell metabolism.
    Keywords:  cancer; electrolytes; metabolism; pH
  17. J Am Soc Mass Spectrom. 2020 Apr 06.
      Ether lipids represent a unique subclass of glycerophospholipid (GPL) that possess a 1-O-alkyl (i.e., plasmanyl subclass) or a 1-O-alk-1'-enyl (i.e., plasmenyl subclass) group linked at the sn-1 position of the glycerol backbone. As changes in ether GPL composition and abundance are associated with numerous human pathologies, analytical strategies capable of providing high-level structural detail are desirable. While mass spectrometry (MS) has emerged as a prominent tool for lipid structural elucidation in biological extracts, distinctions between the various isomeric forms of ether-linked GPLs has remained a significant challenge for tandem-MS, principally due to similarities in the conventional tandem mass spectra obtained from the two ether-linked subclasses. To distinguish plasmanyl and plasmenyl GPL, a multi-stage (i.e., MSn where n = 3 or 4) mass spectrometric approach reliant on low-energy collision induced dissociation (CID) is required. While this method facilitates assignment of the sn-1 bond type (i.e., 1-O-alkyl versus 1-O-alk-1'-enyl), a composite distribution of isomers is left unresolved, as carbon-carbon double bond (C=C) positions cannot be localized in the sn-2 fatty acyl substituent. In this study, we combine a systematic MSn approach with two unique gas-phase charge inversion ion/ion chemistries to elucidate ether GPL structures with high-level detail. Ultimately, we assign both sn-1 bond type and sites of unsaturation in the sn-2 fatty acyl substituent using an entirely gas-phase MS-based workflow. Application of this workflow to human blood plasma extract permitted isomeric resolution and in-depth structural identification of major, and in some cases, minor isomeric contributors to ether GPL that have been previously unresolved when examined via conventional methods.
  18. Anal Chem. 2020 Apr 06.
      Free fatty acids (FFAs) are key intermediates of lipid metabolism that have a crucial role in many critical biological processes. The specific locations of carbon-carbon double bonds (C=C) in FFAs are often associated with distinct biological functions. Despite the rapid development of analytical techniques, identification of C=C locations in FFAs with more than three C=C bonds in complex biological matrices remains a challenge. Herein, we describe a double derivatization strategy, coupled with shotgun-mass spectrometry (MS), for unambiguous and sensitive determination of a high-coverage C=C bond (from 1 to 6) locations of FFAs. Our approach is based on combination of acetone labeling of C=C bonds and N,N-diethyl-1,2-ethanediamine (DEEA) labeling of carboxyl groups within FFAs. Acetone labeling of C=C bonds via photochemical reaction provides diagnostic ions, specific to C=C locations, and DEEA labeling of carboxyl groups significantly enhances MS response of diagnostic ions, by invoking a readily protonated tertiary amine group on FFA analytes. By exploiting this double derivatization strategy, the assignment of C=C locations of FFAs with more than three C=C bonds was achieved with high sensitivity (limit of quantitation (LOQ) 0.1-1.5 nmol/L). In contrast, such assignments were not possible by acetone labeling alone, because of the low sensitivity of diagnostic ions in negative ionization mode of MS. The applicability of our method was demonstrated by profiling of FFAs, including unsaturated FFA C=C positional isomers, in liver samples from mice with nonalcoholic fatty liver disease (NAFLD) and their lean controls. The study showed that the high-specificity and high-sensitivity method developed here is promising for accurate identification and quantitation of a wide array of FFAs in biological samples.
  19. Anal Chim Acta. 2020 May 01. pii: S0003-2670(20)30256-7. [Epub ahead of print]1109 44-52
      The development of quantitative metabolomics approaches for future standardized and translational applications has become increasingly important. Data-independent targeted quantitative metabolomics (DITQM) is a newly proposed method providing ion pair information on 1324 metabolites. However, the quantification of more than 1000 metabolites in large sample sizes has still not been implemented. In this study, on the basis of the DITQM concept, scheduled multiple reaction monitoring (MRM) methods for both high-abundant and low-abundant metabolites were established to broaden the quantification coverage, and an open-source program "Quanter_1.0" was coded to facilitate efficient data handling. Our results demonstrated that 1015 metabolites in human plasma met the quantitative requirements and could be relatively determined in an effective manner. The method was then applied to a large-scale sample study of lung cancer consisting of three distinct analytical batches. It was obvious that data quality that originated from quantitative metabolomics was improved, with substantially lower intra- and inter-batch data variation, resulting in a more effective multivariate statistical model. Finally, 26 potential biomarkers of lung cancer were discovered. Collectively, our approach provides a promising tool for quantitative metabolomics research involving large-scale sample sizes and clinical application.
    Keywords:  High-coverage analysis; Large-scale sample size; Liquid chromatography-tandem mass spectrometry; Relative quantitative metabolomics
  20. Int Rev Cell Mol Biol. 2020 ;pii: S1937-6448(19)30117-0. [Epub ahead of print]351 197-236
      Lipids are fundamental to life as structural components of cellular membranes and for signaling. They are also key regulators of different cellular processes such as cell division, proliferation, and death. Regulated cell death (RCD) requires the engagement of lipids and lipid metabolism for the initiation and execution of its killing machinery. The permeabilization of lipid membranes is a hallmark of RCD that involves, for each kind of cell death, a unique lipid profile. While the permeabilization of the mitochondrial outer membrane allows the release of apoptotic factors to the cytosol during apoptosis, permeabilization of the plasma membrane facilitates the release of intracellular content in other nonapoptotic types of RCD like necroptosis and ferroptosis. Lipids and lipid membranes are important accessory molecules required for the activation of protein executors of cell death such as BAX in apoptosis and MLKL in necroptosis. Peroxidation of membrane phospholipids and the subsequent membrane destabilization is a prerequisite to ferroptosis. Here, we discuss how lipids are essential players in apoptosis, the most common form of RCD, and also their role in necroptosis and ferroptosis. Altogether, we aim to highlight the contribution of lipids and membrane dynamics in cell death regulation.
    Keywords:  Apoptosis; Ferroptosis; Membrane dynamics; Mitochondria; Necroptosis; Plasma membrane; Regulated cell death
  21. Immunity. 2020 Apr 03. pii: S1074-7613(20)30116-3. [Epub ahead of print]
      Innate lymphoid cells (ILCs) play an important role in the control and maintenance of barrier immunity. However, chronic activation of ILCs results in immune-mediated pathology. Here, we show that tissue-resident type 2 ILCs (ILC2s) display a distinct metabolic signature upon chronic activation. In the context of allergen-driven airway inflammation, ILC2s increase their uptake of both external lipids and glucose. Externally acquired fatty acids are transiently stored in lipid droplets and converted into phospholipids to promote the proliferation of ILC2s. This metabolic program is imprinted by interleukin-33 (IL-33) and regulated by the genes Pparg and Dgat1, which are both controlled by glucose availability and mTOR signaling. Restricting dietary glucose by feeding mice a ketogenic diet largely ablated ILC2-mediated airway inflammation by impairing fatty acid metabolism and the formation of lipid droplets. Together, these results reveal that pathogenic ILC2 responses require lipid metabolism and identify ketogenic diet as a potent intervention strategy to treat airway inflammation.
    Keywords:  Dgat1; Pparg; airway inflammation; fatty acids; innate lymphoid cells; ketogenic diet; lipid droplets; metabolism
  22. Lipids. 2020 Apr 07.
      Nonalcoholic fatty liver disease (NAFLD) is associated with an imbalance in fatty acid composition and can progress from simple steatosis to steatohepatitis, liver cirrhosis, and hepatocellular carcinoma. Essential phospholipids (EPL), which contain high levels of 1,2-dilinoleoylphosphatidylcholine, can be used to treat NAFLD. Polyenylphosphatidylcholine (PPC) preparations are external, commercially available EPL products. The lipid composition of five commercially available PPC preparations, including Essentiale Forte, Fortifikat, Hepatoprotect Regenerator, Fortifikat Forte, and Esentin Forte were compared, the outcome of which may impact physician choice in the treatment of NAFLD. Following lipid extraction, a comparative analysis of key lipid content was performed using a QTRAP6500+ triple quadruple ion trap hybrid mass spectrometer (Sciex) in nanoelectrospray ionization mode. The glycerophospholipid composition of each PPC was determined, including levels of phosphatidylcholine (PtdCho), and phosphatidylethanolamine (PtdEtn) species, as well as PtdCho:PtdEtn ratio. Of the five preparations analyzed, Essentiale Forte contained the highest PtdCho levels (61.9 mol%) and lowest PtdEtn levels (4.9 mol%). PtdCho 36:4 levels, a polyunsaturated species of PtdCho, were highest in Esentin Forte (39.3 mol%) and Essentiale Forte (38.3 mol%) compared with other PPCs (28.7-35.8 mol%). Levels of lysophosphatidylcholine, phosphatidylinositol, phosphatidic acid, and phosphatidylglycerol were low in all five preparations. Lipid composition was consistent between the preparations. The high PtdCho:PtdEtn ratio composition of Essentiale Forte compared with the other PPC analyzed, as well as the presence of polyunsaturated fatty acids, suggest it could be the most clinically beneficial commercially available hepatoprotective product in the treatment of NAFLD.
    Keywords:  Essential phospholipids; NAFLD; Phosphatidylcholine; Phosphatidylethanolamine; Polyenylphosphatidylcholine
  23. Cell Chem Biol. 2020 Apr 06. pii: S2451-9456(20)30109-4. [Epub ahead of print]
      The trace elements iron and selenium play decisive roles in a distinct form of necrotic cell death, known as ferroptosis. While iron promotes ferroptosis by contributing to Fenton-type reactions and uncontrolled lipid autoxidation, the hallmark of ferroptosis, selenium in the form of glutathione peroxidase 4 (GPX4), subdues phospholipid peroxidation and associated cell death. Beyond the canonical cystine/glutamate antiporter system xc-/glutathione/GPX4 nexus, recent studies unveiled the second mainstay in ferroptosis entailing extra-mitochondrial ubiquinone, ferroptosis suppressor protein 1, and NAD(P)H as electron donor. Unlike GPX4, this selenium- and thiol-independent system acts on the level of peroxyl radicals in membranes, thereby restraining lipid peroxidation. Therefore, ferroptosis is a multifaceted cell-death paradigm characterized by several metabolic networks, whereby metabolic dyshomeostasis may cause ferroptotic cell death and organ failure. Here, we discuss the basic features of ferroptosis with a focus on selenium, offering exciting opportunities to control diseases linked to ferroptosis, including transient ischemia/reperfusion and neurodegeneration.
    Keywords:  PUFA; ferroptosis; ferroptosis suppressor protein 1; glutathione peroxidase 4; selenium
  24. Eur J Mass Spectrom (Chichester). 2020 Apr 10. 1469066720918446
    Keywords:  Metabolite; normalization; quantification; sample; software
  25. Nat Commun. 2020 Apr 09. 11(1): 1755
      Asparagine synthetase (ASNS) catalyses the ATP-dependent conversion of aspartate to asparagine. However, both the regulation and biological functions of asparagine in tumour cells remain largely unknown. Here, we report that p53 suppresses asparagine synthesis through the transcriptional downregulation of ASNS expression and disrupts asparagine-aspartate homeostasis, leading to lymphoma and colon tumour growth inhibition in vivo and in vitro. Moreover, the removal of asparagine from culture medium or the inhibition of ASNS impairs cell proliferation and induces p53/p21-dependent senescence and cell cycle arrest. Mechanistically, asparagine and aspartate regulate AMPK-mediated p53 activation by physically binding to LKB1 and oppositely modulating LKB1 activity. Thus, we found that p53 regulates asparagine metabolism and dictates cell survival by generating an auto-amplification loop via asparagine-aspartate-mediated LKB1-AMPK signalling. Our findings highlight a role for LKB1 in sensing asparagine and aspartate and connect asparagine metabolism to the cellular signalling transduction network that modulates cell survival.
  26. Proteomics. 2020 Apr 08. e1900351
      A lot of energy in the field of proteomics is dedicated to the application of challenging experimental workflows, which include metaproteomics, proteogenomics, data independent acquisition (DIA), non-specific proteolysis, immunopeptidomics, and open modification searches. These workflows are all challenging because of ambiguity in the identification stage; they either expand the search space and thus increase the ambiguity of identifications, or, in the case of DIA, they generate data that is inherently more ambiguous. In this context, machine learning-based predictive models are now generating considerable excitement in the field of proteomics because these predictive models hold great potential to drastically reduce the ambiguity in the identification process of the above-mentioned workflows. Indeed, the field has already produced classical machine learning and deep learning models to predict almost every aspect of a liquid chromatography-mass spectrometry (LC-MS) experiment. Yet despite all the excitement, thorough integration of predictive models in these challenging LC-MS workflows is still limited, and further improvements to the modeling and validation procedures can still be made. In this viewpoint we therefore point out highly promising recent machine learning developments in proteomics, alongside some of the remaining challenges. This article is protected by copyright. All rights reserved.
    Keywords:  data driven modeling; deep learning; machine learning
  27. Rheumatology (Oxford). 2020 Apr 07. pii: keaa126. [Epub ahead of print]
      OBJECTIVE: The spectrum of clinical manifestations and serological phenomena of SLE is heterogeneous among patients and even changes over time unpredictably in individual patients. For this reason, clinical diagnosis especially in complicated or atypical cases is often difficult or delayed leading to poor prognosis. Despite the medical progress nowadays in the understanding of SLE pathogenesis, disease-specific biomarkers for SLE remain an outstanding challenge. Therefore, we undertook this study to investigate potential biomarkers for SLE diagnosis.METHODS: Serum samples from 32 patients with SLE and 25 gender-matched healthy controls (HCs) were analysed by metabolic profiling based on liquid chromatography-tandem mass spectrometry metabolomics platform. The further validation for the potential biomarker was performed in an independent set consisting of 36 SLE patients and 30 HCs.
    RESULTS: The metabolite profiles of serum samples allowed differentiation of SLE patients from HCs. The levels of arachidonic acid, sphingomyelin (SM) 24:1, monoacylglycerol (MG) 17:0, lysophosphatidyl ethanolamine (lysoPE) 18:0, lysoPE 16:0, lysophosphatidyl choline (lysoPC) 20:0, lysoPC 18:0 and adenosine were significantly decreased in SLE patients, and the MG 20:2 and L-pyroglutamic acid were significantly increased in SLE group. In addition, L-pyroglutamic acid achieved an area under the receiver-operating characteristic curve of 0.955 with high sensitivity (97.22%) and specificity (83.33%) at the cut-off of 61.54 μM in the further targeted metabolism, indicating diagnostic potential.
    CONCLUSION: Serum metabolic profiling is differential between SLE patients and HCs and depicts increased L-pyroglutamic acid as a promising bitformatomarker for SLE.
    Keywords:  L-pyroglutamic acid; biomarker; metabolism; systemic lupus erythematosus
  28. J Biol Chem. 2020 Apr 07. pii: jbc.RA120.012798. [Epub ahead of print]
      The incidence of pancreatic cancer increases with age, suggesting that chronological aging is a significant risk factor for this disease. Fibroblasts are the major non-malignant cell type in the stroma of human pancreatic ductal adenocarcinoma (PDAC). In this study, we investigated whether the chronological aging of normal human fibroblasts (NHFs), a previously underappreciated area in pancreatic cancer research, influences the progression and therapeutic outcomes of PDAC. Results from experiments with murine xenografts and 2D and 3D co-cultures of NHFs and PDAC cells revealed that older NHFs stimulate proliferation of and confer resistance to radiation therapy of PDAC. MS-based metabolite analysis indicated that older NHFs have significantly increased arachidonic acid 12-lipoxygenase (ALOX12) expression and elevated levels of its mitogenic metabolite, 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-(S)-HETE) compared with their younger counterparts. In co-cultures with older rather than with younger NHFs, PDAC cells exhibited increases in mitogen-activated protein kinase (MAPK) signaling and cellular metabolism, as well as a lower oxidation-state that correlated with their enhanced proliferation and resistance to radiation therapy. Expression of ALOX12 was found to be significantly lower in PDAC cell lines and tumor biopsies, suggesting that PDAC cells rely on a stromal supply of mitogens for their proliferative needs. Pharmacological (hydroxytyrosol) and molecular (siRNA) interventions of ALOX12 in older NHFs suppressed their ability to stimulate proliferation of PDAC cells. We conclude that chronological aging of NHFs contributes to PDAC progression and that ALOX12 and 12-(S)-HETE may be potential stromal-targets for interventions that seek to halt progression and improve therapy outcomes.
    Keywords:  aging; arachidonic acid (AA) (ARA); cancer biology; cell proliferation; fibroblast; pancreatic cancer; stromal cell
  29. Proteomics. 2020 Apr 10. e1900276
      This review provides a brief overview of the development of data-independent acquisition (DIA) mass spectrometry-based proteomics and selected DIA data analysis tools. We first summarize various DIA acquisition schemes for proteomics including Shotgun-CID, DIA, MSE , PAcIFIC, AIF, SWATH, MSX, SONAR, WiSIM, BoxCar, Scanning SWATH, diaPASEF and PulseDIA, as well as the mass spectrometers enabling these methods. Next, we categorize the software tools for DIA data analysis into three groups: library-based tools, library-free tools and statistical validation tools. We review the approaches for generating spectral libraries for six selected library-based DIA data analysis software tools which have been tested by the authors, including OpenSWATH, Spectronaut, Skyline, PeakView, DIA-NN and EncyclopeDIA. An increasing number of library-free DIA data analysis tools have been developed including DIA-Umpire, Group-DIA, PECAN, PEAKS, which facilitate identification of novel proteoforms. We share our user experience of when to use DIA-MS, and several selected DIA data analysis software tools. Finally, we summarize the state of the art of DIA mass spectrometry and software tools, and our views of future directions. This article is protected by copyright. All rights reserved.
    Keywords:  Data-independent acquisition; Database search; Software tools; Spectral library; Targeted proteomics
  30. J Oncol. 2020 ;2020 6249829
      Background: Osimertinib is the first-line therapeutic option for the T790M-mutant non-small-cell lung cancer and the acquired resistance obstructs its application. It is an urgent challenge to identify the potential mechanisms of osimertinib resistance for uncovering some novel therapeutic approaches.Methods: In the current study, the cell metabolomics based on ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry and the qualitative and tandem mass tags quantitative proteomics were performed.
    Results: 54 differential metabolites and 195 differentially expressed proteins were, respectively, identified. The amino acids metabolisms were significantly altered. HIF-1 signaling pathway modulating P-glycoproteins expression, PI3K-Akt pathway regulating survivin expression, and oxidative phosphorylation were upregulated, while arginine and proline metabolism regulating NO production and glycolysis/gluconeogenesis were downregulated during osimertinib resistance.
    Conclusion: The regulation of HIF-1 and PI3K-Akt signaling pathway, energy supply process, and amino acids metabolism are the promising therapeutic tactics for osimertinib resistance.