bims-mascan 2020-08-23 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2020–08–23
23 papers selected by
Giovanny Rodriguez Blanco, University of Edinburgh

Integration of GC-MS and LC-MS for untargeted metabolomics profiling.
In-depth mapping carboxylic acid metabolome reveals the potential biomarkers in colorectal cancer through characteristic fragment ions and metabolic flux.
Cellular Fatty Acid Analysis in Macrophage Using Stable Isotope Labeling.
Pieces of the complex puzzle of cancer cell energy metabolism: an overview of energy metabolism and alternatives for targeted cancer therapy.
Automated parallel derivatization of metabolites with SWATH-MS data acquisition for qualitative and quantitative analysis.
Lipidomic-Based Advances in Diagnosis and Modulation of Immune Response to Cancer.
An Optimized Dual Extraction Method for the Simultaneous and Accurate Analysis of Polar Metabolites and Lipids Carried out on Single Biological Samples.
Mitochondrial fatty acid synthesis coordinates oxidative metabolism in mammalian mitochondria.
Age-induced accumulation of methylmalonic acid promotes tumour progression.
Liquid Chromatography-Mass Spectrometry Analysis of Cytosine Modifications.
IL4I1 Is a Metabolic Immune Checkpoint that Activates the AHR and Promotes Tumor Progression.
Oncology Therapeutics Targeting the Metabolism of Amino Acids.
Detection of DNA Methylation in Genomic DNA by UHPLC-MS/MS.
Lipid Metabolism and Cancer Immunotherapy: Immunosuppressive Myeloid Cells at the Crossroad.
Mass Spectrometry-Based Analysis of DNA Modifications: Potential Applications in Basic Research and Clinic.
Lipidome-wide characterization of phosphatidylinositols and phosphatidylglycerols on CC location level.
Heterogeneous adaptation of cysteine reactivity to a covalent oncometabolite.
Quantifications of Lipid Kinetics In Vivo Using Stable Isotope Tracer Methodology.
KRAS controls pancreatic cancer cell lipid metabolism and invasive potential through the lipase HSL.
A comparative study of UniSpray and electrospray sources for the ionization of neuropeptides in liquid chromatography tandem mass spectrometry.
[Blood lipid metabolic profile of overweight/obese boys aged 9-12 years].
The Folate Cycle Enzyme MTHFR is a Critical Regulator of Cell Response to MYC-Targeting Therapies.
Ferroptosis: biological rust of lipid membranes.

J Pharm Biomed Anal. 2020 Aug 02. pii: S0731-7085(20)31395-9. [Epub ahead of print]190 113509

Integration of GC-MS and LC-MS for untargeted metabolomics profiling.

Özge Cansın Zeki, Cemil Can Eylem, Tuba Reçber, Sedef Kır, Emirhan Nemutlu.

  Recently, metabolomics analyses have become increasingly common in the general scientific community as it is applied in several researches relating to diseases diagnosis. Identification and quantification of small molecules belonging to metabolism in biological systems have an important role in diagnosis of diseases. The combination of chromatography with mass spectrometry is used for the accurate and reproducible analysis of hundreds to thousands of metabolites in biological fluids or tissue samples. The number of metabolites that can be identified in biological fluids or tissue varies according to the gas (GC) or liquid (LC) chromatographic techniques used. The cover of these chromatographic techniques also differs from each other based on the metabolite group (polar, lipids, organic acid etc.). Consequently, some of the metabolites can only be analyzed using either GC or LC. However, more than one metabolite or metabolite group may be found altered in a particular disease. Thus, in order to find these alterations, metabolomics analyses that cover a wide range of metabolite groups are usually applied. In this regard, GC-MS and LC-MS techniques are mostly used together to identify completely all the altered metabolites during disease diagnosis. Using these combined techniques also allows identification of metabolite(s) with significantly altered phenotype. This review sheds light on metabolomics studies involving the simultaneous use of GC-MS and LC-MS. The review also discusses the coverage, sample preparation, data acquisition and data preprocessing for untargeted metabolomics studies. Moreover, the advantages and disadvantages of these methods were also evaluated. Finally, precautions and suggestions on how to perform metabolomics studies in an accurate, precise, complete and unbiased way were also outlined.

Keywords:  Data analysis; GC–MS; LC–MS; Metabolomics; Sample preparation

DOI:  https://doi.org/10.1016/j.jpba.2020.113509
Anal Chim Acta. 2020 Sep 01. pii: S0003-2670(20)30713-3. [Epub ahead of print]1128 62-71

In-depth mapping carboxylic acid metabolome reveals the potential biomarkers in colorectal cancer through characteristic fragment ions and metabolic flux.

Xiqing Bian, Yun Qian, Binbin Tan, Kai Li, Xufen Hong, Chi Chun Wong, Li Fu, Jun Zhang, Na Li, Jian-Lin Wu.

  Carboxylic acid metabolome plays vital roles in the study of pathological mechanisms about cancer. This study aimed to find potential biomarkers for colorectal cancer (CRC) using carboxylic acids profiling. However, the identification of much more carboxylic acids was limited due to poor ionization efficiency and lack of characteristic fragment ions. Derivatization-liquid chromatography-mass spectrometry, which contains characteristic MS/MS fragments ions, were performed for carboxylic acid metabolomics analysis in CRC serum samples. 1054 carboxylic acids were quickly and selectively identified after extraction using three characteristic fragment ions and elucidation using the most suitable CE at 30 eV. Among them, 605 carboxylic acids exhibit discriminating levels between healthy and CRC patients in training cohort. Furthermore, the differential metabolites were found to be mainly enriched in amino acid metabolism, fatty acid biosynthesis and TCA cycle by MetaboAnalyst and iPath analysis. Finally, serine, glycine, and methionine were determined as the potential biomarkers after further confirmation using validation cohort and in vitro metabolic flux analysis. The above results collectively demonstrated that a new set of carboxylic acids can be quickly and selectively discovered using characteristic fragment ions.

Keywords:  Carboxylic acid metabolome; Characteristic fragment ions; Colorectal cancer; Metabolic flux; Potential biomarkers

DOI:  https://doi.org/10.1016/j.aca.2020.06.064
Methods Mol Biol. 2020 ;2184 47-60

Cellular Fatty Acid Analysis in Macrophage Using Stable Isotope Labeling.

Kevin J Williams, Steven J Bensinger.

  Fatty acids (FAs) are essential for building complex lipids, posttranslational modifications, and energetics. FAs can be imported from extracellular sources or synthesized by cells. The analysis of fatty acid methyl esters (FAMEs) by gas chromatography-mass spectrometry (GC-MS) allows for the quantitative analysis of long-chain and very-long-chain fatty acid content of cells. When coupled with isotopic labeling, this approach can elucidate the synthetic pathways being engaged by the cells, and the relative contribution of synthesis and import to maintain lipid content. Here, we describe a method for total cellular fatty acid analysis in macrophages.

Keywords:  FAME analysis; Fatty acids; GC–MS; Macrophages; Stable isotope labeling

DOI:  https://doi.org/10.1007/978-1-0716-0802-9_4
Curr Med Chem. 2020 Aug 19.

Pieces of the complex puzzle of cancer cell energy metabolism: an overview of energy metabolism and alternatives for targeted cancer therapy.

Zeinab Ghasemishahrestani, Larissa Maura Melo Mattos, Tatiana Martins Tilli, André Souza Dos Santos, Marcos Dias Pereira.

  Over the past decades, several advances in cancer cell biology have led to relevant details about a phenomenon called "Warburg effect". Currently, it has been accepted that Warburg effect is not anymore compatible with all cancer cells, and thus the process of aerobic glycolysis is now challenged by the knowledge of a large number of cells presenting mitochondrial function. The energy metabolism of cancer cells is focused in the bioenergetic and biosynthetic pathways to meet the requirements of rapid proliferation. Changes in the metabolism of carbohydrate, amino acids and lipids have already been reported in cancer cells and might play relevant roles for cancer progression. To the best of our knowledge, mostly of these changes are established, mainly due to genetic reprogramming that leads to the transformation of a healthy into a cancerous cell. Indeed, several enzymes of high relevance for the energy are targets of oncogenes (ex. PI3K, HIF1 and Myc) and tumor suppressor proteins (ex. p53). As a consequence of the extensive study on cancer cell metabolism, some new therapeutic strategies have appeared that aim to interrupt the aberrant metabolism, as well as the influence of genetic reprogramming in cancer cells. In this perspective, we briefly review the cancer cell metabolism (carbohydrate, amino acid and lipid), and also describe oncogenes and tumor suppressors that affect cancer cell metabolism. We also discuss some potential candidates for target therapy to disrupt the main driven-force for cancer cell metabolism and proliferation.

Keywords:  Carbohydrate metabolism; amino acid metabolism; oncogenes and tumor suppressors; lipid metabolism; target therapy.

DOI:  https://doi.org/10.2174/0929867327999200819123357
Anal Chim Acta. 2020 Aug 29. pii: S0003-2670(20)30678-4. [Epub ahead of print]1127 198-206

Automated parallel derivatization of metabolites with SWATH-MS data acquisition for qualitative and quantitative analysis.

Maria Fernanda Cifuentes Girard, David Ruskic, Günter Böhm, Renzo Picenoni, Gérard Hopfgartner.

  For metabolite profiling chemical derivatization has been used to improve MS sensitivity and LC retention. However, for multi-analytes quantification, the number of commercially available isotopically labelled internal standards is limited. Besides, there is no single workflow which can provide large-scale metabolomics coverage in particular for polar metabolites. To overcome these limitations and to improve reproducibility a fully automated dual derivatization approach was developed. Differential Isotope Labeling (DIL) was adopted by derivatizing carbonyl, amino and phenol metabolites with two isotopic forms. Urine samples were derivatized with 12C-dansyl chloride (DnsCl) and 12C-dansylhydrazine (DnsHz). Suitable quantification standards were generated by derivatized 40 standards including amino acids, sex hormones and other highly polar metabolites with labelled 13C2-dansyl chloride and 13C2-dansylhydrazine. The derivatization of the standards and the urine sample was performed using a PAL RTC autosampler in-line to column-switching LC-HRMS analysis with data independent acquisition (SWATH-MS). The parallel reactions were completed in 15 min inside of two agitators at different conditions overlapping with the LC-MS analysis time which was of 25 min. The column switching setup is critical to remove the excess of reagents which can negatively affect the ionization efficiency and deteriorate the chromatographic performance. The combination of dual DIL with SWATH-MS acquisition enables post-identification of unknown metabolites and quantitation at precursor (MS1) and specific tag fragment (MS2) levels. The inter- and intra-batch accuracy and precision of the method fall in the range ±15% using single point calibration, and at MS1 or MS2 level providing full flexibility. The method was successfully applied to the analysis of human urine samples.

Keywords:  Automation; Derivatization; Differential isotope labeling; LC-MS; Metabolomics; SWATH-MS

DOI:  https://doi.org/10.1016/j.aca.2020.06.030
Metabolites. 2020 Aug 14. pii: E332. [Epub ahead of print]10(8):

Lipidomic-Based Advances in Diagnosis and Modulation of Immune Response to Cancer.

Luis Gil-de-Gómez, David Balgoma, Olimpio Montero.

  While immunotherapies for diverse types of cancer are effective in many cases, relapse is still a lingering problem. Like tumor cells, activated immune cells have an anabolic metabolic profile, relying on glycolysis and the increased uptake and synthesis of fatty acids. In contrast, immature antigen-presenting cells, as well as anergic and exhausted T-cells have a catabolic metabolic profile that uses oxidative phosphorylation to provide energy for cellular processes. One goal for enhancing current immunotherapies is to identify metabolic pathways supporting the immune response to tumor antigens. A robust cell expansion and an active modulation via immune checkpoints and cytokine release are required for effective immunity. Lipids, as one of the main components of the cell membrane, are the key regulators of cell signaling and proliferation. Therefore, lipid metabolism reprogramming may impact proliferation and generate dysfunctional immune cells promoting tumor growth. Based on lipid-driven signatures, the discrimination between responsiveness and tolerance to tumor cells will support the development of accurate biomarkers and the identification of potential therapeutic targets. These findings may improve existing immunotherapies and ultimately prevent immune escape in patients for whom existing treatments have failed.

Keywords:  biomarkers; cancer; immunotherapy; lipids; metabolism

DOI:  https://doi.org/10.3390/metabo10080332
Metabolites. 2020 Aug 19. pii: E338. [Epub ahead of print]10(9):

An Optimized Dual Extraction Method for the Simultaneous and Accurate Analysis of Polar Metabolites and Lipids Carried out on Single Biological Samples.

Joran Villaret-Cazadamont, Nathalie Poupin, Anthony Tournadre, Aurélie Batut, Lara Gales, Daniel Zalko, Nicolas J Cabaton, Floriant Bellvert, Justine Bertrand-Michel.

  The functional understanding of metabolic changes requires both a significant investigation into metabolic pathways, as enabled by global metabolomics and lipidomics approaches, and the comprehensive and accurate exploration of specific key pathways. To answer this pivotal challenge, we propose an optimized approach, which combines an efficient sample preparation, aiming to reduce the variability, with a biphasic extraction method, where both the aqueous and organic phases of the same sample are used for mass spectrometry analyses. We demonstrated that this double extraction protocol allows working with one single sample without decreasing the metabolome and lipidome coverage. It enables the targeted analysis of 40 polar metabolites and 82 lipids, together with the absolute quantification of 32 polar metabolites, providing comprehensive coverage and quantitative measurement of the metabolites involved in central carbon energy pathways. With this method, we evidenced modulations of several lipids, amino acids, and energy metabolites in HepaRG cells exposed to fenofibrate, a model hepatic toxicant, and metabolic modulator. This new protocol is particularly relevant for experiments involving limited amounts of biological material and for functional metabolic explorations and is thus of particular interest for studies aiming to decipher the effects and modes of action of metabolic disrupting compounds.

Keywords:  dual extraction; hepatotoxicity; lipidomics; metabolomics; multi-omics analysis; sample preparation

DOI:  https://doi.org/10.3390/metabo10090338
Elife. 2020 Aug 17. pii: e58041. [Epub ahead of print]9

Mitochondrial fatty acid synthesis coordinates oxidative metabolism in mammalian mitochondria.

Sara M Nowinski, Ashley Solmonson, Scott F Rusin, J Alan Maschek, Claire L Bensard, Sarah Fogarty, Mi-Young Jeong, Sandra Lettlova, Jordan A Berg, Jeffrey T Morgan, Yeyun Ouyang, Bradley C Naylor, Joao A Paulo, Katsuhiko Funai, James E Cox, Steven P Gygi, Dennis R Winge, Ralph J DeBerardinis, Jared Rutter.

  Cells harbor two systems for fatty acid synthesis, one in the cytoplasm (catalyzed by fatty acid synthase, FASN) and one in the mitochondria (mtFAS). In contrast to FASN, mtFAS is poorly characterized, especially in higher eukaryotes, with the major product(s), metabolic roles, and cellular function(s) being essentially unknown. Here we show that hypomorphic mtFAS mutant mouse skeletal myoblast cell lines display a severe loss of electron transport chain (ETC) complexes and exhibit compensatory metabolic activities including reductive carboxylation. This effect on ETC complexes appears to be independent of protein lipoylation, the best characterized function of mtFAS, as mutants lacking lipoylation have an intact ETC. Finally, mtFAS impairment blocks the differentiation of skeletal myoblasts in vitro. Together, these data suggest that ETC activity in mammals is profoundly controlled by mtFAS function, thereby connecting anabolic fatty acid synthesis with the oxidation of carbon fuels.

Keywords:  biochemistry; chemical biology; mouse

DOI:  https://doi.org/10.7554/eLife.58041
Nature. 2020 Aug 19.

Age-induced accumulation of methylmalonic acid promotes tumour progression.

Ana P Gomes, Didem Ilter, Vivien Low, Jennifer E Endress, Juan Fernández-García, Adam Rosenzweig, Tanya Schild, Dorien Broekaert, Adnan Ahmed, Melanie Planque, Ilaria Elia, Julie Han, Charles Kinzig, Edouard Mullarky, Anders P Mutvei, John Asara, Rafael de Cabo, Lewis C Cantley, Noah Dephoure, Sarah-Maria Fendt, John Blenis.

The risk of cancer and associated mortality increases substantially in humans from the age of 65 years onwards1-6. Nonetheless, our understanding of the complex relationship between age and cancer is still in its infancy2,3,7,8. For decades, this link has largely been attributed to increased exposure time to mutagens in older individuals. However, this view does not account for the established role of diet, exercise and small molecules that target the pace of metabolic ageing9-12. Here we show that metabolic alterations that occur with age can produce a systemic environment that favours the progression and aggressiveness of tumours. Specifically, we show that methylmalonic acid (MMA), a by-product of propionate metabolism, is upregulated in the serum of older people and functions as a mediator of tumour progression. We traced this to the ability of MMA to induce SOX4 expression and consequently to elicit transcriptional reprogramming that can endow cancer cells with aggressive properties. Thus, the accumulation of MMA represents a link between ageing and cancer progression, suggesting that MMA is a promising therapeutic target for advanced carcinomas.

DOI: https://doi.org/10.1038/s41586-020-2630-0
Methods Mol Biol. 2021 ;2198 67-78

Liquid Chromatography-Mass Spectrometry Analysis of Cytosine Modifications.

Nan Dai, Ivan R Corrêa.

  Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely used technique in the global analysis of epigenetic DNA modifications. The high-resolution chromatographic separation along with sensitive MS detection permits the identification and quantification of deoxyribonucleosides with precision and reliability. Although there have been tremendous advances in LC and MS instrumentation in recent years, sample preparation has not experienced a similar rate of development and is often a bottleneck to chemical analysis. Here we present a protocol for identification and quantification of cytosine modifications that combines a robust and efficient method to generate single nucleosides from genomic DNA samples followed by direct LC-MS/MS analysis.

Keywords:  Epigenetics; Methylome profiling; Multistage mass spectrometry; Nucleoside digestion; Nucleotide modifications

DOI:  https://doi.org/10.1007/978-1-0716-0876-0_6
Cell. 2020 Aug 17. pii: S0092-8674(20)30946-6. [Epub ahead of print]

IL4I1 Is a Metabolic Immune Checkpoint that Activates the AHR and Promotes Tumor Progression.

Ahmed Sadik, Luis F Somarribas Patterson, Selcen Öztürk, Soumya R Mohapatra, Verena Panitz, Philipp F Secker, Pauline Pfänder, Stefanie Loth, Heba Salem, Mirja Tamara Prentzell, Bianca Berdel, Murat Iskar, Erik Faessler, Friederike Reuter, Isabelle Kirst, Verena Kalter, Kathrin I Foerster, Evelyn Jäger, Carina Ramallo Guevara, Mansour Sobeh, Thomas Hielscher, Gernot Poschet, Annekathrin Reinhardt, Jessica C Hassel, Marc Zapatka, Udo Hahn, Andreas von Deimling, Carsten Hopf, Rita Schlichting, Beate I Escher, Jürgen Burhenne, Walter E Haefeli, Naveed Ishaque, Alexander Böhme, Sascha Schäuble, Kathrin Thedieck, Saskia Trump, Martina Seiffert, Christiane A Opitz.

  Aryl hydrocarbon receptor (AHR) activation by tryptophan (Trp) catabolites enhances tumor malignancy and suppresses anti-tumor immunity. The context specificity of AHR target genes has so far impeded systematic investigation of AHR activity and its upstream enzymes across human cancers. A pan-tissue AHR signature, derived by natural language processing, revealed that across 32 tumor entities, interleukin-4-induced-1 (IL4I1) associates more frequently with AHR activity than IDO1 or TDO2, hitherto recognized as the main Trp-catabolic enzymes. IL4I1 activates the AHR through the generation of indole metabolites and kynurenic acid. It associates with reduced survival in glioma patients, promotes cancer cell motility, and suppresses adaptive immunity, thereby enhancing the progression of chronic lymphocytic leukemia (CLL) in mice. Immune checkpoint blockade (ICB) induces IDO1 and IL4I1. As IDO1 inhibitors do not block IL4I1, IL4I1 may explain the failure of clinical studies combining ICB with IDO1 inhibition. Taken together, IL4I1 blockade opens new avenues for cancer therapy.

Keywords:  AHR; CLL; IL4I1; T cell exhaustion; adaptive immunity; aryl hydrocarbon receptor; interleukin 4 induced 1; kynurenic acid; tryptophan metabolism; tumor micro-environment

DOI:  https://doi.org/10.1016/j.cell.2020.07.038
Cells. 2020 Aug 15. pii: E1904. [Epub ahead of print]9(8):

Oncology Therapeutics Targeting the Metabolism of Amino Acids.

Nefertiti Muhammad, Hyun Min Lee, Jiyeon Kim.

  Amino acid metabolism promotes cancer cell proliferation and survival by supporting building block synthesis, producing reducing agents to mitigate oxidative stress, and generating immunosuppressive metabolites for immune evasion. Malignant cells rewire amino acid metabolism to maximize their access to nutrients. Amino acid transporter expression is upregulated to acquire amino acids from the extracellular environment. Under nutrient depleted conditions, macropinocytosis can be activated where proteins from the extracellular environment are engulfed and degraded into the constituent amino acids. The demand for non-essential amino acids (NEAAs) can be met through de novo synthesis pathways. Cancer cells can alter various signaling pathways to boost amino acid usage for the generation of nucleotides, reactive oxygen species (ROS) scavenging molecules, and oncometabolites. The importance of amino acid metabolism in cancer proliferation makes it a potential target for therapeutic intervention, including via small molecules and antibodies. In this review, we will delineate the targets related to amino acid metabolism and promising therapeutic approaches.

Keywords:  amino acids; cancer metabolism; oncogenic therapeutics

DOI:  https://doi.org/10.3390/cells9081904
Methods Mol Biol. 2021 ;2198 79-90

Detection of DNA Methylation in Genomic DNA by UHPLC-MS/MS.

Konstantinos Boulias, Eric Lieberman Greer.

  DNA methylation serves to mark DNA as either a directed epigenetic signaling modification or in response to DNA lesions. Methods for detecting DNA methylation have become increasingly more specific and sensitive over time. Conventional methods for detecting DNA methylation, ranging from paper chromatography to differential restriction enzyme digestion preference to dot blots, have more recently been supplemented by ultrahigh performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) to accurately quantify specific DNA methylation. Methylated DNA can also be sequenced by either methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) or single-molecule real-time sequencing (SMRTseq) for identifying genomic locations of DNA methylation. Here we describe a protocol for the detection and quantification of epigenetic signaling DNA methylation modifications including, N6-methyladenine (6mA), N4-methylcytosine (4mC) and C5-methylcytosine (5mC) in genomic DNA by triple quadrupole liquid chromatography coupled with tandem mass spectrometry (QQQ-LC-MS/MS). The high sensitivity of the UHPLC-MS/MS methodology and the use of calibration standards of pure nucleosides allow for the accurate quantification of DNA methylation.

Keywords:  4mC; 5mC; 6mA; C5-methylcytosine; N4-methylcytosine; N6-methyladenine; UHPLC-MS/MS; methylated DNA

DOI:  https://doi.org/10.1007/978-1-0716-0876-0_7
Int J Mol Sci. 2020 Aug 14. pii: E5845. [Epub ahead of print]21(16):

Lipid Metabolism and Cancer Immunotherapy: Immunosuppressive Myeloid Cells at the Crossroad.

Augusto Bleve, Barbara Durante, Antonio Sica, Francesca Maria Consonni.

  Cancer progression generates a chronic inflammatory state that dramatically influences hematopoiesis, originating different subsets of immune cells that can exert pro- or anti-tumor roles. Commitment towards one of these opposing phenotypes is driven by inflammatory and metabolic stimuli derived from the tumor-microenvironment (TME). Current immunotherapy protocols are based on the reprogramming of both specific and innate immune responses, in order to boost the intrinsic anti-tumoral activity of both compartments. Growing pre-clinical and clinical evidence highlights the key role of metabolism as a major influence on both immune and clinical responses of cancer patients. Indeed, nutrient competition (i.e., amino acids, glucose, fatty acids) between proliferating cancer cells and immune cells, together with inflammatory mediators, drastically affect the functionality of innate and adaptive immune cells, as well as their functional cross-talk. This review discusses new advances on the complex interplay between cancer-related inflammation, myeloid cell differentiation and lipid metabolism, highlighting the therapeutic potential of metabolic interventions as modulators of anticancer immune responses and catalysts of anticancer immunotherapy.

Keywords:  cancer immunotherapy; cholesterol; fatty acids; lipid metabolism; myeloid-derived suppressor cells (MDSCs); obesity; tumor-associated macrophages (TAMs)

DOI:  https://doi.org/10.3390/ijms21165845
Methods Mol Biol. 2021 ;2198 27-35

Mass Spectrometry-Based Analysis of DNA Modifications: Potential Applications in Basic Research and Clinic.

Ryszard Olinski, Rafal Rozalski, Daniel Gackowski.

  Stable-isotope-dilution tandem mass spectrometry is the most advanced technique used for quantitative determination of a wide spectrum of endogenously generated DNA nucleobase modifications. It is regarded as a gold standard for such analyses. Here, we consider the requirements for reliable identification and quantification of DNA adducts/modifications, whether endogenously derived or not, and discuss how their quantification can provide information on the mechanism of action and the biological relevance of individual nucleobase modifications. A clinical application of such measurements will only be possible after a full validation of the assay and once we have gained a better understanding of the exact role that these DNA modifications play in disease pathogenesis. Once these prerequisites are satisfied, DNA modification measurements may be helpful as clinical parameters for treatment monitoring, for risk group identification and for the development of prevention strategies.

Keywords:  5-carboxycytosine; 5-formylcytosine; 5-hydroxymethylcytosine; 5-hydroxymethyluracil; 5-methylcytosine; 8-oxo-7,8-dihydroguanine; DNA base modifications; Isotope dilution; Tandem mass spectrometry; Uracil; Urinary excretion

DOI:  https://doi.org/10.1007/978-1-0716-0876-0_3
Anal Chim Acta. 2020 Sep 01. pii: S0003-2670(20)30665-6. [Epub ahead of print]1128 107-115

Lipidome-wide characterization of phosphatidylinositols and phosphatidylglycerols on CC location level.

Tian Xia, Hanlin Ren, Wenpeng Zhang, Yu Xia.

  Phosphatidylglycerol (PG) and phosphatidylinositol (PI) are two essential classes of glycerophospholipids (GPs), playing versatile roles such as signalling messengers and lipid-protein interaction ligands in cell. Although a majority of PG and PI molecular species contain unsaturated fatty acyl chain(s), conventional tandem mass spectrometry (MS/MS) methods cannot discern isomers different in carbon-carbon double bond (CC) locations. In this work, we paired phosphate methylation with acetone Paternò-Büchi (PB) reaction, aiming to provide a solution for sensitive and structurally informative analysis of these two important classes of GPs down to the location of CC. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow was established. Offline methylated PG or PI mixtures were subjected to hydrophilic interaction chromatographic separation, online acetone PB reaction, and MS/MS via collision-induced dissociation (CID) for CC location determination in positive ion mode. This method was sensitive, offering limit of identification at 5 nM for both PG and PI standards down to CC locations. On molecular species level, 49 PI and 31 PG were identified from bovine liver, while 61 PIs were identified from human plasma. This workflow also enabled ratiometric comparisons of CC location isomers (C18:1 Δ9 vs. Δ11) of a series of PIs from type 2 diabetes (T2D) plasma to that of normal plasma samples. PI 16:0_18:1 and PI 18:0_18:1 were found to exhibit significant changes in CC isomeric ratios between T2D and normal plasma samples. The above results demonstrate that the developed LC-PB-MS/MS workflow is applicable to different classes of lipids and compatible with other established lipid derivatization methods to achieve comprehensive lipid analysis.

Keywords:  Lipid isomers identification; Methylation derivatization; Paternò–Büchi reaction; Phosphatidylglycerol; Phosphatidylinositol

DOI:  https://doi.org/10.1016/j.aca.2020.06.017
J Biol Chem. 2020 Aug 19. pii: jbc.AC120.014993. [Epub ahead of print]

Heterogeneous adaptation of cysteine reactivity to a covalent oncometabolite.

Minervo Perez, Daniel W Bak, Sarah E Bergholtz, Daniel R Crooks, Bhargav Srinivas Arimilli, Youfeng Yang, Eranthie Weerapana, W Marston Linehan, Jordan L Meier.

  An important context in which metabolism influences tumorigenesis is the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a disease in which mutation of the TCA cycle enzyme fumarate hydratase (FH) causes hyperaccumulation of fumarate. This electrophilic oncometabolite can alter gene activity at the level of transcription, via reversible inhibition of epigenetic dioxygenases, as well as posttranslationally, via covalent modification of cysteine residues. To better understand the potential for metabolites to influence posttranslational modifications important to tumorigenesis and cancer cell growth, here we report a chemoproteomic analysis of a kidney-derived HLRCC cell line. Using a general reactivity probe, we generated a dataset of proteomic cysteine residues sensitive to the reduction in fumarate levels caused by genetic re-introduction of active FH into HLRCC cell lines. This revealed a broad upregulation of cysteine reactivity upon FH rescue, which evidence suggests is caused by an approximately equal proportion of transcriptional and posttranslational modification-mediated regulation. Gene ontology analysis highlighted several new targets and pathways potentially modulated by FH mutation. Comparison of the new dataset to prior studies highlights considerable heterogeneity in the adaptive response of cysteine-containing proteins in different models of HLRCC. This is consistent with emerging studies indicating the existence of cell and tissue-specific cysteine-omes, further emphasizing the need for characterization of diverse models. Our analysis provides a resource for understanding the proteomic adaptation to fumarate accumulation, and a foundation for future efforts to exploit this knowledge for cancer therapy.

Keywords:  chemical biology; inborn error of metabolism; metabolism; mitochondria; post-translational modification (PTM); proteomics; redox signaling; tricarboxylic acid cycle (TCA cycle) (Krebs cycle)

DOI:  https://doi.org/10.1074/jbc.AC120.014993
J Lipid Atheroscler. 2020 Jan;9(1): 110-123

Quantifications of Lipid Kinetics In Vivo Using Stable Isotope Tracer Methodology.

Il-Young Kim, Sanghee Park, Jiwoong Jang, Robert R Wolfe.

  Like other bodily materials, lipids such as plasma triacylglycerol, cholesterols, and free fatty acids are in a dynamic state of constant turnover (i.e., synthesis, breakdown, oxidation, and/or conversion to other compounds) as essential processes for achieving dynamic homeostasis in the body. However, dysregulation of lipid turnover can lead to clinical conditions such as obesity, fatty liver disease, and dyslipidemia. Assessment of "snap-shot" information on lipid metabolism (e.g., tissue contents of lipids, abundance of mRNA and protein and/or signaling molecules) are often used in clinical and research settings, and can help to understand one's health and disease status. However, such "snapshots" do not provide critical information on dynamic nature of lipid metabolism, and therefore may miss "true" origin of the dysregulation implicated in related diseases. In this regard, stable isotope tracer methodology can provide the in vivo kinetic information of lipid metabolism. Combining with "static" information, knowledge of lipid kinetics can enable the acquisition of in depth understanding of lipid metabolism in relation to various health and disease status. This in turn facilitates the development of effective therapeutic approaches (e.g., exercise, nutrition, and/or drugs). In this review we will discuss 1) the importance of obtaining kinetic information for a better understanding of lipid metabolism, 2) basic principles of stable isotope tracer methodologies that enable exploration of "lipid kinetics" in vivo, and 3) quantification of some aspects of lipid kinetics in vivo with numerical examples.

Keywords:  Lipid metabolism; Mass spectrometry; Substrate turnover, Dyslipidemia

DOI:  https://doi.org/10.12997/jla.2020.9.1.110
Cancer Res. 2020 Aug 19. pii: canres.1255.2020. [Epub ahead of print]

KRAS controls pancreatic cancer cell lipid metabolism and invasive potential through the lipase HSL.

Cody N Rozeveld, Katherine M Johnson, Lizhi Zhang, Gina L Razidlo.

Oncogene-induced metabolic reprogramming is a hallmark of pancreatic cancer (PDAC), yet the metabolic drivers of metastasis are unclear. In PDAC, obesity and excess fatty acids accelerate tumor growth and increase metastasis. Here, we report that excess lipids, stored in organelles called lipid droplets (LD), are a key resource to fuel the energy-intensive process of metastasis. The oncogene KRAS controlled the storage and utilization of LD through regulation of hormone sensitive lipase (HSL), which was downregulated in human PDAC. Disruption of the KRAS-HSL axis reduced lipid storage, reprogrammed tumor cell metabolism, and inhibited invasive migration in vitro and metastasis in vivo. Finally, microscopy-based metabolic analysis revealed that migratory cells selectively utilize oxidative metabolism during the process of migration to metabolize stored lipids and fuel invasive migration. Taken together, these results reveal a mechanism that can be targeted to attenuate PDAC metastasis.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-1255
J Chromatogr A. 2020 Aug 11. pii: S0021-9673(20)30738-X. [Epub ahead of print]1628 461462

A comparative study of UniSpray and electrospray sources for the ionization of neuropeptides in liquid chromatography tandem mass spectrometry.

Jana Bongaerts, Karen Segers, Laurence Van Oudenhove, Yannick Van Wanseele, Marijn Van Hulle, Dimitri De Bundel, Debby Mangelings, Ilse Smolders, Yvan Vander Heyden, Ann Van Eeckhaut.

  Despite the extensive use of electrospray ionization (ESI) for the quantification of neuropeptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS), poor ionization and transmission efficiency are described for this ionization interface. A new atmospheric pressure ionization source, named UniSpray, was recently developed and commercialized. In this study, the LC-MS performance of this new ionization interface is evaluated and compared with ESI for the quantification of seven neuropeptides. Besides comparison of signal intensities and charge state distributions, also signal-to-noise (S/N) ratios and accuracy and precision were assessed. Additionally, matrix effects of human precipitated plasma and rat microdialysate were evaluated as well as the effect of three supercharging agents on the ionization of the seven neuropeptides. UniSpray ionization resulted in signal intensities four to eight times higher at the optimal capillary/impactor voltage for all seven neuropeptides. S/N values at the other hand only increased by not more than a twofold when the UniSpray source was used. Moreover, UniSpray ionization resulted in a shift towards lower charge states for some neuropeptides. Evaluation of the matrix effects by a post-column infusion set-up resulted in different infusion profiles between ESI and UniSpray. The charge state distributions of the neuropeptides obtained with UniSpray are highly comparable with ESI. Finally, the effect of the supercharging agents on the ionization of the neuropeptides tends to be peptide-dependent with both ionization sources.

Keywords:  Electrospray ionization; Matrix effect; Neuropeptide; Supercharging agent; UHPLC-MS/MS; UniSpray ionization

DOI:  https://doi.org/10.1016/j.chroma.2020.461462
Zhongguo Dang Dai Er Ke Za Zhi. 2020 Aug;22(8): 874-881

[Blood lipid metabolic profile of overweight/obese boys aged 9-12 years].

Chun-Yu Liang, Yu-Ping Cao, Yi Yan.

OBJECTIVE: To study the features of blood lipid metabolic profile in overweight/obese boys aged 9-12 years and the possible mechanism of overweight/obesity in children.
METHODS: According to body mass index (BMI), 72 boys, aged 9-12 years, were divided into a control group with 42 boys and an overweight/obesity group with 30 boys. Fasting venous blood samples were collected early in the morning. BMI, waist-hip ratio, body composition, and blood lipids were measured. Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry technique was used to analyze the serum lipid compounds. A statistical analysis and visualization of the data were performed.
RESULTS: Compared with the control group, the overweight/obesity group had significantly higher waist-hip ratio, body fat percentage, and triglyceride level (P<0.05) and a significantly lower level of high-density lipoprotein cholesterol (P<0.05). The metabolomic analysis identified 150 differentially expressed lipid compounds between the two groups, mainly glycerolipids (40.7%), glycerophospholipids (24.7%), fatty acyls (10.7%), and sphingolipids (7.3%). The levels of most of glycerolipids were significantly upregulated in the overweight/obesity group, while those of most of glycerophospholipids and sphingolipids were downregulated in this group. Key lipids with differential expression were enriched into two KEGG metabolic pathways, i.e., ether lipid metabolism pathway and terpenoid backbone biosynthesis pathway (P<0.05), and might further affected the biosynthesis and metabolism of downstream coenzyme Q and other terpenoids (P=0.06).
CONCLUSIONS: Disordered lipid metabolic profile is observed in overweight/obese boys aged 9-12 years, with increases in most glycerolipids and reductions in glycerophospholipids and sphingolipids. Overweight/obese boys may have disorders in ether lipid metabolism and biosynthesis of terpenoid and even coenzyme Q.
Cancer Discov. 2020 Aug 21. pii: CD-19-0970. [Epub ahead of print]

The Folate Cycle Enzyme MTHFR is a Critical Regulator of Cell Response to MYC-Targeting Therapies.

Angela Su, Frank Ling, Camille Vaganay, Gaetano Sodaro, Chaima Benaksas, Reinaldo Dal Bello, Antoine Forget, Bryann Pardieu, Kevin H Lin, Justine C Rutter, Christopher F Bassil, Gael Fortin, Justine Pasanisi, Ileana Antony-Debre, Gabriela Alexe, Jean-Francois Benoist, Alain Pruvost, Yana Pikman, Jun Qi, Marie-Helene Schlageter, Jean-Baptiste Micol, Giovanni Roti, Thomas Cluzeau, Herve Dombret, Claude Preudhomme, Nina Fenouille, Lina Benajiba, Hava M Golan, Kimberly Stegmaier, Camille Lobry, Kris C Wood, Raphael Itzykson, Alexandre Puissant.

Deciphering the impact of metabolic intervention on response to anticancer therapy may elucidate a path toward improved clinical responses. Here, we identify amino acid-related pathways connected to the folate cycle whose activation predicts sensitivity to MYC-targeting therapies in acute myeloid leukemia (AML). We establish that folate restriction and deficiency of the rate-limiting folate cycle enzyme, MTHFR - which exhibits reduced-function polymorphisms in about 10% of Caucasians - induce resistance to MYC targeting by BET and CDK7 inhibitors in cell lines, primary patient samples, and syngeneic mouse models of AML. Further, this effect is abrogated by supplementation with the MTHFR enzymatic product, CH3-THF. Mechanistically, folate cycle disturbance reduces H3K27/K9 histone methylation and activates a SPI1 transcriptional program counteracting the effect of BET inhibition. Our data provide a rationale for screening MTHFR polymorphisms and the folate cycle status to nominate patients most likely to benefit from MYC-targeting therapies.

DOI: https://doi.org/10.1158/2159-8290.CD-19-0970
Antioxid Redox Signal. 2020 Aug 18.

Ferroptosis: biological rust of lipid membranes.

Tom Vanden Berghe, Behrouz Hassannia, Samya Van Coillie.

SIGNIFICANCE: Iron is an essential element required for growth and proper functioning of the body. However, an excess of labile ferrous iron increases the risk of oxidative stress-induced injury due to the high reactivity of the unpaired reactive electrons of both ferrous iron and oxygen. This high reactivity can be exemplified in the outside world by one of its consequences, rust formation. In cells, this redox-active iron is involved in the formation of lipid radicals. Recent Advances: Defect or insufficient membrane-protective mechanisms can result in iron-catalyzed excessive lipid peroxidation and subsequent cell death, now conceptualized as ferroptosis. Growing reports propose the detrimental role of iron and ferroptosis in many experimental disease models such as ischemia-reperfusion, acute and chronic organ injuries.
CRITICAL ISSUES: This review first provides a snapshot of iron metabolism, followed by a brief introduction of the molecular mechanisms of ferroptosis, as an iron-dependent lipid peroxidation-driven mode of cell death. Upon describing how iron dysbiosis affects ferroptosis induction, we elaborate on the detrimental role of the iron-ferroptosis axis in several diseases.
FUTURE DIRECTIONS: Despite compelling findings suggesting a role for ferroptosis in experimental animal models, the exact contribution of ferroptosis in human contexts still needs further investigation. Development of reliable ferroptosis biomarkers will be an important step in characterizing ferroptosis in human disease. This can provide therapeutic opportunities aiming at targeting ferroptosis in human diseases.

DOI: https://doi.org/10.1089/ars.2020.8175