bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2019‒11‒03
twenty-one papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research


  1. Cell Rep. 2019 Oct 29. pii: S2211-1247(19)31244-6. [Epub ahead of print]29(5): 1287-1298.e6
    Bott AJ, Shen J, Tonelli C, Zhan L, Sivaram N, Jiang YP, Yu X, Bhatt V, Chiles E, Zhong H, Maimouni S, Dai W, Velasquez S, Pan JA, Muthalagu N, Morton J, Anthony TG, Feng H, Lamers WH, Murphy DJ, Guo JY, Jin J, Crawford HC, Zhang L, White E, Lin RZ, Su X, Tuveson DA, Zong WX.
      Glutamine is thought to play an important role in cancer cells by being deaminated via glutaminolysis to α-ketoglutarate (aKG) to fuel the tricarboxylic acid (TCA) cycle. Supporting this notion, aKG supplementation can restore growth/survival of glutamine-deprived cells. However, pancreatic cancers are often poorly vascularized and limited in glutamine supply, in alignment with recent concerns on the significance of glutaminolysis in pancreatic cancer. Here, we show that aKG-mediated rescue of glutamine-deprived pancreatic ductal carcinoma (PDAC) cells requires glutamate ammonia ligase (GLUL), the enzyme responsible for de novo glutamine synthesis. GLUL-deficient PDAC cells are capable of the TCA cycle but defective in aKG-coupled glutamine biosynthesis and subsequent nitrogen anabolic processes. Importantly, GLUL expression is elevated in pancreatic cancer patient samples and in mouse PDAC models. GLUL ablation suppresses the development of KrasG12D-driven murine PDAC. Therefore, GLUL-mediated glutamine biosynthesis couples the TCA cycle with nitrogen anabolism and plays a critical role in PDAC.
    Keywords:  K-Ras; glutamate ammonia ligase; glutamine; glutamine synthetase; hexosamine; nitrogen metabolism; nucleotide; pancreatic cancer; α-ketoglutarate
    DOI:  https://doi.org/10.1016/j.celrep.2019.09.056
  2. Diagnostics (Basel). 2019 Oct 29. pii: E167. [Epub ahead of print]9(4):
    Evangelista EB, Kwee SA, Sato MM, Wang L, Rettenmeier C, Xie G, Jia W, Wong LL.
      BACKGROUND: Hepatocellular carcinoma (HCC) pathogenesis involves the alteration of multiple liver-specific metabolic pathways. We systematically profiled cancer- and liver-related classes of metabolites in HCC and adjacent liver tissues and applied supervised machine learning to compare their potential yield for HCC biomarkers.METHODS: Tumor and corresponding liver tissue samples were profiled as follows: Bile acids by ultra-performance liquid chromatography (LC) coupled to tandem mass spectrometry (MS), phospholipids by LC-MS/MS, and other small molecules including free fatty acids by gas chromatography-time of flight MS. The overall classification performance of metabolomic signatures derived by support vector machine (SVM) and random forests machine learning algorithms was then compared across classes of metabolite.
    RESULTS: For each metabolite class, there was a plateau in classification performance with signatures of 10 metabolites. Phospholipid signatures consistently showed the highest discrimination for HCC followed by signatures derived from small molecules, free fatty acids, and bile acids with area under the receiver operating characteristic curve (AUC) values of 0.963, 0.934, 0.895, 0.695, respectively, for SVM-generated signatures comprised of 10 metabolites. Similar classification performance patterns were observed with signatures derived by random forests.
    CONCLUSION: Membrane phospholipids are a promising source of tissue biomarkers for discriminating between HCC tumor and liver tissue.
    Keywords:  diagnosis; hepatocellular carcinoma; machine learning; metabolomics; molecular imaging; phospholipids; positron emission tomography
    DOI:  https://doi.org/10.3390/diagnostics9040167
  3. Metabolites. 2019 Oct 28. pii: E252. [Epub ahead of print]9(11):
    García-Jaramillo M, Lytle KA, Spooner MH, Jump DB.
      Nonalcoholic fatty liver disease (NAFLD) is a major public health problem worldwide. NAFLD ranges in severity from benign steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and primary hepatocellular cancer (HCC). Obesity and type 2 diabetes mellitus (T2DM) are strongly associated with NAFLD, and the western diet (WD) is a major contributor to the onset and progression of these chronic diseases. Our aim was to use a lipidomic approach to identify potential lipid mediators of diet-induced NASH. We previously used a preclinical mouse (low density lipoprotein receptor null mouse, Ldlr -/-) model to assess transcriptomic mechanisms linked to WD-induced NASH and docosahexaenoic acid (DHA, 22:6, ω3)-mediated remission of NASH. This report used livers from the previous study to carry out ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography coupled with dynamic multi-reaction monitoring (HPLC-dMRM) to assess the impact of the WD and DHA on hepatic membrane lipid and oxylipin composition, respectively. Feeding mice the WD increased hepatic saturated and monounsaturated fatty acids and arachidonic acid (ARA, 20:4, ω6) in membrane lipids and suppressed ω3 polyunsaturated fatty acids (PUFA) in membrane lipids and ω3 PUFA-derived anti-inflammatory oxylipins. Supplementing the WD with DHA lowered hepatic ARA in membrane lipids and ARA-derived oxylipins and significantly increased hepatic DHA and its metabolites in membrane lipids, as well as C20-22 ω3 PUFA-derived oxylipins. NASH markers of inflammation and fibrosis were inversely associated with hepatic C20-22 ω3 PUFA-derived Cyp2C- and Cyp2J-generated anti-inflammatory oxylipins (false discovery rate adjusted p-value; q ≤ 0.026). Our findings suggest that dietary DHA promoted partial remission of WD-induced NASH, at least in part, by lowering hepatic pro-inflammatory oxylipins derived from ARA and increasing hepatic anti-inflammatory oxylipins derived from C20-22 ω3 PUFA.
    Keywords:  arachidonic acid; docosahexaenoic acid; fibrosis; inflammation; lipidomics; mass spectrometry; nonalcoholic fatty liver disease; nonalcoholic steatohepatitis
    DOI:  https://doi.org/10.3390/metabo9110252
  4. Dev Cell. 2019 Oct 24. pii: S1534-5807(19)30774-9. [Epub ahead of print]
    Juin A, Spence HJ, Martin KJ, McGhee E, Neilson M, Cutiongco MFA, Gadegaard N, Mackay G, Fort L, Lilla S, Kalna G, Thomason P, Koh YWH, Norman JC, Insall RH, Machesky LM.
      Pancreatic ductal adenocarcinoma is one of the most invasive and metastatic cancers and has a dismal 5-year survival rate. We show that N-WASP drives pancreatic cancer metastasis, with roles in both chemotaxis and matrix remodeling. lysophosphatidic acid, a signaling lipid abundant in blood and ascites fluid, is both a mitogen and chemoattractant for cancer cells. Pancreatic cancer cells break lysophosphatidic acid down as they respond to it, setting up a self-generated gradient driving tumor egress. N-WASP-depleted cells do not recognize lysophosphatidic acid gradients, leading to altered RhoA activation, decreased contractility and traction forces, and reduced metastasis. We describe a signaling loop whereby N-WASP and the endocytic adapter SNX18 promote lysophosphatidic acid-induced RhoA-mediated contractility and force generation by controlling lysophosphatidic acid receptor recycling and preventing degradation. This chemotactic loop drives collagen remodeling, tumor invasion, and metastasis and could be an important target against pancreatic cancer spread.
    Keywords:  actin dynamics; cancer cell signaling; cancer invasion; cell migration; chemotaxis; endocytosis; pancreatic cancer metastasis; receptor recycling; self-generated gradients; tumor invasion
    DOI:  https://doi.org/10.1016/j.devcel.2019.09.018
  5. Nat Rev Gastroenterol Hepatol. 2019 Oct 30.
    Satriano L, Lewinska M, Rodrigues PM, Banales JM, Andersen JB.
      Primary liver cancer (PLC) is the fourth most frequent cause of cancer-related death. The high mortality rates arise from late diagnosis and the limited accuracy of diagnostic and prognostic biomarkers. The liver is a major regulator, orchestrating the clearance of toxins, balancing glucose, lipid and amino acid uptake, managing whole-body metabolism and maintaining metabolic homeostasis. Tumour onset and progression is frequently accompanied by rearrangements of metabolic pathways, leading to dysregulation of metabolism. The limitation of current therapies targeting PLCs, such as hepatocellular carcinoma and cholangiocarcinoma, points towards the importance of deciphering this metabolic complexity. In this Review, we discuss the role of metabolic liver disruptions and the implications of these processes in PLCs, emphasizing their clinical relevance and value in early diagnosis and prognosis and as putative therapeutic targets. We also describe system biology approaches able to reconstruct the metabolic complexity of liver diseases. We also discuss whether metabolic rearrangements are a cause or consequence of PLCs, emphasizing the opportunity to clinically exploit the rewired metabolism. In line with this idea, we discuss circulating metabolites as promising biomarkers for PLCs.
    DOI:  https://doi.org/10.1038/s41575-019-0217-8
  6. Proteomes. 2019 Oct 30. pii: E36. [Epub ahead of print]7(4):
    Zhan X, Li B, Zhan X, Schlüter H, Jungblut PR, Coorssen JR.
      Two-dimensional gel electrophoresis (2DE) is an important and well-established technical platform enabling extensive top-down proteomic analysis. However, the long-held but now largely outdated conventional concepts of 2DE have clearly impacted its application to in-depth investigations of proteomes at the level of protein species/proteoforms. It is time to popularize a new concept of 2DE for proteomics. With the development and enrichment of the proteome concept, any given "protein" is now recognized to consist of a series of proteoforms. Thus, it is the proteoform, rather than the canonical protein, that is the basic unit of a proteome, and each proteoform has a specific isoelectric point (pI) and relative mass (Mr). Accordingly, using 2DE, each proteoform can routinely be resolved and arrayed according to its different pI and Mr. Each detectable spot contains multiple proteoforms derived from the same gene, as well as from different genes. Proteoforms derived from the same gene are distributed into different spots in a 2DE pattern. High-resolution 2DE is thus actually an initial level of separation to address proteome complexity and is effectively a pre-fractionation method prior to analysis using mass spectrometry (MS). Furthermore, stable isotope-labeled 2DE coupled with high-sensitivity liquid chromatography-tandem MS (LC-MS/MS) has tremendous potential for the large-scale detection, identification, and quantification of the proteoforms that constitute proteomes.
    Keywords:  SILAC; TMT; bottom-up proteomics; iTRAQ; isoelectric focusing; liquid chromatography; mass spectrometry; post-translational modification; protein speciation; protein species; proteoform; splicing; top-down proteomics; two-dimensional gel electrophoresis
    DOI:  https://doi.org/10.3390/proteomes7040036
  7. Clin Chem Lab Med. 2019 Oct 30. pii: /j/cclm.ahead-of-print/cclm-2019-0858/cclm-2019-0858.xml. [Epub ahead of print]
    Ščupáková K, Balluff B, Tressler C, Adelaja T, Heeren RMA, Glunde K, Ertaylan G.
      Mass spectrometry (MS) is the workhorse of metabolomics, proteomics and lipidomics. Mass spectrometry imaging (MSI), its extension to spatially resolved analysis of tissues, is a powerful tool for visualizing molecular information within the histological context of tissue. This review summarizes recent developments in MSI and highlights current challenges that remain to achieve molecular imaging at the cellular level of clinical specimens. We focus on matrix-assisted laser desorption/ionization (MALDI)-MSI. We discuss the current status of each of the analysis steps and remaining challenges to reach the desired level of cellular imaging. Currently, analyte delocalization and degradation, matrix crystal size, laser focus restrictions and detector sensitivity are factors that are limiting spatial resolution. New sample preparation devices and laser optic systems are being developed to push the boundaries of these limitations. Furthermore, we review the processing of cellular MSI data and images, and the systematic integration of these data in the light of available algorithms and databases. We discuss roadblocks in the data analysis pipeline and show how technology from other fields can be used to overcome these. Finally, we conclude with curative and community efforts that are needed to enable contextualization of the information obtained.
    Keywords:  data analysis; data integration and interpretation; histopathology; mass spectrometry imaging; molecular analysis; spatial resolution
    DOI:  https://doi.org/10.1515/cclm-2019-0858
  8. Methods Enzymol. 2019 ;pii: S0076-6879(19)30257-5. [Epub ahead of print]628 293-307
    Yin R, Prabhakaran V, Laskin J.
      Quantitative mass spectrometry analysis of metabolites at a single-cell level is critical to understanding the cell functionality and heterogeneity. To preserve cell viability after extraction, the extracted volume needs to be precisely controlled at a subpicoliter-to-picoliter level. Recently, we developed a volume-controlled, and highly sensitive approach for live cell analysis at a single-cell level by integrating electroosmotic extraction and nano-electrospray ionization mass spectrometry (nanoESI MS) analysis. Herein, we use outer epidermal cells of Allium cepa as a model system to present the details of our workflow, including detailed descriptions of the experimental setup for live cell analysis, preparation of the extraction nanopipette, establishment of calibration curves, and extraction and quantification of glucose in an individual onion cell. The capability of this procedure for quantitative live cell analysis has been demonstrated by accurate quantification of glucose in Allium cepa. In principle, our approach is applicable to identification and quantification of metabolites in live mammalian cells.
    Keywords:  Electroosmotic extraction; Live cell analysis; Metabolite quantification; Nano-electrospray ionization mass spectrometry (nanoESI MS); Single cell
    DOI:  https://doi.org/10.1016/bs.mie.2019.06.018
  9. Cancer Lett. 2019 Oct 25. pii: S0304-3835(19)30541-5. [Epub ahead of print]
    Eylem CC, Yilmaz M, Derkus B, Nemutlu E, Camci CB, Yilmaz E, Turkoglu MA, Aytac B, Ozyurt N, Emregul E.
      Exosomes are naturally secreted nano-vesicles consisting of biochemical molecules including RNAs, metabolites, lipids, and proteins, that emerge as diagnostic tools and disease-specific reporters. Here we offer a systematic and integrative approach for the simultaneous analysis of altered molecules namely metabolites, lipids, and proteins. These components tend to augment the discovery of low abundance signature components, and assist in explanation of molecular basis of colorectal cancer (CRC). In order to investigate CRC-derived exosomes, we selected mi-R19a, miR-21, miR-92a, and miR-1246 positive exosomes for downstream experiments. The overall multi-omic changes were investigated comparatively in cell culture and serum samples. Following a systematic multi-omic study, 37 (cell culture) and 31 (serum) metabolites; 130 (cell culture) and 56 (serum) lipids; 9 (cell culture) and 13 (serum) proteins were seen to be differentially expressed (p<0.05), enabling discrimination between CRC and control. By using these enriched components, we demonstrated that the joint pathways mainly involving fatty acid and amino acid metabolism related pathways changed in CRC significantly. We conclude that this study increases our understanding of molecular basis of CRC, and provides potential exosomal biomarkers for the non-invasive detection, and discrimination of CRC.
    Keywords:  Exosome; colorectal cancer; metabolomics; miRNA; proteomics
    DOI:  https://doi.org/10.1016/j.canlet.2019.10.038
  10. Nature. 2019 Oct 30.
    Momcilovic M, Jones A, Bailey ST, Waldmann CM, Li R, Lee JT, Abdelhady G, Gomez A, Holloway T, Schmid E, Stout D, Fishbein MC, Stiles L, Dabir DV, Dubinett SM, Christofk H, Shirihai O, Koehler CM, Sadeghi S, Shackelford DB.
      Mitochondria are essential regulators of cellular energy and metabolism, and have a crucial role in sustaining the growth and survival of cancer cells. A central function of mitochondria is the synthesis of ATP by oxidative phosphorylation, known as mitochondrial bioenergetics. Mitochondria maintain oxidative phosphorylation by creating a membrane potential gradient that is generated by the electron transport chain to drive the synthesis of ATP1. Mitochondria are essential for tumour initiation and maintaining tumour cell growth in cell culture and xenografts2,3. However, our understanding of oxidative mitochondrial metabolism in cancer is limited because most studies have been performed in vitro in cell culture models. This highlights a need for in vivo studies to better understand how oxidative metabolism supports tumour growth. Here we measure mitochondrial membrane potential in non-small-cell lung cancer in vivo using a voltage-sensitive, positron emission tomography (PET) radiotracer known as 4-[18F]fluorobenzyl-triphenylphosphonium (18F-BnTP)4. By using PET imaging of 18F-BnTP, we profile mitochondrial membrane potential in autochthonous mouse models of lung cancer, and find distinct functional mitochondrial heterogeneity within subtypes of lung tumours. The use of 18F-BnTP PET imaging enabled us to functionally profile mitochondrial membrane potential in live tumours.
    DOI:  https://doi.org/10.1038/s41586-019-1715-0
  11. Rapid Commun Mass Spectrom. 2019 Oct 31.
    Roggensack T, Merz B, Dick N, Bub A, Krüger R.
      RATIONALE: Methylated amino compounds and basic amino acids are important analyte classes with high relevance in nutrition, physical activity and physiology. Reliable and easy quantification methods covering a variety of metabolites in body fluids are a prerequisite for efficient investigations in the field of food and nutrition.METHODS: Targeted UHPLC/MS analysis was performed using HILIC separation and timed ESI-MRM detection, combined with a short sample preparation. Calibration in urine and blood plasma was achieved by matrix-matched standards, isotope-labelled internal standards and standard addition. The method was fully validated and the performance was evaluated using a subset from the Karlsruhe Metabolomics and Nutrition (KarMeN) study.
    RESULTS: Within this method, a number of 30 compounds could be quantified simultaneously in a short run of 9 minutes in both body fluids. It covers a variety of free amino compounds which are present in very different concentrations. The method is easy, precise and robust, and has a broad working range. As a proof of principle, literature-based associations of certain metabolites with dietary intake of respective foods were clearly confirmed in the KarMeN subset.
    CONCLUSIONS: Overall, the method turned out to be well suited for application in nutrition studies, as shown for the example of food intake biomarkers in KarMeN. Application to a variety of questions such as food-related effects, or physical activity will support future studies in the context of nutrition and health.
    DOI:  https://doi.org/10.1002/rcm.8646
  12. Int J Mol Sci. 2019 Oct 28. pii: E5370. [Epub ahead of print]20(21):
    Bezuidenhout N, Shoshan M.
      Tumor-initiating cells (TICs), or cancer stem cells, constitute highly chemoresistant, asymmetrically dividing, and tumor-initiating populations in cancer and are thought to play a key role in metastatic and chemoresistant disease. Tumor-initiating cells are isolated from cell lines and clinical samples based on features such as sphere formation in stem cell medium and expression of TIC markers, typically a set of outer membrane proteins and certain transcription factors. Although both bulk tumor cells and TICs show an adaptive metabolic plasticity, TIC metabolism is thought to differ and likely in a tumor-specific and growth condition-dependent pattern. In the context of some common solid tumor diseases, we here review reports on how TIC isolation methods and markers associate with metabolic features, with some focus on oxidative metabolism, including fatty acid and lipid metabolism. These have emerged as significant factors in TIC phenotypes, and in tumor biology as a whole. Other sections address mitochondrial biogenesis and dynamics in TICs, and the influence of the tumor microenvironment. Further elucidation of the complex biology of TICs and their metabolism will require advanced methodologies.
    Keywords:  cancer; cellular metabolism; mitochondria; stem cell markers; tumor-initiating cells
    DOI:  https://doi.org/10.3390/ijms20215370
  13. Anal Chim Acta. 2019 Dec 20. pii: S0003-2670(19)30973-0. [Epub ahead of print]1090 23-30
    Wiśniewski JR.
      Filter Aided Sample Preparation (FASP) is a widely used protein processing technique in "bottom-up" proteomics. Its popularity reflects the key features of the method: its applicability to a variety of sample types and the high quality of the released peptides. Successful application of FASP requires optimized properties of sample lysate and its amount, use of ultrafiltration units with membranes having large molecular mass cut-offs and well selected conditions for protein digestion. In contrast to the majority of sample preparation methods, FASP allows digestion of proteins with a variety of enzymes and a straightforward monitoring of protein-to-peptide conversion. A unique feature of FASP is the possibility to cleave proteins in a consecutive way using several proteases and to separate peptide fractions. Understanding principles of the method gives guidance in applying FASP to different types of samples in optimization of conditions of the FASP-workflow.
    Keywords:  Detergent removal; Filter Aided Sample Preparation; Lysate preparation; Multi enzyme digestion filter aided sample preparation; Protein digestion conditions; Proteomic sample preparation
    DOI:  https://doi.org/10.1016/j.aca.2019.08.032
  14. Proteomics. 2019 Oct 28. e1900147
    Perez-Riverol Y, Moreno P.
      The recent improvements in mass spectrometry instruments and new analytical methods are increasing the intersection between proteomics and big data science. In addition, bioinformatics analysis is becoming increasingly complex and convoluted, involving multiple algorithms and tools. A wide variety of methods and software tools have been developed for computational proteomics and metabolomics during recent years, and this trend is likely to continue. However, most of the computational proteomics and metabolomics tools are designed as single-tiered software application where the analytics tasks can't be distributed, limiting the scalability and reproducibility of the data analysis. In this paper we summarise the key steps of metabolomics and proteomics data processing, including the main tools and software used to perform the data analysis. We discuss the combination of software containers with workflows environments for large scale metabolomics and proteomics analysis. Finally, we introduce to the proteomics and metabolomics communities a new approach for reproducible and large-scale data analysis based on BioContainers and two of the most popular workflow environments: Galaxy and Nextflow. This article is protected by copyright. All rights reserved.
    DOI:  https://doi.org/10.1002/pmic.201900147
  15. Mol Metab. 2019 Nov;pii: S2212-8778(19)30620-9. [Epub ahead of print]29 40-54
    Fernández-Tussy P, Fernández-Ramos D, Lopitz-Otsoa F, Simón J, Barbier-Torres L, Gomez-Santos B, Nuñez-Garcia M, Azkargorta M, Gutiérrez-de Juan V, Serrano-Macia M, Rodríguez-Agudo R, Iruzubieta P, Anguita J, Castro RE, Champagne D, Rincón M, Elortza F, Arslanow A, Krawczyk M, Lammert F, Kirchmeyer M, Behrmann I, Crespo J, Lu SC, Mato JM, Varela-Rey M, Aspichueta P, Delgado TC, Martínez-Chantar ML.
      OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolic pathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation and fibrosis. The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, is downregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression.METHODS: miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Different in vitro and in vivo NAFLD murine models were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy.
    RESULTS: We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria. In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondrial functionality in a preclinical murine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating with hepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation and fibrosis by enhancing fatty acid β-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment.
    CONCLUSION: GNMT participates in the regulation of metabolic pathways and mitochondrial functionality through the regulation of Complex II activity in the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment.
    Keywords:  GNMT; Metabolism; Mitochondria; NASH; microRNA; β-oxidation
    DOI:  https://doi.org/10.1016/j.molmet.2019.08.008
  16. J Biomol Tech. 2019 Oct 23. pii: jbt.19-3004-002. [Epub ahead of print]
    Tadi S, Sharp JS.
      Methionine oxidation plays a critical role in many processes of biologic and biomedical importance, including cellular redox responses and stability of protein pharmaceuticals. Bottom-up methods for analysis of methionine oxidation can suffer from incomplete sequence coverage, as well as an inability to readily detect correlated oxidation between 2 or more methionines. However, the methodology for quantifying protein oxidation in top-down analyses is lacking. Previous work has shown that electron transfer dissociation (ETD)-based tandem mass spectrometry (MS/MS) fragmentation offers accurate and precise quantification of amino acid oxidation in peptides, even in complex samples. However, the ability of ETD-based MS/MS fragmentation to accurately quantify amino acid oxidation of proteins in a top-down manner has not been reported. Using apomyoglobin and calmodulin as model proteins, we partially converted methionines into methionine sulfoxide by incubation in H2O2. Using top-down ETD-based fragmentation, we quantified the amount of oxidation of various ETD product ions and compared the quantified values with those from traditional bottom-up analysis. We find that overall quantification of methionine oxidation by top-down MS/MS ranges from good agreement with traditional bottom-up methods to vast differences between the 2 techniques, including missing oxidized product ions and large differences in measured oxidation quantities. Care must be taken in transitioning ETD-based quantitation of oxidation from the peptide level to the intact protein level.
    Keywords:  hydroxyl radical protein footprinting; post-translational modification; proteomics
    DOI:  https://doi.org/10.7171/jbt.19-3004-002
  17. Nat Commun. 2019 Nov 01. 10(1): 5011
    Bueno MJ, Jimenez-Renard V, Samino S, Capellades J, Junza A, López-Rodríguez ML, Garcia-Carceles J, Lopez-Fabuel I, Bolaños JP, Chandel NS, Yanes O, Colomer R, Quintela-Fandino M.
      Upregulation of fatty acid synthase (FASN) is a common event in cancer, although its mechanistic and potential therapeutic roles are not completely understood. In this study, we establish a key role of FASN during transformation. FASN is required for eliciting the anaplerotic shift of the Krebs cycle observed in cancer cells. However, its main role is to consume acetyl-CoA, which unlocks isocitrate dehydrogenase (IDH)-dependent reductive carboxylation, producing the reductive power necessary to quench reactive oxygen species (ROS) originated during the switch from two-dimensional (2D) to three-dimensional (3D) growth (a necessary hallmark of cancer). Upregulation of FASN elicits the 2D-to-3D switch; however, FASN's synthetic product palmitate is dispensable for this process since cells satisfy their fatty acid requirements from the media. In vivo, genetic deletion or pharmacologic inhibition of FASN before oncogenic activation prevents tumor development and invasive growth. These results render FASN as a potential target for cancer prevention studies.
    DOI:  https://doi.org/10.1038/s41467-019-13028-1
  18. Cancers (Basel). 2019 Oct 10. pii: E1523. [Epub ahead of print]11(10):
    Xu Y.
      Since the clear demonstration of lysophosphatidic acid (LPA)'s pathological roles in cancer in the mid-1990s, more than 1000 papers relating LPA to various types of cancer were published. Through these studies, LPA was established as a target for cancer. Although LPA-related inhibitors entered clinical trials for fibrosis, the concept of targeting LPA is yet to be moved to clinical cancer treatment. The major challenges that we are facing in moving LPA application from bench to bedside include the intrinsic and complicated metabolic, functional, and signaling properties of LPA, as well as technical issues, which are discussed in this review. Potential strategies and perspectives to improve the translational progress are suggested. Despite these challenges, we are optimistic that LPA blockage, particularly in combination with other agents, is on the horizon to be incorporated into clinical applications.
    Keywords:  Autotaxin (ATX); G-protein coupled receptor (GPCR); cancer stem cell (CSC); electrospray ionization tandem mass spectrometry (ESI-MS/MS); lipid phosphate phosphatase enzymes (LPPs); lysophosphatidic acid (LPA); nuclear receptor peroxisome proliferator-activated receptor (PPAR); ovarian cancer (OC); phospholipase A2 enzymes (PLA2s); sphingosine-1 phosphate (S1P)
    DOI:  https://doi.org/10.3390/cancers11101523
  19. World J Gastrointest Oncol. 2019 Oct 15. 11(10): 887-897
    Takaya H, Namisaki T, Kitade M, Shimozato N, Kaji K, Tsuji Y, Nakanishi K, Noguchi R, Fujinaga Y, Sawada Y, Saikawa S, Sato S, Kawaratani H, Moriya K, Akahane T, Yoshiji H.
      BACKGROUND: Early diagnosis of hepatocellular carcinoma (HCC) is necessary to improve the prognosis of patients. However, the currently available tumor biomarkers are insufficient for the early detection of HCC. Acylcarnitine is essential in fatty acid metabolic pathways. A recent study reported that a high level of acylcarnitine may serve as a useful biomarker for the early diagnosis of HCC in steatohepatitis (SH) patients. In contrast, another study reported that the level of acetylcarnitine (AC2) - one of the acylcarnitine species - in non-SH patients with HCC was decreased vs that reported in those without HCC.AIM: To investigate the usefulness of acylcarnitine as a biomarker for the early diagnosis of HCC in non-SH patients.
    METHODS: Thirty-three non-SH patients (14 with HCC and 19 without HCC) were enrolled in this study. Blood samples were obtained from patients at the time of admission. The levels of acylcarnitine and AC2 in the serum were determined through tandem mass spectrometry. The levels of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2) were determined by enzyme-linked immunosorbent assay. Univariate and multivariate analyses were used to determine early diagnostic factors of HCC.
    RESULTS: The level of acylcarnitine was significantly lower in non-SH patients with HCC vs those without HCC (P < 0.05). In contrast, the level of lens culinaris agglutinin-reactive fraction of α-fetoprotein (AFP) - AFP-L3% - was significantly higher in non-SH patients with HCC vs those without HCC (P < 0.05). However, the levels of total carnitine, free carnitine, AFP, des-γ-carboxy prothrombin, VEGF, and VEGFR-2 were not different between patients with and without HCC. The multivariate analysis showed that a low level of acylcarnitine was the only independent factor for the early diagnosis of HCC. The patients with a low level of AC2 had a significantly higher level of VEGF vs those with a high level of AC2 (P < 0.05).
    CONCLUSION: The metabolic pathways of fatty acids may differ between SH HCC and non-SH HCC. Further studies are warranted to investigate these differences.
    Keywords:  Acetylcarnitine; Acylcarnitine; Angiogenesis; Biomarker; Carnitine palmitoyltransferase 1; Hepatocellular carcinoma; Oxidative stress
    DOI:  https://doi.org/10.4251/wjgo.v11.i10.887
  20. Cell. 2019 Oct 31. pii: S0092-8674(19)31121-3. [Epub ahead of print]179(4): 813-827
    Gorgoulis V, Adams PD, Alimonti A, Bennett DC, Bischof O, Bishop C, Campisi J, Collado M, Evangelou K, Ferbeyre G, Gil J, Hara E, Krizhanovsky V, Jurk D, Maier AB, Narita M, Niedernhofer L, Passos JF, Robbins PD, Schmitt CA, Sedivy J, Vougas K, von Zglinicki T, Zhou D, Serrano M, Demaria M.
      Cellular senescence is a cell state implicated in various physiological processes and a wide spectrum of age-related diseases. Recently, interest in therapeutically targeting senescence to improve healthy aging and age-related disease, otherwise known as senotherapy, has been growing rapidly. Thus, the accurate detection of senescent cells, especially in vivo, is essential. Here, we present a consensus from the International Cell Senescence Association (ICSA), defining and discussing key cellular and molecular features of senescence and offering recommendations on how to use them as biomarkers. We also present a resource tool to facilitate the identification of genes linked with senescence, SeneQuest (available at http://Senequest.net). Lastly, we propose an algorithm to accurately assess and quantify senescence, both in cultured cells and in vivo.
    DOI:  https://doi.org/10.1016/j.cell.2019.10.005
  21. Cell Metab. 2019 Oct 09. pii: S1550-4131(19)30518-2. [Epub ahead of print]
    Rendina-Ruedy E, Rosen CJ.
      Because of heavy energy demands to maintain bone homeostasis, the skeletal system is closely tied to whole-body metabolism via neuronal and hormonal mediators. Glucose, amino acids, and fatty acids are the chief fuel sources for bone resident cells during its remodeling. Lipids, which can be mobilized from intracellular depots in the bone marrow, can be a potent source of fatty acids. Thus, while it has been suggested that adipocytes in the bone marrow act as "filler" and are detrimental to skeletal homeostasis, we propose that marrow lipids are, in fact, essential for proper bone functioning. As such, we examine the prevailing evidence regarding the storage, use, and export of lipids within the skeletal niche, including from both in vitro and in vivo model systems. We also highlight the numerous challenges that remain to fully appreciate the relationship of lipid turnover to skeletal homeostasis.
    Keywords:  adipocytes; bone; cholesterol; diet; energy; fat; osteoblasts
    DOI:  https://doi.org/10.1016/j.cmet.2019.09.015