bims-mascan 2020-05-10 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2020–05–10
23 papers selected by
Giovanny Rodríguez Blanco, The Beatson Institute for Cancer Research

Formate induces a metabolic switch in nucleotide and energy metabolism.
Combining native and 'omics' mass spectrometry to identify endogenous ligands bound to membrane proteins.
Fructose-1,6-Bisphosphatase 2 Inhibits Sarcoma Progression by Restraining Mitochondrial Biogenesis.
Targeting Pyruvate Carboxylase by a Small Molecule Suppresses Breast Cancer Progression.
FBP1 loss disrupts liver metabolism and promotes tumorigenesis through a hepatic stellate cell senescence secretome.
The tumor microenvironment as a metabolic barrier to effector T cells and immunotherapy.
Microfluidic probe for in-situ extraction of adherent cancer cells to detect heterogeneity difference by electrospray ionization mass spectrometry.
A circulating extracellular vesicles-based novel screening tool for colorectal cancer revealed by shotgun and data-independent acquisition mass spectrometry.
Sphingolipids as prognostic biomarkers of neurodegeneration, neuroinflammation, and psychiatric diseases and their emerging role in lipidomic investigation methods.
High-Throughput Fingerprinting of Rhizobial Free Fatty Acids by Chemical Thin-Film Deposition and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.
Secreted Phospholipase PLA2G2D Contributes to Metabolic Health by Mobilizing ω3 Polyunsaturated Fatty Acids in WAT.
Erianin, a novel dibenzyl compound in Dendrobium extract, inhibits lung cancer cell growth and migration via calcium/calmodulin-dependent ferroptosis.
CDP-diacylglycerol, a critical intermediate in lipid metabolism.
Analysis of vitamin A and retinoids in biological matrices.
Abrogation of ARF6 promotes RSL3-induced ferroptosis and mitigates gemcitabine resistance in pancreatic cancer cells.
Contemporary stable isotope tracer approaches: Insights into skeletal muscle metabolism in health and disease.
PiTMaP: A new analytical platform for high-throughput direct metabolome analysis by probe electrospray ionization/tandem mass spectrometry using an R software-based data pipeline.
The heterocyclic compound Tempol inhibits the growth of cancer cells by interfering with glutamine metabolism.
Direct liquid chromatography tandem mass spectrometry analysis of amino acids in human plasma.
Scientific workflow managers in metabolomics: an overview.
Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing.
Synergism between SLC6A14 blockade and gemcitabine in pancreactic cancer: A 1H-NMR-based metabolomic study in pancreatic cancer cells.
Mass spectrometry imaging and LC-MS reveal decreased cerebellar phosphoinositides in Niemann-Pick type C1-null mice.

Cell Death Dis. 2020 May 04. 11(5): 310

Formate induces a metabolic switch in nucleotide and energy metabolism.

Kristell Oizel, Jacqueline Tait-Mulder, Jorge Fernandez-de-Cossio-Diaz, Matthias Pietzke, Holly Brunton, Sergio Lilla, Sandeep Dhayade, Dimitri Athineos, Giovanny Rodriguez Blanco, David Sumpton, Gillian M Mackay, Karen Blyth, Sara R Zanivan, Johannes Meiser, Alexei Vazquez.

Formate is a precursor for the de novo synthesis of purine and deoxythymidine nucleotides. Formate also interacts with energy metabolism by promoting the synthesis of adenine nucleotides. Here we use theoretical modelling together with metabolomics analysis to investigate the link between formate, nucleotide and energy metabolism. We uncover that endogenous or exogenous formate induces a metabolic switch from low to high adenine nucleotide levels, increasing the rate of glycolysis and repressing the AMPK activity. Formate also induces an increase in the pyrimidine precursor orotate and the urea cycle intermediate argininosuccinate, in agreement with the ATP-dependent activities of carbamoyl-phosphate and argininosuccinate synthetase. In vivo data for mouse and human cancers confirms the association between increased formate production, nucleotide and energy metabolism. Finally, the in vitro observations are recapitulated in mice following and intraperitoneal injection of formate. We conclude that formate is a potent regulator of purine, pyrimidine and energy metabolism.

DOI: https://doi.org/10.1038/s41419-020-2523-z
Nat Methods. 2020 May;17(5): 505-508

Combining native and 'omics' mass spectrometry to identify endogenous ligands bound to membrane proteins.

Joseph Gault, Idlir Liko, Michael Landreh, Denis Shutin, Jani Reddy Bolla, Damien Jefferies, Mark Agasid, Hsin-Yung Yen, Marcus J G W Ladds, David P Lane, Syma Khalid, Christopher Mullen, Philip M Remes, Romain Huguet, Graeme McAlister, Michael Goodwin, Rosa Viner, John E P Syka, Carol V Robinson.

Ligands bound to protein assemblies provide critical information for function, yet are often difficult to capture and define. Here we develop a top-down method, 'nativeomics', unifying 'omics' (lipidomics, proteomics, metabolomics) analysis with native mass spectrometry to identify ligands bound to membrane protein assemblies. By maintaining the link between proteins and ligands, we define the lipidome/metabolome in contact with membrane porins and a mitochondrial translocator to discover potential regulators of protein function.

DOI: https://doi.org/10.1038/s41592-020-0821-0
Cell Metab. 2020 May 05. pii: S1550-4131(20)30191-1. [Epub ahead of print]31(5): 1032

Fructose-1,6-Bisphosphatase 2 Inhibits Sarcoma Progression by Restraining Mitochondrial Biogenesis.

Peiwei Huangyang, Fuming Li, Pearl Lee, Itzhak Nissim, Aalim M Weljie, Anthony Mancuso, Bo Li, Brian Keith, Sam S Yoon, M Celeste Simon.

DOI: https://doi.org/10.1016/j.cmet.2020.04.009
Adv Sci (Weinh). 2020 May;7(9): 1903483

Targeting Pyruvate Carboxylase by a Small Molecule Suppresses Breast Cancer Progression.

Qingxiang Lin, Yuan He, Xue Wang, Yong Zhang, Meichun Hu, Weikai Guo, Yundong He, Tao Zhang, Li Lai, Zhenliang Sun, Zhengfang Yi, Mingyao Liu, Yihua Chen.

  Rapid metabolism differentiates cancer cells from normal cells and relies on anaplerotic pathways. However, the mechanisms of anaplerosis-associated enzymes are rarely understood. The lack of potent and selective antimetabolism drugs restrains further clinical investigations. A small molecule ZY-444 ((N 4-((5-(4-(benzyloxy)phenyl)-2-thiophenyl)methyl)-N 2-isobutyl-2,4-pyrimidinediamine) is discovered to inhibit cancer cell proliferation specifically, having potent efficacies against tumor growth, metastasis, and recurrence. ZY-444 binds to cellular pyruvate carboxylase (PC), a key anaplerotic enzyme of the tricarboxylic acid cycle, and inactivates its catalytic activity. PC inhibition suppresses breast cancer growth and metastasis through inhibiting the Wnt/β-catenin/Snail signaling pathway. Lower PC expression in patient tumors is correlated with significant survival benefits. Comparative profiles of PC expression in cancer versus normal tissues implicate the tumor selectivity of ZY-444. Overall, ZY-444 holds promise therapeutically as an anti-cancer metabolism agent.

Keywords:  Wnt/β‐catenin/Snail pathway; breast cancer; cancer metabolism; pyruvate carboxylase; small molecule ZY‐444

DOI:  https://doi.org/10.1002/advs.201903483
Nat Cell Biol. 2020 May 04.

FBP1 loss disrupts liver metabolism and promotes tumorigenesis through a hepatic stellate cell senescence secretome.

Fuming Li, Peiwei Huangyang, Michelle Burrows, Kathy Guo, Romain Riscal, Jason Godfrey, Kyoung Eun Lee, Nan Lin, Pearl Lee, Ian A Blair, Brian Keith, Bo Li, M Celeste Simon.

The crosstalk between deregulated hepatocyte metabolism and cells within the tumour microenvironment, as well as the consequent effects on liver tumorigenesis, are not completely understood. We show here that hepatocyte-specific loss of the gluconeogenic enzyme fructose 1,6-bisphosphatase 1 (FBP1) disrupts liver metabolic homeostasis and promotes tumour progression. FBP1 is universally silenced in both human and murine liver tumours. Hepatocyte-specific Fbp1 deletion results in steatosis, concomitant with activation and senescence of hepatic stellate cells (HSCs), exhibiting a senescence-associated secretory phenotype. Depleting senescent HSCs by 'senolytic' treatment with dasatinib/quercetin or ABT-263 inhibits tumour progression. We further demonstrate that FBP1-deficient hepatocytes promote HSC activation by releasing HMGB1; blocking its release with the small molecule inflachromene limits FBP1-dependent HSC activation, the subsequent development of the senescence-associated secretory phenotype and tumour progression. Collectively, these findings provide genetic evidence for FBP1 as a metabolic tumour suppressor in liver cancer and establish a critical crosstalk between hepatocyte metabolism and HSC senescence that promotes tumour growth.

DOI: https://doi.org/10.1038/s41556-020-0511-2
Elife. 2020 May 05. pii: e55185. [Epub ahead of print]9

The tumor microenvironment as a metabolic barrier to effector T cells and immunotherapy.

Aaron R Lim, W Kimryn Rathmell, Jeffrey C Rathmell.

  Breakthroughs in anti-tumor immunity have led to unprecedented advances in immunotherapy, yet it is now clear that the tumor microenvironment (TME) restrains immunity. T cells must substantially increase nutrient uptake to mount a proper immune response and failure to obtain sufficient nutrients or engage the appropriate metabolic pathways can alter or prevent effector T cell differentiation and function. The TME, however, can be metabolically hostile due to insufficient vascular exchange and cancer cell metabolism that leads to hypoxia, depletion of nutrients, and accumulation of waste products. Further, inhibitory receptors present in the TME can inhibit T cell metabolism and alter T cell signaling both directly and through release of extracellular vesicles such as exosomes. This review will discuss the metabolic changes that drive T cells into different stages of their development and how the TME imposes barriers to the metabolism and activity of tumor infiltrating lymphocytes.

Keywords:  cancer; cancer biology; immunology; immunometabolism; immunotherapy; inflammation; t cells; tumor microenvironment; tumor-infiltrating lymphocytes

DOI:  https://doi.org/10.7554/eLife.55185
Anal Chem. 2020 May 04.

Microfluidic probe for in-situ extraction of adherent cancer cells to detect heterogeneity difference by electrospray ionization mass spectrometry.

Qiushi Huang, Mashooq Khan, Ning Xu, Hong-Ren Yao, Jin-Ming Lin.

The pathological studies of cancer tissues and cell molecular could provide early diagnosis for the treatment of cancer. In this work, we have designed a microfluidic surface extractor (MSE). MSE has been coupled with electrospray mass spectrometry (Extraction reagent, methanol; Optimum flow rate,0.5mL/h) to analyze the phospholipid content of different tumor cells. Three types of cancer cell lines including A549 cell, HepG2 cell, and U87 cell were investigated and the principle component analysis (PCA (LDA, PC1 97.2%; PC2, 2.8%)) was carried out to analyze the difference in the lipid contents. The LDA revealed heterogeneity among the cancer cells. The designed MSE could have potential applications in the clinical analysis of cancer tissue.

DOI: https://doi.org/10.1021/acs.analchem.0c01200
J Extracell Vesicles. 2020 ;9(1): 1750202

A circulating extracellular vesicles-based novel screening tool for colorectal cancer revealed by shotgun and data-independent acquisition mass spectrometry.

Xi Zheng, Kailun Xu, Biting Zhou, Ting Chen, Yanqin Huang, Qilong Li, Fei Wen, Weiting Ge, Jian Wang, Shaojun Yu, Lifeng Sun, Liang Zhu, Wei Liu, Huanhuan Gao, Liang Yue, Xue Cai, Qiushi Zhang, Guan Ruan, Tiansheng Zhu, Zhicheng Wu, Yi Zhu, Yingkuan Shao, Tiannan Guo, Shu Zheng.

  Background: Early screening for colorectal cancer (CRC) is essential to improve its prognosis. Liquid biopsies are increasingly being considered for diagnosing cancer due to low invasiveness and high reproducibility. In addition, circulating extracellular vesicles (crEVs, extracellular vesicles isolated from plasma) expressing tumour-specific proteins are potential biomarkers for various cancers. Here, we present a data-independent acquisition (DIA)-mass spectrometry (MS)-based diagnostic method for liquid biopsies. Methods: Extracellular vesicles (EVs) were isolated from culture supernatants of human CRC cell lines, and plasma of patients with CRC at different tumour stages, by overnight ultracentrifugation coupled with sucrose density gradient centrifugation. Tumour-specific EV proteins were prioritized using Tandem Mass Tag (TMT)-based shotgun proteomics and phosphoproteomics. The results were verified in a second independent cohort and a mouse tumour-bearing model using Western blotting (WB). The candidate biomarkers were further validated in a third cohort by DIA-MS. Finally, the DIA-MS methodology was accelerated to permit high-throughput detection of EV biomarkers in another independent cohort of patients with CRC and healthy controls. Results: High levels of total and phosphorylated fibronectin 1 (FN1) in crEVs, haptoglobin (HP), S100A9 and fibrinogen α chain (FGA) were significantly associated with cancer progression. FGA was the most dominant biomarker candidate. Analysis of the human CRC cell lines and the mouse model indicated that FGA+ crEVs were likely released by CRC cells. Furthermore, fast DIA-MS and parallel reaction monitoring (PRM)-MS both confirmed that FGA+ crEVs could distinguish colon adenoma with an area of curve (AUC) in the receiver operating characteristic (ROC) curve of 0.949 and patients with CRC (AUC of ROC is 1.000) from healthy individuals. The performance outperformed conventional tumour biomarkers. The DIA-MS quantification of FGA+ crEVs among three groups agreed with that from PRM-MS. Conclusion: DIA-MS detection of FGA+ crEVs is a potential rapid and non-invasive screening tool to identify early stage CRC. Abbreviations: FGA: fibrinogen α chain; CRC: colorectal cancer; crEVs: circulating extracellular vesicles; EV: extracellular vesicles;MS: mass spectrometry; WB: Western blotting; ROC: receiver operating characteristic; PRM: Parallel Reaction Monitoring; GPC1: Glypican-1; GO: Gene ontology; TEM: transmission electron microscopy; FN1: Fibronectin 1; HP: haptoglobin; TMT: Tandem Mass Tag; LC-MS/MS: liquid chromatography coupled to tandem mass spectrometry; DIA: data-independent acquisition; DDA: data-dependent acquisition; CiRT: Common internal Retention Time standards;AGC: Automatic gain control; AUC: area under curve.

Keywords:  Colorectal Cancer; DIA Mass Spectrometry; biomarker; extracellular vesicle; proteome

DOI:  https://doi.org/10.1080/20013078.2020.1750202
Adv Drug Deliv Rev. 2020 Apr 28. pii: S0169-409X(20)30029-6. [Epub ahead of print]

Sphingolipids as prognostic biomarkers of neurodegeneration, neuroinflammation, and psychiatric diseases and their emerging role in lipidomic investigation methods.

Daan van Kruining, Qian Luo, Gerhild van Echten-Deckert, Michelle M Mielke, Andrew Bowman, Shane Ellis, Tiago Gil Oliveira, Pilar Martinez-Martinez.

  Lipids play an important role in neurodegeneration, neuroinflammation, and psychiatric disorders and an imbalance in sphingolipid levels is associated to disease. Although early diagnosis and intervention of these disorders would clearly have favorable long-term outcomes, no diagnostic tests currently exist that can accurately identify people at risk. Reliable prognostic biomarkers that are easily accessible would be beneficial to determine therapy and treatment response in clinical trials. Recent advances in lipidomic investigation methods have greatly progressed the knowledge of sphingolipids in neurodegenerative and psychiatric disorders over the past decades although more longitudinal studies are needed to understand its exact role in these disorders to be used as potential tools in the clinic. In this review, we give an overview of the current knowledge of sphingolipids in neurodegenerative and psychiatric disorders and explore recent advances in investigation methods. Finally, the potential of sphingolipid metabolism products and signaling molecules as potential biomarkers for diagnosis, prognostic, or surrogate markers of treatment response is discussed.

Keywords:  Biomarkers; Lipidomics; Neurodegeneration; Neuroinflammation; Psychiatric diseases; Sphingolipids; Surrogate markers

DOI:  https://doi.org/10.1016/j.addr.2020.04.009
Methods Protoc. 2020 May 04. pii: E36. [Epub ahead of print]3(2):

High-Throughput Fingerprinting of Rhizobial Free Fatty Acids by Chemical Thin-Film Deposition and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Aleksey Gladchuk, Julia Shumilina, Alena Kusnetsova, Ksenia Bureiko, Susan Billig, Alexander Tsarev, Irina Alexandrova, Larisa Leonova, Vladimir A Zhukov, Igor A Tikhonovich, Claudia Birkemeyer, Ekaterina Podolskaya, Andrej Frolov.

  Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and signaling compounds in cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative content of free fatty acids (FFAs). In this context, nitrogen-fixing bacteria (order Rhizobiales), the symbionts of legumes, are particularly interesting. Indeed, the FA profiles influence the structure of rhizobial nodulation factors, required for successful infection of plant root. Although FA patterns can be assessed by gas chromatography-(GC-) and liquid chromatography-mass spectrometry (LC-MS), sample preparation for these methods is time-consuming and quantification suffers from compromised sensitivity, low stability of derivatives and artifacts. In contrast, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by combination of Langmuir technology and MALDI-TOF-MS featuring a high sensitivity, accuracy and precision of quantification. We describe a step-by-step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. As a case study, a comparison of the FFA metabolomes of two rhizobia species-Rhizobium leguminosarum and Sinorhizobium meliloti, demonstrates the analytical potential of the protocol.

Keywords:  Langmuir film technology; bacteria; barium monocarboxylates; chemical deposition technique; free fatty acids (FFAs); matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS); metabolic fingerprinting; rhizobia

DOI:  https://doi.org/10.3390/mps3020036
Cell Rep. 2020 May 05. pii: S2211-1247(20)30528-3. [Epub ahead of print]31(5): 107579

Secreted Phospholipase PLA2G2D Contributes to Metabolic Health by Mobilizing ω3 Polyunsaturated Fatty Acids in WAT.

Hiroyasu Sato, Yoshitaka Taketomi, Yoshimi Miki, Remi Murase, Kei Yamamoto, Makoto Murakami.

  Polyunsaturated fatty acids (PUFAs) confer health benefits by preventing inflammation and obesity and by increasing thermogenesis in brown and beige adipocytes. As well as being supplied exogenously as nutrients, PUFAs are largely stored in membrane glycerophospholipids and released by phospholipase A2s (PLA2s). However, the molecular identity of the PLA2 subtype(s) that supplies endogenous PUFAs for metabolic homeostasis remains unclear. Here we show that PLA2G2D, a secreted PLA2 isoform, is constitutively expressed in M2-type macrophages in white adipose tissue (WAT) and shows a reciprocal correlation with obesity. Studies using global and macrophage-specific Pla2g2d-deficient mice reveal that PLA2G2D increases energy expenditure and thermogenesis by facilitating adipocyte browning, thereby ameliorating diet-induced obesity, insulin resistance, and WAT inflammation. Mechanistically, PLA2G2D constitutively supplies a pool of PUFAs, ω3 in particular, in WAT. Thus, our present findings underscore the contribution of the macrophage-driven PLA2G2D-ω3 PUFA axis to metabolic health.

Keywords:  beige adipocyte; inflammation; lipidomics; macrophage; metabolic health; obesity; phospholipase A(2); polyunsaturated fatty acid; thermogenesis

DOI:  https://doi.org/10.1016/j.celrep.2020.107579
Signal Transduct Target Ther. 2020 May 08. 5(1): 51

Erianin, a novel dibenzyl compound in Dendrobium extract, inhibits lung cancer cell growth and migration via calcium/calmodulin-dependent ferroptosis.

Peng Chen, Qibiao Wu, Jiao Feng, Lili Yan, Yitian Sun, Shuiping Liu, Yu Xiang, Mingming Zhang, Ting Pan, Xiaying Chen, Ting Duan, Lijuan Zhai, Bingtao Zhai, Wengang Wang, Ruonan Zhang, Bi Chen, Xuemeng Han, Yicong Li, Liuxi Chen, Ying Liu, Xingxing Huang, Ting Jin, Wenzheng Zhang, Hong Luo, Xiaohui Chen, Yongqiang Li, Qiujie Li, Guohua Li, Qin Zhang, Lvjia Zhuo, Zuyi Yang, Huifen Tang, Tian Xie, Xiaoping Ouyang, Xinbing Sui.

Ferroptosis, a novel form of programmed cell death, is characterized by iron-dependent lipid peroxidation and has been shown to be involved in multiple diseases, including cancer. Stimulating ferroptosis in cancer cells may be a potential strategy for cancer therapy. Therefore, ferroptosis-inducing drugs are attracting more attention for cancer treatment. Here, we showed that erianin, a natural product isolated from Dendrobium chrysotoxum Lindl, exerted its anticancer activity by inducing cell death and inhibiting cell migration in lung cancer cells. Subsequently, we demonstrated for the first time that erianin induced ferroptotic cell death in lung cancer cells, which was accompanied by ROS accumulation, lipid peroxidation, and GSH depletion. The ferroptosis inhibitors Fer-1 and Lip-1 but not Z-VAD-FMK, CQ, or necrostatin-1 rescued erianin-induced cell death, indicating that ferroptosis contributed to erianin-induced cell death. Furthermore, we demonstrated that Ca2+/CaM signaling was a critical mediator of erianin-induced ferroptosis and that blockade of this signaling significantly rescued cell death induced by erianin treatment by suppressing ferroptosis. Taken together, our data suggest that the natural product erianin exerts its anticancer effects by inducing Ca2+/CaM-dependent ferroptosis and inhibiting cell migration, and erianin will hopefully serve as a prospective compound for lung cancer treatment.

DOI: https://doi.org/10.1038/s41392-020-0149-3
Chem Phys Lipids. 2020 Apr 28. pii: S0009-3084(20)30045-1. [Epub ahead of print] 104914

CDP-diacylglycerol, a critical intermediate in lipid metabolism.

William Jennings, Richard M Epand.

  The roles of lipids expand beyond the basic building blocks of biological membranes. In addition to forming complex and dynamic barriers, the thousands of different lipid species in the cell contribute to essentially all the processes of life. Specific lipids are increasingly identified in cellular processes, including signal transduction, membrane trafficking, metabolic control and protein regulation. Tight control of their synthesis and degradation is essential for homeostasis. Most of the lipid molecules in the cell originate from a small number of critical intermediates. Thus, regulating the synthesis of intermediates is essential for lipid homeostasis and optimal biological functions. Cytidine diphosphate diacylglycerol (CDP-DAG) is an intermediate which occupies a branch point in lipid metabolism. CDP-DAG is incorporated into different synthetic pathways to form distinct phospholipid end-products depending on its location of synthesis. Identification and characterization of CDP-DAG synthases which catalyze the synthesis of CDP-DAG has been hampered by difficulties extracting these membrane-bound enzymes for purification. Recent developments have clarified the cellular localization of the CDP-DAG synthases and identified a new unrelated CDP-DAG synthase enzyme. These findings have contributed to a deeper understanding of the extensive synthetic and signaling networks stemming from this key lipid intermediate.

Keywords:  CDP-DAG; Lipid biosynthesis; Lipid intermediates; Lipid transport; Membrane contact sites; Signaling lipids

DOI:  https://doi.org/10.1016/j.chemphyslip.2020.104914
Methods Enzymol. 2020 ;pii: S0076-6879(20)30088-4. [Epub ahead of print]637 309-340

Analysis of vitamin A and retinoids in biological matrices.

Lindsay C Czuba, Guo Zhong, King C Yabut, Nina Isoherranen.

  Vitamin A signaling pathways are predominantly driven by the cellular concentrations of all-trans-retinoic acid (atRA), as the main mechanism of retinoid signaling is via activation of retinoic acid receptors. atRA concentrations are in turn controlled by the storage of vitamin A and enzymatic processes that synthesize and clear atRA. This has resulted in the need for robust and highly specific analytical methods to accurately quantify retinoids in diverse biological matrices. Tissue-specific differences in both the quantity of retinoids and background matrix interferences can confound the quantification of retinoids, and the bioanalysis requires high performance instrumentation, such as liquid chromatography mass-spectrometry (LC-MS). Successful bioanalysis of retinoids is further complicated by the innate structural instability of retinoids and their relatively high lipophilicity. Further, in vitro experiments with retinoids require attention to experimental design and interpretation to account for the instability of retinoids due to isomerization and degradation, sequential metabolism to numerous structurally similar metabolites, and substrate depletion during experiments. In addition, in vitro biological activity is often confounded by residual presence of retinoids in common biological reagents such as cell culture media. This chapter identifies common biological and analytical complexities in retinoid bioanalysis in diverse biological matrices, and in the use of retinoids in cell culture and metabolic incubations. In addition, this chapter highlights best practices for the successful detection and quantification of the vitamin A metabolome in a wide range of biological matrices.

Keywords:  Liquid chromatography; Mass spectrometry; Retinoic acid; Retinoid; Retinoid bioanalysis

DOI:  https://doi.org/10.1016/bs.mie.2020.02.010
Am J Cancer Res. 2020 ;10(4): 1182-1193

Abrogation of ARF6 promotes RSL3-induced ferroptosis and mitigates gemcitabine resistance in pancreatic cancer cells.

Zeng Ye, Qiangsheng Hu, Qifeng Zhuo, Yuemeng Zhu, Guixiong Fan, Mengqi Liu, Qiqing Sun, Zheng Zhang, Wensheng Liu, Wenyan Xu, Shunrong Ji, Xianjun Yu, Xiaowu Xu, Yi Qin.

ADP Ribosylation Factor 6 (ARF6) is a part of the RAS superfamily and regulates vesicular trafficking, remodeling of membrane lipids, and signaling pathways. Our previous study has found that ARF6, functioned as a downstream of Kras/ERK signaling pathway, could promote proliferation and Warburg effect in pancreatic cancer cells. Moreover, ARF6 is promising to be a biomarker for predicting prognosis of pancreatic cancer. Ferroptosis is a new defined iron-dependent form of nonapoptotic cell death, which is closely related to Kras mutation. Therefore, it is urgent to further explore the relationship between ARF6 and ferroptosis. Our study demonstrated that ARF6 did not directly regulate lipid peroxidation, but endowed pancreatic cancer cells to a status that is sensitive to oxidative stress, especially RSL3-induced lipid peroxidation. Further study revealed that ARF6 could also regulate gemcitabine resistance via multiple pathways. In conclusion, ARF6 has a profound effect on pancreatic cancer development.

Keywords: ACSL4; ARF6; Ferroptosis; gemcitabine; pancreatic cancer
Exp Physiol. 2020 May 02.

Contemporary stable isotope tracer approaches: Insights into skeletal muscle metabolism in health and disease.

Matthew S Brook, Daniel J Wilkinson.

   NEW FINDINGS: What is the topic of this review? This review discusses the application of novel stable isotope tracer techniques in understanding the control of skeletal muscle mass What advances does it highlight? This review highlights current advances in stable isotope tracer techniques through their incorporation with high throughput proteomics technologies.
ABSTRACT: Beyond its primary locomotory and key structural functions, skeletal muscle provides additional vital roles for maintaining metabolic health, acting as a storage point for glucose and intramuscular lipids for energy production, alongside being the body's largest reservoir for amino acids. Therefore, maintenance of muscle mass is key to the promotion of health and wellbeing across the life span, and in number of different disease states. As such when skeletal muscle is lost, in either clinical (cancer, organ failure etc.) or non-clinical (ageing, inactivity) situations, there are potentially devastating consequences attached, with robust links being shown between muscle mass loss and mortality. Great efforts are being made to reverse or slow muscle mass declines in health and disease, through combinations of lifestyle changes, nutritional and/or pharmaceutical intervention. However, despite this comprehensive research effort, the underlying metabolic and molecular mechanisms have yet to be properly defined. However, with the rapid acceleration of analytical developments over recent years, the application of stable isotope tracers to the study of human muscle metabolism are providing unique insights into the mechanisms controlling skeletal muscle loss and allowing more targeted therapeutic strategies to be developed. This review aims to highlight the technical breakthroughs in our understanding of muscle wasting in health and disease, and how future directions and developments incorporating OMICs with stable isotope tracers will allow for a more personalized and stratified therapeutic approach. This article is protected by copyright. All rights reserved.

Keywords:  muscle; protein turnover; stable isotope tracers

DOI:  https://doi.org/10.1113/EP087492
Anal Chem. 2020 May 06.

PiTMaP: A new analytical platform for high-throughput direct metabolome analysis by probe electrospray ionization/tandem mass spectrometry using an R software-based data pipeline.

Kei Zaitsu, Seiichiro Eguchi, Tomomi Ohara, Kenta Kondo, Akira Ishii, Hitoshi Tsuchihashi, Takakazu Kawamata, Akira Iguchi.

A new analytical platform called PiTMaP was developed for high-throughput direct metabolome analysis by probe electrospray ionization/tandem mass spectrometry (PESI/MS/MS) using an R software-based data pipeline. PESI/MS/MS was used as the data acquisition technique, applying a scheduled-selected reaction monitoring method to expand the targeted metabolites. Seventy-two metabolites mainly related to the central energy metabolism were selected; data acquisition time was optimized using mouse liver and brain samples, indicating that the 2.4 min data acquisition method had a higher repeatability than the 1.2 min and 4.8 min methods. A data pipeline was constructed using the R software, and it was proven that it can i) automatically generate box-and-whisker plots for all metabolites, ii) perform multivariate analyses such as principal component analysis (PCA) and projections to latent structures-discriminant analysis (PLS-DA), iii) generate score and loading plots of PCA and PLS-DA, iv) calculate variable importance of projection (VIP) values, v) determine a statistical family by VIP value criterion, vi) perform tests of significance with the false discovery rate (FDR) correction method, and vii) draw box-and-whisker plots only for significantly changed metabolites. These tasks could be completed within ca. 1 min. Finally, PiTMaP was applied to two cases: 1) an acetaminophen (APAP)-induced acute liver injury model and control mice and 2) human meningioma samples with different grades (G1-G3), demonstrating the feasibility of PiTMaP. PiTMaP was found to perform data acquisition without tedious sample preparation and a post-hoc data analysis within ca. 1 min. Thus, it would be a universal platform to perform rapid metabolic profiling of biological samples.

DOI: https://doi.org/10.1021/acs.analchem.0c01271
Cell Death Dis. 2020 May 04. 11(5): 312

The heterocyclic compound Tempol inhibits the growth of cancer cells by interfering with glutamine metabolism.

Shuangyan Ye, Pengfei Xu, Mengqiu Huang, Xi Chen, Sisi Zeng, Qianli Wang, Jianping Chen, Keyi Li, Wenwen Gao, Ruiyuan Liu, Jingxian Liu, Yihao Shao, Hui Zhang, Yang Xu, Qianbing Zhang, Zhuo Zhong, Zibo Wei, Jiale Wang, Bingtao Hao, Wenhua Huang, Qiuzhen Liu.

Tempol (4-hydroxy-2,2,6,6-Tetramethylpiperidine-1-oxyl, TPL), a nitroxide compound, inhibits proliferation and increases the vulnerability of cancer cells to apoptosis induced by cytotoxic agents. However, the molecular mechanism of TPL inhibiting cancer cell proliferation has not been fully understood. In this study, we evaluated the metabolic effect of TPL on cancer cells and explored its cancer therapeutic potential. Extracellular flow assays showed that TPL inhibited cellular basal and maximal oxygen consumption rates of mitochondrial. 13C metabolic flux analysis showed that TPL treatment had minimal effect on glycolysis. However, we found that TPL inhibits glutamine metabolism by interfering with the oxidative tricarboxylic acid cycle (TCA) process and reductive glutamine process. We found that the inhibitory effect of TPL on metabolism occurs mainly on the step from citrate to α-ketoglutarate or vice versa. We also found that activity of isocitrate dehydrogenase IDH1 and IDH2, the key enzymes in TCA, were inhibited by TPL treatment. In xenograft mouse model, TPL treatment reduced tumor growth by inhibiting cellular proliferation of xenograft tumors. Thus, we provided a mechanism of TPL inhibiting cancer cell proliferation by interfering with glutamine utilization that is important for survival and proliferation of cancer cells. The study may help the development of a therapeutic strategy of TPL combined with other anticancer medicines.

DOI: https://doi.org/10.1038/s41419-020-2499-8
J Chromatogr A. 2020 Apr 17. pii: S0021-9673(20)30364-2. [Epub ahead of print] 461135

Direct liquid chromatography tandem mass spectrometry analysis of amino acids in human plasma.

Aurore Desmons, Elizabeth Thioulouse, Jean-Yves Hautem, Anne Saintier, Bruno Baudin, Antonin Lamazière, Claude Netter, Fathi Moussa.

  Here we describe a new HPLC-MS/MS method using a mixed mode stationary phase and a binary gradient of elution for the rapid separation and quantification of AAs in human plasma without derivatization or ion pairing reagent addition. The sample preparation procedure consists in a single dilution step after protein precipitation with sulfosalicylic acid. The proposed method allows for the unambiguous identification and analysis of 52 AAs and related compounds including the separation of isomers and isobars in an 18 min chromatographic run including the conditioning and the equilibration times. AAs were detected by selective reaction monitoring. Internal calibration was used for the quantification of 37 AAs, including 25 using the corresponding isotopically labeled internal standards. External calibration (no internal standard) was used for five additional analytes. Qualitative detection was achieved for the remaining compounds. Validation studies evaluated accuracy, linearity, within- and between-run precision, lower limits of detection and quantification for 37 amino acids present in commonly used quality control samples. For within-run precision CVs averaged 3.8 % (n = 30) for all compounds. For between-run precision, CVs averaged 8.6 % for all compounds (n = 20). Correlation with the common standard ion-exchange chromatography with post-column derivatization method was also performed for 32 plasma samples. While the proposed method is at least 50 times more sensitive, the data showed good correlation with slopes equal or higher than 0.9 and correlation coefficients mostly higher than 0.90. The method was successfully applied for analysis of plasma samples for detection of inherited disorders of amino acid metabolism.

Keywords:  Amino acid profiling; Human plasma; LC-MS/MS; Mixed-mode stationary phases

DOI:  https://doi.org/10.1016/j.chroma.2020.461135
Analyst. 2020 May 06.

Scientific workflow managers in metabolomics: an overview.

Aswin Verhoeven, Martin Giera, Oleg A Mayboroda.

Providing maximum information on the provenance of scientific results in life sciences is getting considerable attention since the widely publicized reproducibility crisis. Improving the reproducibility of data processing and analysis workflows is part of this movement and may help achieve clinical deployment quicker. Scientific workflow managers can be valuable tools towards achieving this goal. Although these platforms are already well established in the field of genomics and other omics fields, in metabolomics scripts and dedicated software packages are still more popular. However, versatile workflows for metabolomics exist in the KNIME and Galaxy platforms. We will here summarize the available options of scientific workflow managers dedicated to metabolomics analysis.

DOI: https://doi.org/10.1039/d0an00272k
Nat Commun. 2020 May 08. 11(1): 2284

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing.

K K Khoo, I Galleano, F Gasparri, R Wieneke, H Harms, M H Poulsen, H C Chua, M Wulf, R Tampé, S A Pless.

Manipulation of proteins by chemical modification is a powerful way to decipher their function. However, most ribosome-dependent and semi-synthetic methods have limitations in the number and type of modifications that can be introduced, especially in live cells. Here, we present an approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into proteins expressed in eukaryotic cells. We insert synthetic peptides into GFP, NaV1.5 and P2X2 receptors via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating protein function. We anticipate the approach will overcome some drawbacks of existing protein enigineering methods.

DOI: https://doi.org/10.1038/s41467-020-16208-6
Biochem J. 2020 May 07. pii: BCJ20200275. [Epub ahead of print]

Synergism between SLC6A14 blockade and gemcitabine in pancreactic cancer: A 1H-NMR-based metabolomic study in pancreatic cancer cells.

Aimin Cai, Hailun Zheng, Zhiwei Chen, Xinlu Lin, Chen Li, Qing Yao, Yangzom Doma Bhutia, Vadivel Ganapathy, Ruijie Chen, Longfa Kou.

  Gemcitabine is the first-line chemotherapy for pancreatic cancer. To overcome the often-acquired gemcitabine resistance, other drugs are used in combination with gemcitabine. It is well-known that cancer cells reprogram cellular metabolism, coupled with the upregulation of selective nutrient transporters to feed into the altered metabolic pathways. Our previous studies have demonstrated that the amino acid transporter SLC6A14 is markedly upregulated in pancreatic cancer and that it is a viable therapeutic target. α-Methyltryptophan (α-MT) is a blocker of SLC6A14 and is effective against pancreatic cancer in vitro and in vivo. In the present study, we tested the hypothesis that α-MT could synergize with gemcitabine in the treatment of pancreatic cancer. We investigated the effects of combination of α-MT and gemcitabine on proliferation, migration, and apoptosis in a human pancreatic cancer cell line, and examined the underlying mechanisms using 1H-NMR-based metabolomic analysis. These studies examined the intracellular metabolite profile and the extracellular metabolite profile separately. Combination of α-MT with gemcitabine elicited marked changes in a wide variety of metabolic pathways, particularly amino acid metabolism with notable alterations in pathways involving tryptophan, branched-chain amino acids, ketone bodies, and membrane phospholipids. The metabolomic profiles of untreated control cells and cells treated with gemcitabine or α-MT were distinctly separable, and the combination regimen showed a certain extent of overlap with the individual α-MT and gemcitabine groups. This represents the first study detailing the metabolomic basis of the anti-cancer efficacy of gemcitabine, α-MT and their combination.

Keywords:  1H-NMR-based metabolomics; SLC6A14; combination therapy; gemcitabine; pancreatic cancer; α-methyltryptophan

DOI:  https://doi.org/10.1042/BCJ20200275
J Lipid Res. 2020 May 05. pii: jlr.RA119000606. [Epub ahead of print]

Mass spectrometry imaging and LC-MS reveal decreased cerebellar phosphoinositides in Niemann-Pick type C1-null mice.

Koralege C Pathmasiri, Melissa R Pergande, Fernando Tobias, Rima Rebiai, Avia Rosenhouse-Dantsker, Ernesto R Bongarzone, Stephanie M Cologna.

  Niemann-Pick disease, type C1 (NPC1) is a lipid storage disorder in which cholesterol and glycosphingolipids accumulate in late endosomal/lysosomal compartments because of mutations in the NPC1 gene. A hallmark of NPC1 is progressive neurodegeneration of the cerebellum as well as visceral organ damage; however, the mechanisms driving this disease pathology are not fully understood. Phosphoinositides are phospholipids that play distinct roles in signal transduction and vesicle trafficking. Here, we utilized consensus spectra analysis of MS imaging datasets and orthogonal LC-MS analyses to evaluate the spatial distribution of phosphoinositides and quantify them in cerebellar tissue from Npc1-null mice. Our results suggest significant depletion of multiple phosphoinositide species, including phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP), and bisphosphate (PIP2), in the cerebellum of the Npc1-null mice in both whole-tissue lysates and myelin-enriched fractions. Additionally, we observed altered levels of the regulatory enzyme phosphatidylinositol 4-kinase type 2 α (PI4K2A) in Npc1-null mice. In contrast, the levels of related kinases, phosphatases, and transfer proteins were unaltered in the Npc1-null mouse model as observed by Western blot analysis. Our discovery of phosphoinositide lipid biomarkers for NPC1 opens new perspectives on the pathophysiology underlying this fatal neurodegenerative disease.

Keywords:  Cholesterol; Genetics; Lipidomics; Mass spectrometry; Niemann-Pick disease; lysosomal storage disorder; mass spectrometry imaging; neurodegeneration; phosphoinositide signaling; phospholipid

DOI:  https://doi.org/10.1194/jlr.RA119000606