bims-mascan 2021-04-18 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2021‒04‒18
twenty-one papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh

Ferroptotic cell death triggered by conjugated linolenic acids is mediated by ACSL1.
Role of LncRNAs in regulating cancer amino acid metabolism.
Lessons from Cancer Metabolism for Pulmonary Arterial Hypertension and Fibrosis.
Changes in phospholipid metabolism in exosomes of hormone-sensitive and hormone-resistant prostate cancer cells.
Perspective on Emerging Mass Spectrometry Technologies for Comprehensive Lipid Structural Elucidation.
Targeted urine metabolomics with a graphical reporting tool for rapid diagnosis of inborn errors of metabolism.
Investigation of Obesity-Alleviation Effect of Eurycoma longifolia on Mice Fed with a High-Fat Diet through Metabolomics Revealed Enhanced Decomposition and Inhibition of Accumulation of Lipids.
Defining intermediates and redundancies in coenzyme Q precursor biosynthesis.
Novel Strategy for Untargeted Chiral Metabolomics using Liquid Chromatography-High Resolution Tandem Mass Spectrometry.
Isotope tracing reveals glycolysis and oxidative metabolism in childhood tumors of multiple histologies.
Spectrally Accurate Quantitative Analysis of Isotope Labeled Compounds.
Metabolomics bridging proteomics along metabolites/oncometabolites and protein modifications: Paving the way toward integrative multiomics.
The role of S-nitrosylation of PFKM in regulation of glycolysis in ovarian cancer cells.
Optimizing sulfate pyrolysis triple oxygen isotope analysis for samples from desert environments.
An improved strategy for analysis of lipid molecules utilising a reversed phase C30 UHPLC column and scheduled MS/MS acquisition.
A methionine-Mettl3-N6-methyladenosine axis promotes polycystic kidney disease.
The lipidome in nonalcoholic fatty liver disease: Actionable targets.
Loss of function of lysosomal acid lipase (LAL) profoundly impacts osteoblastogenesis and increases fracture risk in humans.
Sensitive immunopeptidomics by leveraging available large-scale multi-HLA spectral libraries, data-independent acquisition and MS/MS prediction.
The mTORC1 pathway stimulates glutamine metabolism and cell proliferation by repressing SIRT4.
Proteome characterisation of extracellular vesicles isolated from heart.

Nat Commun. 2021 04 14. 12(1): 2244

Ferroptotic cell death triggered by conjugated linolenic acids is mediated by ACSL1.

Alexander Beatty, Tanu Singh, Yulia Y Tyurina, Vladimir A Tyurin, Svetlana Samovich, Emmanuelle Nicolas, Kristen Maslar, Yan Zhou, Kathy Q Cai, Yinfei Tan, Sebastian Doll, Marcus Conrad, Aravind Subramanian, Hülya Bayır, Valerian E Kagan, Ulrike Rennefahrt, Jeffrey R Peterson.

Ferroptosis is associated with lipid hydroperoxides generated by the oxidation of polyunsaturated acyl chains. Lipid hydroperoxides are reduced by glutathione peroxidase 4 (GPX4) and GPX4 inhibitors induce ferroptosis. However, the therapeutic potential of triggering ferroptosis in cancer cells with polyunsaturated fatty acids is unknown. Here, we identify conjugated linoleates including α-eleostearic acid (αESA) as ferroptosis inducers. αESA does not alter GPX4 activity but is incorporated into cellular lipids and promotes lipid peroxidation and cell death in diverse cancer cell types. αESA-triggered death is mediated by acyl-CoA synthetase long-chain isoform 1, which promotes αESA incorporation into neutral lipids including triacylglycerols. Interfering with triacylglycerol biosynthesis suppresses ferroptosis triggered by αESA but not by GPX4 inhibition. Oral administration of tung oil, naturally rich in αESA, to mice limits tumor growth and metastasis with transcriptional changes consistent with ferroptosis. Overall, these findings illuminate a potential approach to ferroptosis, complementary to GPX4 inhibition.

DOI: https://doi.org/10.1038/s41467-021-22471-y
Cancer Cell Int. 2021 Apr 13. 21(1): 209

Role of LncRNAs in regulating cancer amino acid metabolism.

Yuhong Guo, Bin Lv, Renfeng Liu, Zhengzai Dai, Feifei Zhang, Yiping Liang, Bo Yu, Duo Zeng, Xiao-Bin Lv, Zhiping Zhang.

  The metabolic change of tumor cells is an extremely complicated process that involves the intersection and integration of various signal pathways. Compared with normal tissues, cancer cells show distinguished metabolic characteristics called metabolic reprogramming, which has been considered as a sign of cancer occurrence. With the deepening of tumor research in recent years, people gradually found that amino acid metabolism played crucial roles in cancer progression. Long non-coding RNAs (lncRNAs), which are implicated in many important biological processes, were firstly discovered dysregulating in cancer tissues and participating in extensive regulation of tumorigenesis. This review focuses on the reprogramming of amino acid metabolism in cancers and how lncRNAs participate in the regulatory network by interacting with other macromolecular substances. Understanding the functions of lncRNA in amino acid reprogramming in tumors might provide a new vision on the mechanisms of tumorigenesis and the development of new approaches for cancer therapy.

Keywords:  Amino acid; Cancer; Glutamine; Metabolism; lncRNA

DOI:  https://doi.org/10.1186/s12935-021-01926-8
Am J Respir Cell Mol Biol. 2021 Apr 12.

Lessons from Cancer Metabolism for Pulmonary Arterial Hypertension and Fibrosis.

SeungHye Han, Navdeep S Chandel.

  Metabolism is essential for a living-organism to sustain life. It provides energy to a cell by breaking down compounds (catabolism), and supplies building blocks for the synthesis of macromolecules (anabolism). Signal transduction pathways tightly regulate mammalian cellular metabolism. Simultaneously, metabolism itself serves as a signaling pathway to control many cellular processes such as proliferation, differentiation, cell death, gene expression, and adaptation to stress. Considerable progress in the metabolism field has come from understanding how cancer cells co-opt metabolic pathways for growth and survival. Recent data also show that several metabolic pathways may participate in the pathogenesis of lung diseases, some of which could be promising therapeutic targets. In this translational review, we will outline the basic metabolic principles learned from the cancer metabolism field as it applies to the pathogenesis of pulmonary arterial hypertension and fibrosis with an emphasis on therapeutic potential.

Keywords:  Metabolism; metformin; pulmonary arterial hypertension; pulmonary fibrosis

DOI:  https://doi.org/10.1165/rcmb.2020-0550TR
J Cancer. 2021 ;12(10): 2893-2902

Changes in phospholipid metabolism in exosomes of hormone-sensitive and hormone-resistant prostate cancer cells.

Xianlin Yi, You Li, XiaoGang Hu, FuBing Wang, Tiangang Liu.

  Background: To explore the changes in lipids in exosomes of hormone-sensitive and hormone-resistant prostate cancer cells and develop an inexpensive and rapid technique for screening lipid-based biomarkers of prostate cancer. Methods: Exosomes were extracted from LnCap, PC3 and DU-145 cells, and their lipid composition was analyzed quantitatively using high-throughput mass spectrometry. Exosomes released by LnCap prostate cancer cells were also purified using a modified procedure based on polyethylene glycol (PEG) precipitation. Results: Exosomes extracted from LnCap cells contained higher proportions of phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl inositol lipids than whole LnCap cells. Lysophosphatidylcholine, a harmful intermediate product of phosphatidylcholine metabolism in vivo, was not found in LnCap cells but in exosomes. Phospholipids were different in exosomes from LnCap, PC3 and DU-145 prostate cancer cells. The main lipid pathways involved, i.e., glycerophospholipid metabolism, autophagy, and ferroptosis pathways, were also different in these cells. Exosomes isolated by this modified PEG precipitation technique were similar in purity to those obtained using a commercial kit. Conclusions: This study demonstrates that phosphatidylcholine and its harmful product lysophosphatidylcholine may play important roles in hormone-sensitive prostate cancer. Phospholipid exosome metabolism was changed in hormone-sensitive and hormone-resistant prostate cancer cells. The LPC, lipid pathway of autophagy and ferroptosis may act as therapeutic targets. The possibility of purifying prostate cancer cell exosomes using modified PEG precipitation is suitable for cancer screening.

Keywords:  lipidomics, exosome; prostate cancer

DOI:  https://doi.org/10.7150/jca.48906
Anal Chem. 2021 Apr 15.

Perspective on Emerging Mass Spectrometry Technologies for Comprehensive Lipid Structural Elucidation.

Julia R Bonney, Boone M Prentice.

Lipids and metabolites are of interest in many clinical and research settings because it is the metabolome that is increasingly recognized as a more dynamic and sensitive molecular measure of phenotype. The enormous diversity of lipid structures and the importance of biological structure-function relationships in a wide variety of applications makes accurate identification a challenging yet crucial area of research in the lipid community. Indeed, subtle differences in the chemical structures of lipids can have important implications in cellular metabolism and many disease pathologies. The speed, sensitivity, and molecular specificity afforded by modern mass spectrometry has led to its widespread adoption in the field of lipidomics on many different instrument platforms and experimental workflows. However, unambiguous and complete structural identification of lipids by mass spectrometry remains challenging. Increasingly sophisticated tandem mass spectrometry (MS/MS) approaches are now being developed and seamlessly integrated into lipidomics workflows to meet this challenge. These approaches generally either (i) alter the type of ion that is interrogated or (ii) alter the dissociation method in order to improve the structural information obtained from the MS/MS experiment. In this Perspective, we highlight recent advances in both ion type alteration and ion dissociation methods for lipid identification by mass spectrometry. This discussion is aimed to engage investigators involved in fundamental ion chemistry and technology developments as well as practitioners of lipidomics and its many applications. The rapid rate of technology development in recent years has accelerated and strengthened the ties between these two research communities. We identify the common characteristics and practical figures of merit of these emerging approaches and discuss ways these may catalyze future directions of lipid structural elucidation research.

DOI: https://doi.org/10.1021/acs.analchem.1c00061
J Inherit Metab Dis. 2021 Apr 11.

Targeted urine metabolomics with a graphical reporting tool for rapid diagnosis of inborn errors of metabolism.

Laura Km Steinbusch, Ping Wang, Huub Wah Waterval, Fons Apm Stassen, Karlien Lm Coene, Udo Fh Engelke, Daphna Dj Habets, Jörgen Bierau, Irene Mlw Körver-Keularts.

The current diagnostic work-up of inborn errors of metabolism (IEM) is rapidly moving towards integrative analytical approaches. We aimed to develop an innovative, targeted urine metabolomics (TUM) screening procedure to accelerate the diagnosis of patients with IEM. Urinary samples, spiked with 3 stable isotope-labelled internal standards, were analysed for 258 diagnostic metabolites with an UHPLC-QTOF-MS configuration run in positive and negative ESI modes. The software automatically annotated peaks, corrected for peak overloading, and reported peak quality and shifting. Robustness and reproducibility were satisfactory for most metabolites. Z-scores were calculated against 4 age-group matched control cohorts. Disease phenotypes were scored based on database metabolite matching. Graphical reports comprised a needle plot, annotating abnormal metabolites, and a heatmap showing the prioritised disease phenotypes. In the clinical validation we analysed samples of 289 patients covering 78 OMIM phenotypes from 12 of the 15 SSIEM disease groups. The disease groups include disorders in the metabolism of amino acids, fatty acids, ketones, purines and pyrimidines, carbohydrates, porphyrias, neurotransmitters, vitamins, cofactors, and creatine. The reporting tool easily and correctly diagnosed most samples. Even subtle aberrant metabolite patterns as seen in mild multiple acyl-CoA dehydrogenase deficiency (, GAII) and maple syrup urine disease (MSUD) were correctly called without difficulty. Others, like creatine transporter deficiency, are illustrative of IEM that remain difficult to diagnose. We present TUM as a powerful diagnostic screening tool that that merges most urinary diagnostic assays expediting the diagnostics for patients suspected of an IEM. This article is protected by copyright. All rights reserved.

DOI: https://doi.org/10.1002/jimd.12385
J Proteome Res. 2021 Apr 15.

Investigation of Obesity-Alleviation Effect of Eurycoma longifolia on Mice Fed with a High-Fat Diet through Metabolomics Revealed Enhanced Decomposition and Inhibition of Accumulation of Lipids.

Dingkun Zhang, Wen Zheng, Xin Li, Ge Liang, Nan Ye, Yueqiu Liu, Ang Li, Xin Liu, Rui Zhang, Jingqiu Cheng, Hao Yang, Meng Gong.

  The metabolic and bioactivity effects of Eurycoma longifolia (Eucalyptus longifolia) in obesity treatment were studied in mice fed with a high-fat diet using a metabolomics approach. Aqueous extracts of E. longifolia were obtained via grinding, dissolving, and freeze-drying. The hepatic steatosis effect of E. longifolia was characterized by hematoxylin and eosin histological staining. External performance of the obesity-alleviation effect was monitored by measuring body and food weight. In addition, the metabolomics analysis of the E. longifolia-mice interaction system was performed using the established platform combining liquid chromatography-tandem mass spectrometry with statistical analysis. The presence and spatial distribution patterns of differential molecules were further evaluated through desorption electrospray ionization-mass spectrometry imaging. The results showed that E. longifolia played a vital role in downregulating lipid accumulation (especially triacylglycerols) and fatty acids biosynthesis together with enhanced lipid decomposition and healing in Bagg albino mice. During such a process, E. longifolia mainly induced metabolomic alterations of amino acids, organic acids, phospholipids, and glycerolipids. Moreover, under the experimental concentrations, E. longifolia induced more fluctuations of aqueous-soluble metabolites in the plasma and lipids in the liver than in the kidneys. This study provides an advanced alternative to traditional E. longifolia-based studies for evaluating the metabolic effects and bioactivity of E. longifolia through metabolomics technology, revealing potential technological improvement and clinical application.

Keywords:  Bagg albino mice; Eurycoma longifolia; bioactivity assessment; metabolite analysis; metabolomics approach

DOI:  https://doi.org/10.1021/acs.jproteome.1c00015
J Biol Chem. 2021 Apr 14. pii: S0021-9258(21)00429-4. [Epub ahead of print] 100643

Defining intermediates and redundancies in coenzyme Q precursor biosynthesis.

Kyle P Robinson, Adam Jochem, Sheila E Johnson, Thiruchelvi R Reddy, Jason D Russell, Joshua J Coon, David J Pagliarini.

  Coenzyme Q (CoQ), a redox-active lipid essential for oxidative phosphorylation, is synthesized by virtually all cells, but how eukaryotes make the universal CoQ head group precursor 4-hydroxybenzoate (4-HB) from tyrosine is unknown. The first and last steps of this pathway have been defined in Saccharomyces cerevisiae, but the intermediates and enzymes involved in converting 4-hydroxyphenylpyruvate (4-HPP) to 4-hydroxybenzaldehyde (4-HBz) have not been described. Here, we interrogate this pathway with genetic screens, targeted LC-MS, and chemical genetics. We identify three redundant aminotransferases (Bna3, Bat2, and Aat2) that support CoQ biosynthesis in the absence of the established pathway tyrosine aminotransferases, Aro8 and Aro9. We use isotope labeling to identify bona fide tyrosine catabolites, including 4-hydroxyphenylacetate (4-HPA) and 4-hydroxyphenyllactate (4-HPL). Additionally, we find multiple compounds that rescue this pathway when exogenously supplemented, most notably 4-hydroxyphenylacetaldehyde (4-HPAA) and 4-hydroxymandelate (4-HMA). Finally, we show that the Ehrlich pathway decarboxylase Aro10 is dispensable for 4-HB production. These results define new features of 4-HB synthesis in yeast, demonstrate the redundant nature of this pathway, and provide a foundation for further study.

Keywords:  4-hydroxybenzoate; Saccharomyces cerevisiae; biosynthesis; coenzyme Q; mass spectrometry (MS); metabolism; ubiquinone

DOI:  https://doi.org/10.1016/j.jbc.2021.100643
Anal Chem. 2021 Apr 13. 93(14): 5805-5814

Novel Strategy for Untargeted Chiral Metabolomics using Liquid Chromatography-High Resolution Tandem Mass Spectrometry.

Renu Pandey, Meghan Collins, Xiyuan Lu, Shannon R Sweeney, Jennifer Chiou, Alessia Lodi, Stefano Tiziani.

Stereospecific recognition of metabolites plays a significant role in the detection of potential disease biomarkers thereby providing new insights in diagnosis and prognosis. D-Hdroxy/amino acids are recognized as potential biomarkers in several metabolic disorders. Despite continuous advances in metabolomics technologies, the simultaneous measurement of different classes of enantiomeric metabolites in a single analytical run remains challenging. Here, we develop a novel strategy for untargeted chiral metabolomics of hydroxy/amine groups (-OH/-NH2) containing metabolites, including all hydroxy acids (HAs) and amino acids (AAs), by chiral derivatization coupled with liquid chromatography-high resolution tandem mass spectrometry (LC-HR-MS/MS). Diacetyl-tartaric anhydride (DATAN) was used for the simultaneous derivatization of-OH/-NH2 containing metabolites as well as the resulting diastereomers, and all the derivatized metabolites were resolved in a single analytical run. Data independent MS/MS acquisition (DIA) was applied to positively identify DATAN-labeled metabolites based on reagent specific diagnostic fragment ions. We discriminated chiral from achiral metabolites based on the reversal of elution order of D and L isomers derivatized with the enantiomeric pair (±) of DATAN in an untargeted manner. Using the developed strategy, a library of 301 standards that consisted of 214 chiral and 87 achiral metabolites were separated and detected in a single analytical run. This approach was then applied to investigate the enantioselective metabolic profile of the bone marrow (BM) and peripheral blood (PB) plasma samples from patients with acute myeloid leukemia (AML) at diagnosis and following completion of the induction phase of chemotherapeutic treatment. The sensitivity and selectivity of the developed method enabled the detection of trace levels of the D-enantiomer of HAs and AAs in primary plasma patient samples. Several of these metabolites were significantly altered in response to chemotherapy. The developed LC-HR-MS method entails a valuable step forward in chiral metabolomics.

DOI: https://doi.org/10.1021/acs.analchem.0c05325
Med (N Y). 2021 Apr 09. 2(4): 395-410

Isotope tracing reveals glycolysis and oxidative metabolism in childhood tumors of multiple histologies.

Kendra Johnston, Panayotis Pachnis, Alpaslan Tasdogan, Brandon Faubert, Lauren G Zacharias, Hieu Sy Vu, Laurie Rodgers-Augustyniak, Allison Johnson, Fang Huang, Sean Ricciardio, Zhiyu Zhao, Thomas P Mathews, Tanya Watt, Patrick Leavey, Ralph J DeBerardinis.

  Background: Survival among children with high-risk solid tumors remains poor. Reprogrammed metabolism promotes tumor growth and may contain therapeutic liabilities. Tumor metabolism has been assessed in adults using intra-operative 13C-glucose infusions. Pediatric tumors differ from adult cancers in their low mutational burden and derivation from embryonic tissues. Here we used 13C infusions to examine tumor metabolism in children, comparing phenotypes among tumor types and between childhood and adult cancers.Methods: Patients recruited to study NCT03686566 received an intra-operative infusion of [U-13C]glucose during tumor resection to evaluate central carbon pathways in the tumor, with concurrent metabolomics to provide a broad overview of metabolism. Differential characteristics were determined using multiple comparison tests and mixed effect analyses.
Findings: We studied 23 tumors from 22 patients. All tumors analyzed by [U-13C]glucose contained labeling in glycolytic and tricarboxylic acid (TCA) cycle intermediates. Labeling in the TCA cycle indicated activity of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), with PDH predominating. Neuroblastomas had high lactate labeling relative to other childhood cancers and lung cancer, and were distinguished by abundant tyrosine catabolites consistent with catecholamine synthesis.
Conclusions: Intra-operative [U13C]glucose infusions are safe and informative in pediatric cancer. Tumors of various histologies use glycolysis and oxidative metabolism, with subtype-selective differences evident from this small cohort. Expanding this cohort may uncover predictive biomarkers and therapeutic targets from tumor metabolism.
Funding: N.C.I grants to P.L. (R21CA220090-01A1) and R.J.D. (R35CA22044901); H.H.M.I. funding to R.J.D.; Children's Clinical Research Advisory Committee funding to K.J.

Keywords:  Cancer; glucose; isotopes; metabolism; metabolomics; neuroblastoma; sarcoma

DOI:  https://doi.org/10.1016/j.medj.2021.01.002
Rapid Commun Mass Spectrom. 2021 Apr 13. e9103

Spectrally Accurate Quantitative Analysis of Isotope Labeled Compounds.

Don Kuehl, Yongdong Wang, Peter L Wang, Dawei Zhou.

Calculated profile mode mass spectrometry (MS) data are fitted to lineshape calibrated LC/MS data using a Multiple Linear Regression (MLR) model to quantitate the relative concentrations of stable or radio labeled compound mixtures. This alternative approach significantly improves the precision and accuracy over existing MS methods while providing the much-needed statistical diagnostics on the goodness of the fitting model and thus reliability of the quantitative results obtained. Test compound data containing S/Cl atoms have been measured with either stable deuterium labeling or radioisotope uniform 14 C labeling onto an aromatic ring. Since the entire relative distribution of variously labeled compounds is automatically obtained through this approach, it is feasible to directly calculate the Specific Activity (SA) from such mass spectral analysis without radioactivity detection and the usual standard curve quantitation. The applicability of this approach to systematically and accurately accommodate and account for incomplete labeling chemistry or other impurities is also discussed, with wide-ranging implications including metabolic flux, HDX (Hydrogen-Deuterium Exchange), and quantitative proteomics.

DOI: https://doi.org/10.1002/rcm.9103
J Pharm Biomed Anal. 2021 Mar 19. pii: S0731-7085(21)00143-6. [Epub ahead of print]199 114031

Metabolomics bridging proteomics along metabolites/oncometabolites and protein modifications: Paving the way toward integrative multiomics.

Sinem Nalbantoglu, Abdullah Karadag.

  Systems biology adopted functional and integrative multiomics approaches enable to discover the whole set of interacting regulatory components such as genes, transcripts, proteins, metabolites, and metabolite dependent protein modifications. This interactome build up the midpoint of protein-protein/PTM, protein-DNA/RNA, and protein-metabolite network in a cell. As the key drivers in cellular metabolism, metabolites are precursors and regulators of protein post-translational modifications [PTMs] that affect protein diversity and functionality. The precisely orchestrated core pattern of metabolic networks refer to paradigm 'metabolites regulate PTMs, PTMs regulate enzymes, and enzymes modulate metabolites' through a multitude of feedback and feed-forward pathway loops. The concept represents a flawless PTM-metabolite-enzyme(protein) regulomics underlined in reprogramming cancer metabolism. Immense interconnectivity of those biomolecules in their spectacular network of intertwined metabolic pathways makes integrated proteomics and metabolomics an excellent opportunity, and the central component of integrative multiomics framework. It will therefore be of significant interest to integrate global proteome and PTM-based proteomics with metabolomics to achieve disease related altered levels of those molecules. Thereby, present update aims to highlight role and analysis of interacting metabolites/oncometabolites, and metabolite-regulated PTMs loop which may function as translational monitoring biomarkers along the reprogramming continuum of oncometabolism.

Keywords:  Integrative multiomics; Metabolite; Metabolomics; Oncometabolite; PTMs; Proteomics; Reprogramming cancer metabolism; Systems biology

DOI:  https://doi.org/10.1016/j.jpba.2021.114031
Cell Death Dis. 2021 Apr 15. 12(4): 408

The role of S-nitrosylation of PFKM in regulation of glycolysis in ovarian cancer cells.

Wenwen Gao, Mengqiu Huang, Xi Chen, Jianping Chen, Zhiwei Zou, Linlin Li, Kaiyuan Ji, Zhirui Nie, Bingsheng Yang, Zibo Wei, Pengfei Xu, Junshuang Jia, Qianbing Zhang, Hongfen Shen, Qianli Wang, Keyi Li, Lingqun Zhu, Meng Wang, Shuangyan Ye, Sisi Zeng, Ying Lin, Zhili Rong, Yang Xu, Peng Zhu, Hui Zhang, Bingtao Hao, Qiuzhen Liu.

One of the malignant transformation hallmarks is metabolism reprogramming, which plays a critical role in the biosynthetic needs of unchecked proliferation, abrogating cell death programs, and immunologic escape. However, the mechanism of the metabolic switch is not fully understood. Here, we found that the S-nitrosoproteomic profile of endogenous nitrogen oxide in ovarian cancer cells targeted multiple components in metabolism processes. Phosphofructokinase (PFKM), one of the most important regulatory enzymes of glycolysis, was S-nitrosylated by nitric oxide synthase NOS1 at Cys351. S-nitrosylation at Cys351 stabilized the tetramer of PFKM, leading to resist negative feedback of downstream metabolic intermediates. The PFKM-C351S mutation decreased the proliferation rate of cultured cancer cells, and reduced tumor growth and metastasis in the mouse xenograft model. These findings indicated that S-nitrosylation at Cys351 of PFKM by NOS1 contributes to the metabolic reprogramming of ovarian cancer cells, highlighting a critical role of endogenous nitrogen oxide on metabolism regulations in tumor progression.

DOI: https://doi.org/10.1038/s41419-021-03681-0
Rapid Commun Mass Spectrom. 2021 Apr 17. e9102

Optimizing sulfate pyrolysis triple oxygen isotope analysis for samples from desert environments.

Swea Klipsch, Daniel Herwartz, Michael Staubwasser.

RATIONALE: The triple oxygen isotope composition of sulfate may reveal formation pathway and depositional sources, and indicate slow biologic cycling in the environment. Pyrolysis mass spectrometry is better suited for large sample workloads during environmental profiling but sufficient precision and a thorough verification of accuracy are required for comparison with higher precision laser fluorination data.METHODS: Quantitative sulfate extraction from soil samples at neutral pH, purification, conversation to Ag-sulfate, and pyrolysis mass spectrometry were modified for high sample throughput. Samples were analyzed after pyrolysis in quartz cups and gold capsules in a modified EuroVector Elemental Analyzer 3000. Sample O2 was measured in continuous He-flow after purification by cryo-trapping and chromatography on a Thermo Finnigan MAT253 isotope ratio mass spectrometer. A protocol for routine quality control and data normalization ensures long-term accuracy of the pyrolysis method.
RESULTS: The 1σ SD external reproducibility is 0.12‰ for Δ17 OSO4 values on 30 μmol samples. Careful normalization for daily analytical session accounts for changing pyrolysis conditions over the course of multiple sessions. The precision and accuracy obtained with quartz cups are comparable with those obtained with gold capsules. Pyrolysis and fluorination data for in-house standards from four laboratories and from an Atacama Desert gypsum-soil profile are identical and demonstrate the accuracy of our simplified method.
CONCLUSIONS: Pyrolysis of sulfate in quartz cups and a modified simple elemental analyzer setup allows for accurate, precise, fast, cost-efficient, and non-hazardous mass spectrometry analysis. Exchangeability of data from pyrolysis and laser fluorination methods was demonstrated by repeat analysis of standards and natural samples despite high contents of interfering, easily soluble nitrates and chlorides.

DOI: https://doi.org/10.1002/rcm.9102
Talanta. 2021 Jul 01. pii: S0039-9140(21)00183-1. [Epub ahead of print]229 122262

An improved strategy for analysis of lipid molecules utilising a reversed phase C30 UHPLC column and scheduled MS/MS acquisition.

Andris Jankevics, Amelia Jenkins, Warwick B Dunn, Lukáš Najdekr.

  Measuring physiochemically diverse molecules (including lipids) which vary significantly in their concentrations poses a great analytical challenge. In untargeted lipidomics studies, reversed phase chromatography coupled with data-dependent MS/MS acquisition (DDA) is frequently applied. The optimal assay should deliver a high number of detected compounds with associated fragmentation data. In this work, we introduce novel 30 and 50 min UHPLC assays utilising lipid separation on a C30 stationary phase with a modified DDA strategy using smaller precursor m/z ranges scheduled for different lipid classes across the retention time range (defined as scheduled MS/MS). To evaluate the efficiency of the novel assays, mammalian tissue extracts (lamb liver, kidney and heart) were analysed and data were compared to a 15 min reversed phase C18 assay with multiple traditional DDA injections. The 30 min C30 assay detected double the number of detected compounds compared to the 15 min C18 assay. Applying the scheduled MS/MS DDA strategy with a single injection, a similar number of annotated lipids were reported compared to the traditional DDA strategy applied with five replicate injections on a C18 column. A longer 50 min C30 chromatographic assay did not result in an expected improvement in the chromatographic separation of overlapping isomer peaks compared to the 30 min method but did result in loss of accuracy of peak picking algorithms. We recommend the 30 min C30 assay with scheduled MS/MS acquisition as an efficient tool to analyse complex biological matrices and to annotate lipid species based on MS/MS data.

Keywords:  C(18) reversed phase; C(30) reversed phase; Intelligent DDA; Lipidomics; Scheduled MS/MS

DOI:  https://doi.org/10.1016/j.talanta.2021.122262
Cell Metab. 2021 Apr 09. pii: S1550-4131(21)00131-5. [Epub ahead of print]

A methionine-Mettl3-N6-methyladenosine axis promotes polycystic kidney disease.

Harini Ramalingam, Sonu Kashyap, Patricia Cobo-Stark, Andrea Flaten, Chun-Mien Chang, Sachin Hajarnis, Kyaw Zaw Hein, Jorgo Lika, Gina M Warner, Jair M Espindola-Netto, Ashwani Kumar, Mohammed Kanchwala, Chao Xing, Eduardo N Chini, Vishal Patel.

  Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic disorder marked by numerous progressively enlarging kidney cysts. Mettl3, a methyltransferase that catalyzes the abundant N6-methyladenosine (m6A) RNA modification, is implicated in development, but its role in most diseases is unknown. Here, we show that Mettl3 and m6A levels are increased in mouse and human ADPKD samples and that kidney-specific transgenic Mettl3 expression produces tubular cysts. Conversely, Mettl3 deletion in three orthologous ADPKD mouse models slows cyst growth. Interestingly, methionine and S-adenosylmethionine (SAM) levels are also elevated in ADPKD models. Moreover, methionine and SAM induce Mettl3 expression and aggravate ex vivo cyst growth, whereas dietary methionine restriction attenuates mouse ADPKD. Finally, Mettl3 activates the cyst-promoting c-Myc and cAMP pathways through enhanced c-Myc and Avpr2 mRNA m6A modification and translation. Thus, Mettl3 promotes ADPKD and links methionine utilization to epitranscriptomic activation of proliferation and cyst growth.

Keywords:  AVPR2; METTL3; N(6)-methyladenosine; S-adenosylmethionine; c-Myc; m6A mRNA methylation; mRNA translation; methionine; polycystic kidney disease

DOI:  https://doi.org/10.1016/j.cmet.2021.03.024
J Lipid Res. 2021 Apr 09. pii: S0022-2275(21)00055-9. [Epub ahead of print] 100073

The lipidome in nonalcoholic fatty liver disease: Actionable targets.

Carlos J Pirola, Silvia Sookoian.

  Nonalcoholic fatty liver disease (NAFLD) has become the most prevalent chronic liver disease. Recent technological advances, combined with OMICs experiments and explorations involving different biological samples, have uncovered vital aspects of NAFLD biology. In this review, we summarize recent work by our group and others that expands what is known about the role of lipidome in NAFLD pathogenesis. We discuss how pathway and enrichment analyses were performed by integrating a list of query metabolites derived from text-mining existing NAFLD-lipidomics studies, resulting in the identification of nine KEGG dysregulated pathways, including biosynthesis of unsaturated fatty acids, butanoate metabolism, synthesis and degradation of ketone bodies, sphingolipid, arachidonic acid and pyruvate metabolism, and numerous nonsteroidal anti-inflammatory drug pathways predicted from The Small Molecule Pathway Database. We also summarize an integrated pathway-level analysis of genes and lipid-related metabolites associated with NAFLD, which shows overrepresentation of signal transduction, selenium micronutrient network, Class A/1Rhodopsin-like receptors and GPCR ligand binding, and GPCR-downstream signaling. Generated gene-metabolite-disease interaction networks indicate that NAFLD and arterial hypertension are interlinked by molecular signatures. Finally, we discuss how mining pathways and associations among metabolites, lipids, genes, and proteins can be exploited to infer networks and potential pharmacological targets and how lipidomic studies may provide insight into the interrelationships among metabolite clusters thatmodify NAFLD biology, genetic susceptibility, diet, and the gut microbiome.

Keywords:  Lipidomics; NAFLD; NASH; OMICS; PUFA; butanoate; microbiome; systems biology

DOI:  https://doi.org/10.1016/j.jlr.2021.100073
Bone. 2021 Apr 07. pii: S8756-3282(21)00108-3. [Epub ahead of print]148 115946

Loss of function of lysosomal acid lipase (LAL) profoundly impacts osteoblastogenesis and increases fracture risk in humans.

Ron C Helderman, Daniel G Whitney, Madalina Duta-Mare, Alena Akhmetshina, Nemanja Vujic, Shobana Jayapalan, Jeffry S Nyman, Biswapriya B Misra, Clifford J Rosen, Michael P Czech, Dagmar Kratky, Elizabeth Rendina-Ruedy.

  Lysosomal acid lipase (LAL) is essential for cholesteryl ester (CE) and triacylglycerol (TAG) hydrolysis in the lysosome. Clinically, an autosomal recessive LIPA mutation causes LAL deficiency (LALD), previously described as Wolman Disease or Cholesteryl Ester Storage Disease (CESD). LAL-D is associated with ectopic lipid accumulation in the liver, small intestine, spleen, adrenal glands, and blood. Considering the importance of unesterified cholesterol and fatty acids in bone metabolism, we hypothesized that LAL is essential for bone formation, and ultimately, skeletal health. To investigate the role of LAL in skeletal homeostasis, we used LAL-deficient (-/-) mice, in vitro osteoblast cultures, and novel clinical data from LAL-D patients. Both male and female LAL-/- mice demonstarted lower trabecular and cortical bone parameters , which translated to reduced biomechanical properties. Further histological analyses revealed that LAL-/- mice had fewer osteoblasts, with no change in osteoclast or marrow adipocyte numbers. In studying the cell-autonomous role of LAL, we observed impaired differentiation of LAL-/- calvarial osteoblasts and in bone marrow stromal cells treated with the LAL inhibitor lalistat. Consistent with LAL's role in other tissues, lalistat resulted in profound lipid puncta accumulation and an altered intracellular lipid profile. Finally, we analyzed a large de-identified national insurance database (i.e. 2016/2017 Optum Clinformatics®) which revealed that adults (≥18 years) with CESD (n = 3076) had a higher odds ratio (OR = 1.21; 95% CI = 1.03-1.41) of all-cause fracture at any location compared to adults without CESD (n = 13.7 M) after adjusting for demographic variables and osteoporosis. These data demonstrate that alterations in LAL have significant clinical implications related to fracture risk and that LAL's modulation of lipid metabolism is a critical for osteoblast function.

Keywords:  Bone; Cholesterol; Lipid; Lipophagy; Metabolism; Osteoblast; Skeleton

DOI:  https://doi.org/10.1016/j.bone.2021.115946
Mol Cell Proteomics. 2021 Apr 09. pii: S1535-9476(21)00053-0. [Epub ahead of print] 100080

Sensitive immunopeptidomics by leveraging available large-scale multi-HLA spectral libraries, data-independent acquisition and MS/MS prediction.

HuiSong Pak, Justine Michaux, Florian Huber, Chloe Chong, Brian J Stevenson, Markus Müller, George Coukos, Michal Bassani-Sternberg.

Mass spectrometry is the state-of-the-art methodology for capturing the breadth and depth of the immunopeptidome across HLA allotypes and cell types. The majority of studies in the immunopeptidomics field are discovery-driven. Hence, data-dependent tandem mass spectrometry acquisition (DDA MS/MS) is widely used, as it generates high quality references of peptide fingerprints. However, DDA suffers from the stochastic selection of abundant ions that impairs sensitivity and reproducibility. In contrast, in data-independent acquisition (DIA), the systematic fragmentation and acquisition of all fragment ions within a given isolation m/z windows yield a comprehensive map for a given sample. However, many DIA approaches commonly require generating comprehensive DDA-based spectrum libraries, which can become impractical for studying non-canonical and personalized neoantigens. Since the amount of HLA peptides eluted from biological samples such small tissue biopsies is typically not sufficient for acquiring both meaningful DDA data necessary for generating comprehensive spectral libraries and DIA MS measurements, the implementation of DIA in the immunopeptidomics translational research domain has remained limited. We implemented a DIA immunopeptidomics workflow and assessed its sensitivity and accuracy by matching DIA data against libraries with growing complexity - from sample-specific libraries to libraries combining two to forty different immunopeptidomics samples. Analyzing DIA immunopeptidomics data against a complex multi-HLA spectral library resulted in a two-fold increase in peptide identification compared with sample-specific library and in a three-fold increase compared with DDA measurements, yet with no detrimental effect on the specificity. Furthermore, we demonstrated the implementation of DIA for sensitive personalized neoantigen discovery through the analysis of DIA data with predicted MS/MS spectra of clinically relevant HLA ligands. We conclude that a comprehensive multi-HLA library for DIA approach in combination with MS/MS prediction is highly advantageous for the clinical immunopeptidomics especially when low amounts of biological samples are available for immunopeptidomics.

DOI: https://doi.org/10.1016/j.mcpro.2021.100080
Cell. 2021 Apr 15. pii: S0092-8674(21)00432-3. [Epub ahead of print]184(8): 2256

The mTORC1 pathway stimulates glutamine metabolism and cell proliferation by repressing SIRT4.

Alfred Csibi, Sarah-Maria Fendt, Chenggang Li, George Poulogiannis, Andrew Y Choo, Douglas J Chapski, Seung Min Jeong, Jamie M Dempsey, Andrey Parkhitko, Tasha Morrison, Elizabeth P Henske, Marcia C Haigis, Lewis C Cantley, Gregory Stephanopoulos, Jane Yu, John Blenis.

DOI: https://doi.org/10.1016/j.cell.2021.03.059
Proteomics. 2021 Apr 16. e2100026

Proteome characterisation of extracellular vesicles isolated from heart.

Bethany Claridge, Alin Rai, Haoyun Fang, Aya Matsumoto, Jieting Luo, Julie R McMullen, David W Greening.

  Cardiac intercellular communication is critical for heart function and often dysregulated in cardiovascular diseases. While cardiac extracellular vesicles (cEVs) are emerging mediators of signalling, their isolation remains a technical challenge hindering our understanding of cEV protein composition. Here, we utilised Langendorff-collagenase-based enzymatic perfusion and differential centrifugation to isolate cEVs from mouse heart (yield 3-6 μg/heart). cEVs are ∼200 nm, express classical EV markers (Cd63/81/9+ , Tsg101+ , Pdcd6ip/Alix+ ), and are depleted of blood (Alb/Fga/Hba) and cardiac damage markers (Mb, Tnnt2, Ldhb). Comparison with mechanically-derived EVs revealed greater detection of EV markers and decreased cardiac damage contaminants. Mass spectrometry-based proteomic profiling revealed 1,721 proteins in cEVs, implicated in proteasomal and autophagic proteostasis, glycolysis and fatty acid metabolism; essential functions often disrupted in cardiac pathologies. There was striking enrichment of 942 proteins in cEVs compared to mouse heart tissue - implicated in EV biogenesis, antioxidant activity, and lipid transport, suggesting active cargo selection and specialised function. Interestingly, cEVs contain marker proteins for cardiomyocytes, cardiac progenitors, B-cells, T-cells, macrophages, smooth muscle cells, endothelial cells, and cardiac fibroblasts, suggesting diverse cellular origin. We present a method of cEV isolation and provide insight into potential functions, enabling future studies into EV roles in cardiac physiology and disease. This article is protected by copyright. All rights reserved.

Keywords:  extracellular vesicles; heart; isolation; proteomics; tissue

DOI:  https://doi.org/10.1002/pmic.202100026