bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2020‒02‒23
twenty-four papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research
  1. Quantitative amino acid analysis by liquid chromatography-tandem mass spectrometry using low cost derivatization and an automated liquid handler.
  2. Lipidomic Analysis of Cells and Extracellular Vesicles from High- and Low-Metastatic Triple-Negative Breast Cancer.
  3. Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysis.
  4. The Aging Metabolome- Biomarkers to Hub Metabolites.
  5. Redox lipid reprogramming commands susceptibility of macrophages and microglia to ferroptotic death.
  6. A computational platform for high-throughput analysis of RNA sequences and modifications by mass spectrometry.
  7. Validation of lipidomic analysis of human plasma and serum by supercritical fluid chromatography-mass spectrometry and hydrophilic interaction liquid chromatography-mass spectrometry.
  8. Targeting Hepatic Glutaminase 1 Ameliorates Non-alcoholic Steatohepatitis by Restoring Very-Low-Density Lipoprotein Triglyceride Assembly.
  9. Simultaneous quantification of free fatty acids and acylcarnitines in plasma samples using dansylhydrazine labeling and liquid chromatography-triple quadrupole mass spectrometry.
  10. Serum Metabolomics of Early Postoperative Cognitive Dysfunction in Elderly Patients Using Liquid Chromatography and Q-TOF Mass Spectrometry.
  11. Recent progresses of derivatization approaches in the targeted lipidomics analysis by mass spectrometry.
  12. Blood Metabolite Signature of Metabolic Syndrome Implicates Alterations in Amino Acid Metabolism: Findings from the Baltimore Longitudinal Study of Aging (BLSA) and the Tsuruoka Metabolomics Cohort Study (TMCS).
  13. Induction of the nicotinamide riboside kinase NAD+ salvage pathway in a model of sarcoplasmic reticulum dysfunction.
  14. MDM2 and MDMX promote ferroptosis by PPARα-mediated lipid remodeling.
  15. FLASHDeconv: Ultrafast, High-Quality Feature Deconvolution for Top-Down Proteomics.
  16. Glutamine metabolism in the proliferation of GS-expression pituitary tumor cells.
  17. Mass Spectrometry of Human Transporters.
  18. Paper spray mass spectrometry for the analysis of picoliter droplets.
  19. COLMAR Lipids Web Server and Ultrahigh-Resolution Methods for 2D NMR- and MS-Based Lipidomics.
  20. Progress and pitfalls of using isobaric mass tags for proteome profiling.
  21. Lipidomics reveals the dynamics of lipid profile altered by omega-3 polyunsaturated fatty acid supplementation in healthy people.
  22. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay to profile 20 plasma steroids in endocrine disorders.
  23. Cytochrome P450 oxidoreductase contributes to phospholipid peroxidation in ferroptosis.
  24. At-line high throughput site-specific glycan profiling using targeted mass spectrometry.
  1. JIMD Rep. 2020 Jan;51(1): 62-69
    Quantitative amino acid analysis by liquid chromatography-tandem mass spectrometry using low cost derivatization and an automated liquid handler.
    William S Phipps, Eric Crossley, Richard Boriack, Patricia M Jones, Khushbu Patel.
      Amino acid analysis is central to newborn screening and the investigation of inborn errors of metabolism. Ion-exchange chromatography with ninhydrin derivatization remains the reference method for quantitative amino acid analysis but offers slow chromatography and is susceptible to interference from other co-eluting compounds. Liquid-chromatography tandem mass spectrometry (LC-MS/MS) provides a rapid and highly specific alternative, but sample preparation is frequently laborious and sometimes cost prohibitive. To address these limitations, we validated an LC-MS/MS method using the aTRAQ Reagents Application Kit with a modified protocol consuming only half reagents. Adequate performance for clinical specimen measurement of 26 amino acids with high clinical relevance was achieved. An automated liquid handler and modified calibration and normalization approaches were used to ensure reproducible assay performance. Linear measurement between 5 and 2000 μM was achieved for most analytes despite use of a small, 10 μl sample size. Overall the method achieved near substantially improved throughput and enabled use of smaller samples volumes for batched analyses of clinical samples.
    Keywords:  amino acid; inborn errors of metabolism; isotope‐coded derivatization; liquid chromatography; mass spectrometry; triple quadrupole
    DOI:  https://doi.org/10.1002/jmd2.12080
  2. Metabolites. 2020 Feb 13. pii: E67. [Epub ahead of print]10(2):
    Lipidomic Analysis of Cells and Extracellular Vesicles from High- and Low-Metastatic Triple-Negative Breast Cancer.
    Nao Nishida-Aoki, Yoshihiro Izumi, Hiroaki Takeda, Masatomo Takahashi, Takahiro Ochiya, Takeshi Bamba.
      Extracellular vesicles (EVs) are lipid bilayer nanovesicles secreted from almost all cells including cancer. Cancer-derived EVs contribute to cancer progression and malignancy via educating the surrounding normal cells. In breast cancer, epidemiological and experimental observations indicated that lipids are associated with cancer malignancy. However, lipid compositions of breast cancer EVs and their contributions to cancer progression are unexplored. In this study, we performed a widely targeted quantitative lipidomic analysis in cells and EVs derived from high- and low-metastatic triple-negative breast cancer cell lines, using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry. We demonstrated the differential lipid compositions between EVs and cells of their origin, and between high- and low-metastatic cell lines. Further, we demonstrated EVs from highly metastatic breast cancer accumulated unsaturated diacylglycerols (DGs) compared with EVs from lower-metastatic cells, without increasing the amount in cells. The EVs enriched with DGs could activate the protein kinase D signaling pathway in endothelial cells, which can lead to stimulated angiogenesis. Our results indicate that lipids are selectively loaded into breast cancer EVs to support tumor progression.
    Keywords:  angiogenesis; diacylglycerol; endothelial cells; exosomes; extracellular vesicles; lipidomics; protein kinase D
    DOI:  https://doi.org/10.3390/metabo10020067
  3. Nat Immunol. 2020 Feb 17.
    Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysis.
    Florian J Weisel, Steven J Mullett, Rebecca A Elsner, Ashley V Menk, Nikita Trivedi, Wei Luo, Daniel Wikenheiser, William F Hawse, Maria Chikina, Shuchi Smita, Laura J Conter, Stephen M Joachim, Stacy G Wendell, Michael J Jurczak, Thomas H Winkler, Greg M Delgoffe, Mark J Shlomchik.
      Germinal center B cells (GCBCs) are critical for generating long-lived humoral immunity. How GCBCs meet the energetic challenge of rapid proliferation is poorly understood. Dividing lymphocytes typically rely on aerobic glycolysis over oxidative phosphorylation for energy. Here we report that GCBCs are exceptional among proliferating B and T cells, as they actively oxidize fatty acids (FAs) and conduct minimal glycolysis. In vitro, GCBCs had a very low glycolytic extracellular acidification rate but consumed oxygen in response to FAs. [13C6]-glucose feeding revealed that GCBCs generate significantly less phosphorylated glucose and little lactate. Further, GCBCs did not metabolize glucose into tricarboxylic acid (TCA) cycle intermediates. Conversely, [13C16]-palmitic acid labeling demonstrated that GCBCs generate most of their acetyl-CoA and acetylcarnitine from FAs. FA oxidation was functionally important, as drug-mediated and genetic dampening of FA oxidation resulted in a selective reduction of GCBCs. Hence, GCBCs appear to uncouple rapid proliferation from aerobic glycolysis.
    DOI:  https://doi.org/10.1038/s41590-020-0598-4
  4. Proteomics. 2020 Feb 18. e1800407
    The Aging Metabolome- Biomarkers to Hub Metabolites.
    Rishi Sharma, Arvind Ramanathan.
      Aging biology is intimately associated with dysregulated metabolism, which is one of the hallmarks of aging. Aging related pathways such as mTOR and AMPK which are major targets of anti-aging interventions including rapamcyin, metformin and exercise, either directly regulate or intersect with metabolic pathways. In this review we outline numerous candidate bio-markers of aging that have emerged using metabolomics. Metabolomics studies also reveal that not all metabolites are created equally. A set of core 'hub' metabolites are emerging as central mediators of aging. The hub metabolites reviewed here are- Nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide dinucleotide phosphate (NADPH), alpha ketoglutarate (aKG) and beta-hydroxybutyrate (βHB). These 'hub' metabolites have signaling and epigenetic roles along with their canonical roles as co-factors or intermediates of carbon metabolism. Together these hub metabolites suggest a central role of the TCA cycle in signaling and metabolic dysregulation associated with aging. This article is protected by copyright. All rights reserved.
    Keywords:  ageing; hub metabolites; metabolomics
    DOI:  https://doi.org/10.1002/pmic.201800407
  5. Nat Chem Biol. 2020 Mar;16(3): 278-290
    Redox lipid reprogramming commands susceptibility of macrophages and microglia to ferroptotic death.
    Alexandr A Kapralov, Qin Yang, Haider H Dar, Yulia Y Tyurina, Tamil S Anthonymuthu, Rina Kim, Claudette M St Croix, Karolina Mikulska-Ruminska, Bing Liu, Indira H Shrivastava, Vladimir A Tyurin, Hsiu-Chi Ting, Yijen L Wu, Yuan Gao, Galina V Shurin, Margarita A Artyukhova, Liubov A Ponomareva, Peter S Timashev, Rosario M Domingues, Detcho A Stoyanovsky, Joel S Greenberger, Rama K Mallampalli, Ivet Bahar, Dmitry I Gabrilovich, Hülya Bayır, Valerian E Kagan.
      Ferroptotic death is the penalty for losing control over three processes-iron metabolism, lipid peroxidation and thiol regulation-that are common in the pro-inflammatory environment where professional phagocytes fulfill their functions and yet survive. We hypothesized that redox reprogramming of 15-lipoxygenase (15-LOX) during the generation of pro-ferroptotic signal 15-hydroperoxy-eicosa-tetra-enoyl-phosphatidylethanolamine (15-HpETE-PE) modulates ferroptotic endurance. Here, we have discovered that inducible nitric oxide synthase (iNOS)/NO•-enrichment of activated M1 (but not alternatively activated M2) macrophages/microglia modulates susceptibility to ferroptosis. Genetic or pharmacologic depletion/inactivation of iNOS confers sensitivity on M1 cells, whereas NO• donors empower resistance of M2 cells to ferroptosis. In vivo, M1 phagocytes, in comparison to M2 phagocytes, exert higher resistance to pharmacologically induced ferroptosis. This resistance is diminished in iNOS-deficient cells in the pro-inflammatory conditions of brain trauma or the tumour microenvironment. The nitroxygenation of eicosatetraenoyl (ETE)-PE intermediates and oxidatively truncated species by NO• donors and/or suppression of NO• production by iNOS inhibitors represent a novel redox mechanism of regulation of ferroptosis in pro-inflammatory conditions.
    DOI:  https://doi.org/10.1038/s41589-019-0462-8
  6. Nat Commun. 2020 Feb 17. 11(1): 926
    A computational platform for high-throughput analysis of RNA sequences and modifications by mass spectrometry.
    Samuel Wein, Byron Andrews, Timo Sachsenberg, Helena Santos-Rosa, Oliver Kohlbacher, Tony Kouzarides, Benjamin A Garcia, Hendrik Weisser.
      The field of epitranscriptomics continues to reveal how post-transcriptional modification of RNA affects a wide variety of biological phenomena. A pivotal challenge in this area is the identification of modified RNA residues within their sequence contexts. Mass spectrometry (MS) offers a comprehensive solution by using analogous approaches to shotgun proteomics. However, software support for the analysis of RNA MS data is inadequate at present and does not allow high-throughput processing. Existing software solutions lack the raw performance and statistical grounding to efficiently handle the numerous modifications found on RNA. We present a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), that addresses these shortcomings. We demonstrate the capability of NASE to reliably identify a wide range of modified RNA sequences in four original datasets of varying complexity. In human tRNA, we characterize over 20 different modification types simultaneously and find many cases of incomplete modification.
    DOI:  https://doi.org/10.1038/s41467-020-14665-7
  7. Anal Bioanal Chem. 2020 Feb 20.
    Validation of lipidomic analysis of human plasma and serum by supercritical fluid chromatography-mass spectrometry and hydrophilic interaction liquid chromatography-mass spectrometry.
    Denise Wolrab, Michaela Chocholoušková, Robert Jirásko, Ondřej Peterka, Michal Holčapek.
      Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) has a great potential for the high-throughput lipidomic quantitation of biological samples; therefore, the full optimization and method validation of UHPSFC/MS is compared here with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) in hydrophilic interaction liquid chromatography (HILIC) mode as the second powerful technique for the lipid class separation. First, the performance of six common extraction protocols is investigated, where the Folch procedure yields the best results with regard to recovery rate, matrix effect, and precision. Then, the full optimization and analytical validation for eight lipid classes using UHPSFC/MS and HILIC-UHPLC/MS methods are performed for the same sample set and applied for the lipidomic characterization of pooled samples of human plasma, human serum, and NIST SRM 1950 human plasma. The choice of appropriate internal standards (IS) for individual lipid classes has a key importance for reliable quantitative workflows illustrated by the selectivity while validation and the calculation of the quantitation error using multiple internal standards per lipid class. Validation results confirm the applicability of both methods, but UHPSFC/MS provides some distinct advantages, such as the successful separation of both non-polar and polar lipid classes unlike to HILIC-UHPLC/MS, shorter total run times (8 vs. 10.5 min), and slightly higher robustness. Various types of correlations between methods (UHPSFC/MS and HILIC-UHPLC/MS), biological material (plasma and serum), IS (laboratory and commercially mixtures), and literature data on the standard reference material show the intra- and inter-laboratory comparison in the quantitation of lipid species from eight lipid classes, the concentration differences in serum and plasma as well as the applicability of non-commercially available internal standard mixtures for lipid quantitation.
    Keywords:  Hydrophilic interaction liquid chromatography; Lipidomics; Mass spectrometry; Matrix effect; Plasma; Quantitation; Serum; Supercritical fluid chromatography; Validation
    DOI:  https://doi.org/10.1007/s00216-020-02473-3
  8. Cell Metab. 2020 Feb 20. pii: S1550-4131(20)30056-5. [Epub ahead of print]
    Targeting Hepatic Glutaminase 1 Ameliorates Non-alcoholic Steatohepatitis by Restoring Very-Low-Density Lipoprotein Triglyceride Assembly.
    Jorge Simon, Maitane Nuñez-García, Pablo Fernández-Tussy, Lucía Barbier-Torres, David Fernández-Ramos, Beatriz Gómez-Santos, Xabier Buqué, Fernando Lopitz-Otsoa, Naroa Goikoetxea-Usandizaga, Marina Serrano-Macia, Rubén Rodriguez-Agudo, Maider Bizkarguenaga, Imanol Zubiete-Franco, Virginia Gutiérrez-de Juan, Diana Cabrera, Cristina Alonso, Paula Iruzubieta, Manuel Romero-Gomez, Sebastiaan van Liempd, Azucena Castro, Ruben Nogueiras, Marta Varela-Rey, Juan Manuel Falcón-Pérez, Erica Villa, Javier Crespo, Shelly C Lu, Jose M Mato, Patricia Aspichueta, Teresa C Delgado, María Luz Martínez-Chantar.
      Non-alcoholic steatohepatitis (NASH) is characterized by the accumulation of hepatic fat in an inflammatory/fibrotic background. Herein, we show that the hepatic high-activity glutaminase 1 isoform (GLS1) is overexpressed in NASH. Importantly, GLS1 inhibition reduces lipid content in choline and/or methionine deprivation-induced steatotic mouse primary hepatocytes, in human hepatocyte cell lines, and in NASH mouse livers. We suggest that under these circumstances, defective glutamine fueling of anaplerotic mitochondrial metabolism and concomitant reduction of oxidative stress promotes a reprogramming of serine metabolism, wherein serine is shifted from the generation of the antioxidant glutathione and channeled to provide one-carbon units to regenerate the methionine cycle. The restored methionine cycle can induce phosphatidylcholine synthesis from the phosphatidylethanolamine N-methyltransferase-mediated and CDP-choline pathways as well as by base-exchange reactions between phospholipids, thereby restoring hepatic phosphatidylcholine content and very-low-density lipoprotein export. Overall, we provide evidence that hepatic GLS1 targeting is a valuable therapeutic approach in NASH.
    Keywords:  GLS1; GLS2; NAFLD; NASH; TCA cycle; VLDL; folate cycle; glutaminase; methionine cycle; phospholipids
    DOI:  https://doi.org/10.1016/j.cmet.2020.01.013
  9. Anal Bioanal Chem. 2020 Feb 20.
    Simultaneous quantification of free fatty acids and acylcarnitines in plasma samples using dansylhydrazine labeling and liquid chromatography-triple quadrupole mass spectrometry.
    Guan-Yuan Chen, Qibin Zhang.
      Free fatty acid (FFA) and acylcarnitine (AcCar) are key elements of energy metabolism. Dysregulated levels of FFA and AcCar are associated with genetic defects and other metabolic disorders. Due to differences in the physicochemical properties of these two classes of compounds, it is challenging to quantify FFA and AcCar in human plasma using a single method. In this work, we developed a chemical isotope labeling (CIL)-based liquid chromatography-multiple reaction monitoring (LC-MRM) method to simultaneously quantify FFA and AcCar. Dansylhydrazine (DnsHz) was used to label the carboxylic acid moiety on FFA and AcCar. This resulted in the formation of a permanently charged ammonium ion for facile ionization in positive ionization mode and higher hydrophobicity for enhanced retention of short-chain analogs on reversed-phase LC columns and enabled absolute quantification by using heavy labeled DnsHz analogs as internal standards. Labeling conditions including the concentration and freshness of cross-linker, reaction time, and temperature were optimized. This method can successfully quantify all short-, medium- and long-chain FFAs and AcCars with greatly enhanced sensitivity. Using this method, 25 FFAs and 13 AcCars can be absolutely quantified and validated in human plasma samples within 12 min. Simultaneous quantification of FFA and AcCar enabled by this CIL-based LC-MRM method facilitates the investigation of fatty acid metabolism and has potential in clinical applications.
    Keywords:  Acylcarnitine; Chemical isotope labeling; Free fatty acid; Human plasma; LC-MRM-MS; Targeted metabolomics
    DOI:  https://doi.org/10.1007/s00216-020-02514-x
  10. Oxid Med Cell Longev. 2020 ;2020 8957541
    Serum Metabolomics of Early Postoperative Cognitive Dysfunction in Elderly Patients Using Liquid Chromatography and Q-TOF Mass Spectrometry.
    Gang Qian, YueLan Wang.
      Postoperative cognitive dysfunction (POCD) is a common postoperative complication observed in elderly patients. However, the diagnosis of POCD is not very satisfactory as no specific biomarkers have been classified. It is necessary to identify new diagnostic markers to better understand the pathogenesis of POCD. We performed liquid chromatography with a time-of-flight mass spectrometer- (LC/Q-TOF-MS-) based metabolomics study to investigate POCD. A total of 40 metabolites were differentially expressed between POCD and non-POCD patients. In this study, we investigated whether phosphatidylserine (PS) (17:2/0:0), with an area under the curve value of 0.966, was a potential sensitive and specific biomarker for the diagnosis and prognosis of POCD. Pathway analysis showed that fatty acid metabolism, lipid metabolism, and carnitine metabolism were significantly altered in POCD. Network analysis indicated that nitric oxide signaling, PI3K-AKT signaling, mTOR signaling, and mitochondrial dysfunction were related to the pathogenesis of POCD. This study showed that metabolic profiling was meaningful when studying the diagnosis and pathogenesis of POCD.
    DOI:  https://doi.org/10.1155/2020/8957541
  11. J Sep Sci. 2020 Feb 18.
    Recent progresses of derivatization approaches in the targeted lipidomics analysis by mass spectrometry.
    Xian-En Zhao, Shuyun Zhu, Huwei Liu.
      Lipidomics plays an essential role in the development of an improved understanding of lipids metabolism and the identification of new biomarkers or therapeutic targets of related diseases. The strong analytical power of mass spectrometry and its rapid developments in the respect of instruments and techniques have significantly accelerated the emerging lipidomics and related application fields in biology, medicine and pharmacy. The strategy of chemical derivatization can remarkably improve the shortcomings of mass spectrometric analytical technologies of shotgun lipidomics and liquid chromatography mass spectrometry, and in the past decade many related studies have been reported for fatty acids, glycerophospholipids, sphingomyelins, monoglycerides, diacylglycerols, long-chain bases, steroids and so on. Therefore, this review will focus on new chemical derivatization approaches about the research progresses of shotgun- and liquid chromatography mass spectrometry-based targeted lipidomics (from 2005 to July 2019, most of reports emerged in the past five years), and put forward the problems and prospects in this field. It is expected to be helpful for the design and synthesis of new derivatization reagents especially the outstanding stable isotope labeling derivatization reagents, and the development and application of new chemical derivatization strategies and matched mass spectrometric analysis methods. This article is protected by copyright. All rights reserved.
    Keywords:  chemical derivatization; lipidomics; liquid chromatography mass spectrometry; shotgun lipidomics; stable isotope labeling
    DOI:  https://doi.org/10.1002/jssc.201901346
  12. Int J Mol Sci. 2020 Feb 13. pii: E1249. [Epub ahead of print]21(4):
    Blood Metabolite Signature of Metabolic Syndrome Implicates Alterations in Amino Acid Metabolism: Findings from the Baltimore Longitudinal Study of Aging (BLSA) and the Tsuruoka Metabolomics Cohort Study (TMCS).
    Jackson A Roberts, Vijay R Varma, Chiung-Wei Huang, Yang An, Anup Oommen, Toshiko Tanaka, Luigi Ferrucci, Palchamy Elango, Toru Takebayashi, Sei Harada, Miho Iida, Madhav Thambisetty.
      Rapid lifestyle and dietary changes have contributed to a rise in the global prevalence of metabolic syndrome (MetS), which presents a potential healthcare crisis, owing to its association with an increased burden of multiple cardiovascular and neurological diseases. Prior work has identified the role that genetic, lifestyle, and environmental factors can play in the prevalence of MetS. Metabolomics is an important tool to study alterations in biochemical pathways intrinsic to the pathophysiology of MetS. We undertook a metabolomic study of MetS in serum samples from two ethnically distinct, well-characterized cohorts-the Baltimore Longitudinal Study of Aging (BLSA) from the U.S. and the Tsuruoka Metabolomics Cohort Study (TMCS) from Japan. We used multivariate logistic regression to identify metabolites that were associated with MetS in both cohorts. Among the top 25 most significant (lowest p-value) metabolite associations with MetS in each cohort, we identified 18 metabolites that were shared between TMCS and BLSA, the majority of which were classified as amino acids. These associations implicate multiple biochemical pathways in MetS, including branched-chain amino acid metabolism, glutathione production, aromatic amino acid metabolism, gluconeogenesis, and the tricarboxylic acid cycle. Our results suggest that fundamental alterations in amino acid metabolism may be central features of MetS.
    Keywords:  amino acids; blood pressure; fasting glucose; metabolic syndrome; metabolomics; triglycerides; waist circumference
    DOI:  https://doi.org/10.3390/ijms21041249
  13. Skelet Muscle. 2020 Feb 19. 10(1): 5
    Induction of the nicotinamide riboside kinase NAD+ salvage pathway in a model of sarcoplasmic reticulum dysfunction.
    Craig L Doig, Agnieszka E Zielinska, Rachel S Fletcher, Lucy A Oakey, Yasir S Elhassan, Antje Garten, David Cartwright, Silke Heising, Ahmed Alsheri, David G Watson, Cornelia Prehn, Jerzy Adamski, Daniel A Tennant, Gareth G Lavery.
      BACKGROUND: Hexose-6-Phosphate Dehydrogenase (H6PD) is a generator of NADPH in the Endoplasmic/Sarcoplasmic Reticulum (ER/SR). Interaction of H6PD with 11β-hydroxysteroid dehydrogenase type 1 provides NADPH to support oxo-reduction of inactive to active glucocorticoids, but the wider understanding of H6PD in ER/SR NAD(P)(H) homeostasis is incomplete. Lack of H6PD results in a deteriorating skeletal myopathy, altered glucose homeostasis, ER stress and activation of the unfolded protein response. Here we further assess muscle responses to H6PD deficiency to delineate pathways that may underpin myopathy and link SR redox status to muscle wide metabolic adaptation.METHODS: We analysed skeletal muscle from H6PD knockout (H6PDKO), H6PD and NRK2 double knockout (DKO) and wild-type (WT) mice. H6PDKO mice were supplemented with the NAD+ precursor nicotinamide riboside. Skeletal muscle samples were subjected to biochemical analysis including NAD(H) measurement, LC-MS based metabolomics, Western blotting, and high resolution mitochondrial respirometry. Genetic and supplement models were assessed for degree of myopathy compared to H6PDKO.
    RESULTS: H6PDKO skeletal muscle showed adaptations in the routes regulating nicotinamide and NAD+ biosynthesis, with significant activation of the Nicotinamide Riboside Kinase 2 (NRK2) pathway. Associated with changes in NAD+ biosynthesis, H6PDKO muscle had impaired mitochondrial respiratory capacity with altered mitochondrial acylcarnitine and acetyl-CoA metabolism. Boosting NAD+ levels through the NRK2 pathway using the precursor nicotinamide riboside elevated NAD+/NADH but had no effect to mitigate ER stress and dysfunctional mitochondrial respiratory capacity or acetyl-CoA metabolism. Similarly, H6PDKO/NRK2 double KO mice did not display an exaggerated timing or severity of myopathy or overt change in mitochondrial metabolism despite depression of NAD+ availability.
    CONCLUSIONS: These findings suggest a complex metabolic response to changes in muscle SR NADP(H) redox status that result in impaired mitochondrial energy metabolism and activation of cellular NAD+ salvage pathways. It is possible that SR can sense and signal perturbation in NAD(P)(H) that cannot be rectified in the absence of H6PD. Whether NRK2 pathway activation is a direct response to changes in SR NAD(P)(H) availability or adaptation to deficits in metabolic energy availability remains to be resolved.
    Keywords:  Endoplasmic/sarcoplasmic reticulum; Hexose-6-phosphate dehydrogenase; Nicotinamide adenine dinucleotide; Nicotinamide riboside; Skeletal muscle
    DOI:  https://doi.org/10.1186/s13395-019-0216-z
  14. Genes Dev. 2020 Feb 20.
    MDM2 and MDMX promote ferroptosis by PPARα-mediated lipid remodeling.
    Divya Venkatesh, Nicholas A O'Brien, Fereshteh Zandkarimi, David R Tong, Michael E Stokes, Denise E Dunn, Everett S Kengmana, Allegra T Aron, Alyssa M Klein, Joleen M Csuka, Sung-Hwan Moon, Marcus Conrad, Christopher J Chang, Donald C Lo, Angelo D'Alessandro, Carol Prives, Brent R Stockwell.
      MDM2 and MDMX, negative regulators of the tumor suppressor p53, can work separately and as a heteromeric complex to restrain p53's functions. MDM2 also has pro-oncogenic roles in cells, tissues, and animals that are independent of p53. There is less information available about p53-independent roles of MDMX or the MDM2-MDMX complex. We found that MDM2 and MDMX facilitate ferroptosis in cells with or without p53. Using small molecules, RNA interference reagents, and mutant forms of MDMX, we found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. We observed that MDM2 and MDMX alter the lipid profile of cells to favor ferroptosis. Inhibition of MDM2 or MDMX leads to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q10, an endogenous lipophilic antioxidant. This suggests that MDM2 and MDMX normally prevent cells from mounting an adequate defense against lipid peroxidation and thereby promote ferroptosis. Moreover, we found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis, suggesting that the MDM2-MDMX complex regulates lipids through altering PPARα activity. These findings reveal the complexity of cellular responses to MDM2 and MDMX and suggest that MDM2-MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Furthermore, they suggest that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers.
    Keywords:  CoQ10; FSP1; MDM2; MDMX; PPARα; cancer; ferroptosis; lipid metabolism; p53-independent
    DOI:  https://doi.org/10.1101/gad.334219.119
  15. Cell Syst. 2020 Feb 12. pii: S2405-4712(20)30030-2. [Epub ahead of print]
    FLASHDeconv: Ultrafast, High-Quality Feature Deconvolution for Top-Down Proteomics.
    Kyowon Jeong, Jihyung Kim, Manasi Gaikwad, Siti Nurul Hidayah, Laura Heikaus, Hartmut Schlüter, Oliver Kohlbacher.
      Top-down mass spectrometry (TD-MS)-based proteomics analyzes intact proteoforms and thus preserves information about individual protein species. The MS signal of these high-mass analytes is complex and challenges the accurate determination of proteoform masses. Fast and accurate feature deconvolution (i.e., the determination of intact proteoform masses) is, therefore, an essential step for TD data analysis. Here, we present FLASHDeconv, an algorithm achieving higher deconvolution quality, with an execution speed two orders of magnitude faster than existing approaches. FLASHDeconv transforms peak positions (m/z) within spectra into log m/z space. This simple transformation turns the deconvolution problem into a search for constant patterns, thereby greatly accelerating the process. In both simple and complex samples, FLASHDeconv reports more genuine feature masses and substantially fewer artifacts than other existing methods. FLASHDeconv is freely available for download here: https://www.openms.org/flashdeconv/. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.
    Keywords:  feature deconvolution; intact protein analysis; mass deconvolution; proteoform characterization; top-down proteomics
    DOI:  https://doi.org/10.1016/j.cels.2020.01.003
  16. Endocr Connect. 2020 Feb 01. pii: EC-19-0515.R1. [Epub ahead of print]
    Glutamine metabolism in the proliferation of GS-expression pituitary tumor cells.
    Jintao Hu, Qingbo Chen, Xiao Ding, Xin Zheng, Xuefeng Tang, Song Li, Hui Yang.
      OBJECTIVE: Many cancer cells cannot survive without exogenous glutamine (Gln)，however, cancer cells expressed glutamine synthetase (GS) do not have this restriction. Previous metabolomics studies have indicated that glutamine metabolism is altered during pituitary tumorigenesis. However, the main role of Gln in pituitary adenoma (PA) pathophysiology remains unknown. The aim of this study was to evaluate the expression of GS and the main role of Gln in human PAs.METHODS: We used cell proliferation assay and flow cytometry to assess the effect of Gln depletion on three different pituitary cell lines and human primary PA cells. Then investigated the expression level of Gln synthetase (GS) in 24 human PA samples. At last, we used LC-MS/MS to identify the differences in metabolites of PA cells after the blockage of both endogenous and exogenous Gln.
    RESULTS: PA cell lines showed different sensitivities to Gln starvation, and the sensitivity is correlated with GS expression level. GS expressed in 21 out of the 24 human PA samples. Furthermore, a positive p53 and ki-67 index was correlated with a higher GS expression level (p<0.05). Removal of both endogenous and exogenous Gln from GS-expressing PA cells resulted in blockage of nucleotide metabolism and cell cycle arrest.
    CONCLUSIONS: Our data indicate that GS is needed for PA cells to proliferation during Gln deprivation, and most human PA cells express GS and might have a negative response to exogenous Gln depletion. Moreover, Gln is mainly responsible for nucleotide metabolism in the proliferation of GS-expressing pituitary tumor cells.
    DOI:  https://doi.org/10.1530/EC-19-0515
  17. Annu Rev Anal Chem (Palo Alto Calif). 2020 Feb 21.
    Mass Spectrometry of Human Transporters.
    Brahim Achour, Zubida Al-Majdoub, Amin Rostami-Hodjegan, Jill Barber.
      Transporters are key to understanding how an individual will respond a particular dose of a drug. Two patients with similar systemic concentrations may have quite different local concentrations of a drug at the required site. The transporter profile of any individual depends upon a variety of genetic and environmental factors, including genotype, age, and diet status. Robust models (virtual patients) are therefore required and these models are data hungry. Necessary data include quantitative transporter profiles at the relevant organ. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is currently the most powerful method available for obtaining this information. Challenges include sourcing the tissue, isolating the hydrophobic membrane-embedded transporter proteins, preparing the samples for MS (including proteolytic digestion), choosing appropriate quantification methodology, and optimizing the LC-MS/MS conditions. Great progress has been made with all of these, especially within the last few years, and is discussed here. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 13 is June 12, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-anchem-091719-024553
  18. Analyst. 2020 Feb 17.
    Paper spray mass spectrometry for the analysis of picoliter droplets.
    Ravleen Kaur Kohli, James F Davies.
      Recent experimental efforts have shown that single particle levitation methods may be effectively coupled with mass spectrometry (MS) using paper spray (PS) ionization for compositional analysis of picoliter droplets. In this work, we characterize the response of PS-MS to analytes delivered in the form of picoliter droplets and explore its potential for identification and quantification of these samples. Using a microdroplet dispenser to generate droplets, we demonstrate sensitivity to a range of oxygenated organic molecules typical of compounds found in atmospheric secondary organic aerosol. We assess experimental factors that influence the reproducibility and sensitivity of the method and explore the linearity of the system response to increasing analyte mass in droplets containing single or multicomponent analytes. We show that the ratio of analyte signal from multicomponent samples may be used to characterize the relative composition of the system. These measurements demonstrate that the droplet PS-MS method is an effective tool for qualitative and quantitative analysis of single picoliter droplets containing picogram levels of analyte. The potential applications of this technique for characterizing the composition of levitated particles will be discussed.
    DOI:  https://doi.org/10.1039/c9an02534k
  19. J Proteome Res. 2020 Feb 19.
    COLMAR Lipids Web Server and Ultrahigh-Resolution Methods for 2D NMR- and MS-Based Lipidomics.
    Cheng Wang, Istvan Timari, Bo Zhang, Da-Wei Li, Abigail Leggett, Amal O Amer, Lei Bruschweiler-Li, Rachel E Kopec, Rafael Bruschweiler.
      Accurate identification of lipids in biological samples is a key step in lipidomics studies. Multidimensional NMR spectroscopy is a powerful analytical tool for this purpose as it provides comprehensive structural information on lipid composition at atomic resolution. However, the interpretation of NMR spectra of complex lipids mixtures is currently hampered by limited spectral resolution and the absence of a customized lipids NMR databases along with user-friendly spectral analysis tools. We introduce a new 2D HSQC metabolite database "COLMAR Lipids" that was specifically curated for hydrophobic metabolites presently containing 501 compounds with accurate experimental 2D 13C-1H HSQC chemical shift data measured in CDCl3. A new module in the public COLMAR suite of NMR web servers was developed for the semi-automated analysis of complex lipidomics mixtures (http://spin.ccic.osu.edu/index.php/colmarm/index2). To obtain 2D HSQC spectra with the necessary high spectral resolution along both 13C and 1H dimensions, non-uniform sampling (NUS) in combination with pure shift spectroscopy was applied allowing the extraction of an abundance of unique cross-peaks belonging to hydrophobic compounds in complex lipidomics mixtures. As shown here, this information is critical for the unambiguous identification of underlying lipid molecules by means of the new COLMAR Lipids web server as is demonstrated for Caco-2 cell and lung tissue cell extracts.
    DOI:  https://doi.org/10.1021/acs.jproteome.9b00845
  20. Expert Rev Proteomics. 2020 Feb 20. 1-13
    Progress and pitfalls of using isobaric mass tags for proteome profiling.
    Loïc Dayon, Michael Affolter.
      Introduction: Quantitative proteomics using mass spectrometry is performed via label-free or label-based approaches. Labeling strategies rely on the incorporation of stable heavy isotopes by metabolic, enzymatic, or chemical routes. Isobaric labeling uses chemical labels of identical masses but of different fragmentation behaviors to allow the relative quantitative comparison of peptide/protein abundances between biological samples.Areas covered: We have carried out a systematic review on the use of isobaric mass tags in proteomic research since their inception in 2003. We focused on their quantitative performances, their multiplexing evolution, as well as their broad use for relative quantification of proteins in pre-clinical models and clinical studies. Current limitations, primarily linked to the quantitative ratio distortion, as well as state-of-the-art and emerging solutions to improve their quantitative readouts are discussed.Expert opinion: The isobaric mass tag technology offers a unique opportunity to compare multiple protein samples simultaneously, allowing higher sample throughput and internal relative quantification for improved trueness and precision. Large studies can be performed when shared reference samples are introduced in multiple experiments. The technology is well suited for proteome profiling in the context of proteomic discovery studies.
    Keywords:  Proteomics; quantification; tagging
    DOI:  https://doi.org/10.1080/14789450.2020.1731309
  21. Clin Exp Pharmacol Physiol. 2020 Feb 18.
    Lipidomics reveals the dynamics of lipid profile altered by omega-3 polyunsaturated fatty acid supplementation in healthy people.
    Meng Yan, Wen-Bin Cai, Tong Hua, Qian Cheng, Ding Ai, Hong-Feng Jiang, Xu Zhang.
      Glycerophospholipids (GPs) and sphingolipids (SPs) are important lipid components in the body and play biological functions. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are important nutrients, and their supplements are commonly used for preventing some diseases. However, the effect of n-3 PUFAs on the human glycerophospholipidome and sphingolipidome is unclear. We used targeted lipidomics to study the GP and SP profile of healthy individuals after supplementation with n-3 PUFAs for 3, 7, 14 and 21 days. Fuzzy c-means clustering was used to cluster the lipid species into 6 classes reflecting different changed-content patterns after n-3 PUFA supplementation. Among the species with significantly changed content, lysophospholipids were the most sensitive; their content started to increase on day 3. The content of phosphatidylserines increased at a later stage. The content of most of the phosphatidylcholines and alkylphosphatidylcholines decreased on day 21. A correlation network analysis of lipid species suggested that some enzymes involved in the metabolism of lysophospholipids and phosphatidylserines were regulated by n-3 PUFAs. Levels of creatine kinase-MB (CK-MB), urea, glucose, triglycerides and total bilirubin were altered by n-3 PUFA at 21 days. Correlation analysis revealed that the level of CK-MB was negatively correlated with those of species in lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine and phosphatidylserine classes, which were increased by n-3 PUFA supplementation. With the analysis in this work, we demonstrated the regular pattern of n-3 PUFAs on GP and SP metabolism, which provides a pharmacological basis for n-3 PUFAs for clinical application.
    Keywords:  glycerophospholipids; lipidomic profile; omega-3 polyunsaturated fatty acid; sphingolipids
    DOI:  https://doi.org/10.1111/1440-1681.13285
  22. Clin Chem Lab Med. 2020 Feb 21. pii: /j/cclm.ahead-of-print/cclm-2019-0869/cclm-2019-0869.xml. [Epub ahead of print]
    A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay to profile 20 plasma steroids in endocrine disorders.
    Zhenxin Wang, Hao Wang, Yingfei Peng, Fangjun Chen, Lin Zhao, Xiaomu Li, Jiaqian Qin, Qianqian Li, Beili Wang, Baishen Pan, Wei Guo.
      Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assays are employed in more and more clinical laboratories to quantify steroids. The steroid quantification by LC-MS/MS shows great value in screening or diagnosing endocrine disorders; however, the number of functional steroids included in the LC-MS/MS methods is still limited. Methods Here, we describe the performance and validation of a 20-steroid plasma panel by LC-MS/MS. The panel included progestogens (including mineralocorticoids and glucocorticoids), androgens and estrogens biosynthesized in steroid metabolic pathways. The LC-MS/MS method was validated according to guidance documents, and subsequently employed to profile steroid changes in endocrine disorders. Results Using LC-MS/MS, 20 steroids were separated and quantified in 8 min. Coefficients of variation (CVs) of the 20 analytes at the lower limit of quantification (LLoQ) were all less than 15% (ranging from 1.84% to 14.96%). The linearity of the assay was demonstrated by all the R2 values greater than 0.995. Individual plasma steroids changed significantly in patients with subclinical Cushing's syndrome (SCS) and polycystic ovary syndrome (PCOS) - 17-hydroxypregnenolone (17-OH-PR), testosterone (T) and dihydrotestosterone (DHT) were significantly decreased in SCS patients, while in PCOS patients, pregnenolone, corticosterone (CORT), androstenedione (A4) and T were significantly increased and DHT was decreased. Conclusions The LC-MS/MS method we developed for the quantification of 20 plasma steroids is clinical practicable. The steroid profiling data using this assay indicate its screening value for endocrine disorders. To further explore the value of the assay, more investigations are however needed.
    Keywords:  endocrine disorders; liquid chromatography-tandem mass spectrometry (LC-MS/MS); panel; pathway; steroids
    DOI:  https://doi.org/10.1515/cclm-2019-0869
  23. Nat Chem Biol. 2020 Mar;16(3): 302-309
    Cytochrome P450 oxidoreductase contributes to phospholipid peroxidation in ferroptosis.
    Yilong Zou, Haoxin Li, Emily T Graham, Amy A Deik, John K Eaton, Wenyu Wang, Gerardo Sandoval-Gomez, Clary B Clish, John G Doench, Stuart L Schreiber.
      Ferroptosis is widely involved in degenerative diseases in various tissues including kidney, liver and brain, and is a targetable vulnerability in multiple primary and therapy-resistant cancers. Accumulation of phospholipid hydroperoxides in cellular membranes is the hallmark and rate-limiting step of ferroptosis; however, the enzymes contributing to lipid peroxidation remain poorly characterized. Using genome-wide, CRISPR-Cas9-mediated suppressor screens, we identify cytochrome P450 oxidoreductase (POR) as necessary for ferroptotic cell death in cancer cells exhibiting inherent and induced susceptibility to ferroptosis. By genetic depletion of POR in cancer cells, we reveal that POR contributes to ferroptosis across a wide range of lineages and cell states, and in response to distinct mechanisms of ferroptosis induction. Using systematic lipidomic profiling, we further map POR's activity to the lipid peroxidation step in ferroptosis. Hence, our work suggests that POR is a key mediator of ferroptosis and potential druggable target for developing antiferroptosis therapeutics.
    DOI:  https://doi.org/10.1038/s41589-020-0472-6
  24. Biotechnol Rep (Amst). 2020 Mar;25 e00424
    At-line high throughput site-specific glycan profiling using targeted mass spectrometry.
    Bertie Chi, Christel Veyssier, Toyin Kasali, Faisal Uddin, Christopher A Sellick.
      Protein post-translational modification (PTM) plays an important role in many biological processes; of which glycosylation is arguably one of the most complex and diverse modifications and is crucial for the safety and efficacy of biotherapeutic proteins. Mass spectrometric characterization of protein glycosylation is well established with clear advantages and disadvantages; on one hand it is precise and information-rich, as well as being relative inexpensive in terms of the reagents and consumables despite the instrumentation cost and, depending on the method, can give site specific information; on the other hand it generally suffers from low throughput, restriction to largely purified samples and is less quantitative, especially for sialylated glycan species. Here, we describe a high throughput, site-specific, targeted mass spectrometric peptide mapping approach to quickly screen/rank candidate production cell lines and culture conditions that give favourable glycosylation profiles directly from conditioned culture media for an Fc-fusion protein. The methodology is fully compatible with automation and combines the speed of 'top-down' mass spectrometry with the site-specific information of 'bottom-up' mass spectrometry. In addition, this strategy can be used for multi-attribute product quality screening/monitoring as an integral part of cell line selection and process development.
    Keywords:  2-AB, 2-aminobenzamide labelled UPLC glycan analysis; CM, conditioned media; Cell line selection; DoE, design of experiment; ESI, electrospray ionization; Glycan profiling; Glycosylation; PKPD, pharmacokinetic pharmacodynamic; PTM, post-translation modification; QBD, quality by design; TQ-MS, triple quadrupole mass spectrometry; Targeted mass spectrometry
    DOI:  https://doi.org/10.1016/j.btre.2020.e00424
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