bims-mascan 2019-07-07 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2019–07–07
25 papers selected by
Giovanny Rodriguez Blanco, The Beatson Institute for Cancer Research

Blood Vessel Proximity Shapes Cancer Cell Metabolism.
Metabolite Exchange between Mammalian Organs Quantified in Pigs.
Steroyl-CoA Desaturase 1 (SCD1) protects ovarian cancer cells from ferroptotic cell death.
Changes in whole metabolites after exenatide treatment in overweight/obese polycystic ovary syndrome patients.
Inhibition of Pancreatic Carcinoma Growth Through Enhancing ω-3 Epoxy Polyunsaturated Fatty Acid Profile by Inhibition of Soluble Epoxide Hydrolase.
Iron-dependent histone 3 lysine 9 demethylation controls B cell proliferation and humoral immune responses.
Yin-Yang Mechanisms Regulating Lipid Peroxidation of Docosahexaenoic Acid and Arachidonic Acid in the Central Nervous System.
Adipocyte and lipid metabolism in cancer drug resistance.
Targeted metabolomics for serum amino acids and acylcarnitines in patients with lung cancer.
Nutrient availability shapes methionine metabolism in p16/MTAP-deleted cells.
Metabolomic profiling suggests systemic signatures of premature aging induced by Hutchinson-Gilford progeria syndrome.
Tissue-Specific Sample Dilution: An Important Parameter to Optimise Prior to Untargeted LC-MS Metabolomics.
Oxylipin profiling in endothelial cells in vitro - Effects of DHA and hydrocortisone upon an inflammatory challenge.
AP3: An Advanced Proteotypic Peptide Predictor for Targeted Proteomics by Incorporating Peptide Digestibility.
Small Molecule Isotope Resolved Formula Enumeration: A Methodology for Assigning Isotopologues and Metabolite Formulas in Fourier Transform Mass Spectra.
Advances in the Regulatory Effects of Bioactive Peptides on Metabolic Signaling Pathways in Tumor Cells.
The urinary bile acid profiling analysis of asymptomatic hypercholanemia of pregnancy: A pseudo-targeted metabolomics study.
LC-MS bioanalysis of intact proteins and peptides.
Reprogramming of Amino Acid Transporters to Support Aspartate and Glutamate Dependency Sustains Endocrine Resistance in Breast Cancer.
Systems structural biology measurements by in vivo cross-linking with mass spectrometry.
Potential Use of Gluconate in Cancer Therapy.
Acyl-Coenzyme A: Cholesterol Acyltransferase Inhibition in Cancer Treatment.
Quantification of Drug Molecules in Live Single Cells Using the Single-Probe Mass Spectrometry Technique.
Quantitation of Super Basic Peptides in Biological Matrices by a Generic Perfluoropentanoic Acid-Based Liquid Chromatography-Mass Spectrometry Method.
Human Plasma Metabolomics in Age-Related Macular Degeneration: Meta-Analysis of Two Cohorts.

Cell Metab. 2019 Jul 02. pii: S1550-4131(19)30314-6. [Epub ahead of print]30(1): 16-18

Blood Vessel Proximity Shapes Cancer Cell Metabolism.

Federico Virga, Manuel Ehling, Massimiliano Mazzone.

Cancer cells are highly heterogeneous, and their features markedly vary within different areas of the tumor microenvironment. In this issue, Kumar et al. (2019) identified perivascular tumor cells, derived from mouse glioblastoma xenografts, as the fraction that displays the highest mTOR-dependent anabolic metabolism, aggressiveness, and resistance to therapy.

DOI: https://doi.org/10.1016/j.cmet.2019.06.011
Cell Metab. 2019 Jun 26. pii: S1550-4131(19)30305-5. [Epub ahead of print]

Metabolite Exchange between Mammalian Organs Quantified in Pigs.

Cholsoon Jang, Sheng Hui, Xianfeng Zeng, Alexis J Cowan, Lin Wang, Li Chen, Raphael J Morscher, Jorge Reyes, Christian Frezza, Ho Young Hwang, Akito Imai, Yoshiaki Saito, Keitaro Okamoto, Christine Vaspoli, Loewe Kasprenski, Gerald A Zsido, Joseph H Gorman, Robert C Gorman, Joshua D Rabinowitz.

  Mammalian organs continually exchange metabolites via circulation, but systems-level analysis of this shuttling process is lacking. Here, we compared, in fasted pigs, metabolite concentrations in arterial blood versus draining venous blood from 11 organs. Greater than 90% of metabolites showed arterial-venous differences across at least one organ. Surprisingly, the liver and kidneys released not only glucose but also amino acids, both of which were consumed primarily by the intestine and pancreas. The liver and kidneys exhibited additional unexpected activities: liver preferentially burned unsaturated over more atherogenic saturated fatty acids, whereas the kidneys were unique in burning circulating citrate and net oxidizing lactate to pyruvate, thereby contributing to circulating redox homeostasis. Furthermore, we observed more than 700 other cases of tissue-specific metabolite production or consumption, such as release of nucleotides by the spleen and TCA intermediates by pancreas. These data constitute a high-value resource, providing a quantitative atlas of inter-organ metabolite exchange.

Keywords:  circulating metabolite; flux; fuel; inter-organ exchange; isotope tracing; mammalian organ-specific metabolism; metabolomics; pig; tissue; uptake and release

DOI:  https://doi.org/10.1016/j.cmet.2019.06.002
Cancer Res. 2019 Jul 03. pii: canres.0369.2019. [Epub ahead of print]

Steroyl-CoA Desaturase 1 (SCD1) protects ovarian cancer cells from ferroptotic cell death.

Lia Tesfay, Bibbin T Paul, Anna Konstorum, Zhiyong Deng, Anderson O Cox, Jingyun Lee, Cristina M Furdui, Poornima Hegde, Frank M Torti, Suzy V Torti.

Activation of ferroptosis, a recently described mechanism of regulated cell death, dramatically inhibits growth of ovarian cancer cells. Given the importance of lipid metabolism in ferroptosis and the key role of lipids in ovarian cancer, we examined the contribution to ferroptosis of steroyl CoA desaturase (SCD1), an enzyme that catalyzes the rate-limiting step in monounsaturated fatty acid synthesis, in ovarian cancer cells. SCD1 was highly expressed in ovarian cancer tissue, cell lines, and a genetic model of ovarian cancer stem cells. Inhibition of SCD1 induced lipid oxidation and cell death. Conversely, over-expression of SCD1 or exogenous administration of its C16:1 and C18:1 products, palmitoleicic acid or oleate, protected cells from death. Inhibition of SCD1 induced both ferroptosis and apoptosis: inhibition of SCD1 decreased CoQ10, an endogenous membrane antioxidant whose depletion has been linked to ferroptosis, while concomitantly decreasing unsaturated fatty acyl chains in membrane phospholipids and increasing long chain saturated ceramides, changes previously linked to apoptosis. Simultaneous triggering of two death pathways suggests SCD1 inhibition may be an effective component of anti-tumor therapy, since overcoming this dual mechanism of cell death may present a significant barrier to the emergence of drug resistance. Supporting this concept, we observed that inhibition of SCD1 significantly potentiated the anti-tumor effect of ferroptosis inducers in both ovarian cancer cell lines and a mouse orthotopic xenograft model. Our results suggest that the use of combined treatment with SCD1 inhibitors and ferroptosis inducers may provide a new therapeutic strategy for patients with ovarian cancer.

DOI: https://doi.org/10.1158/0008-5472.CAN-19-0369
Clin Endocrinol (Oxf). 2019 Jul 04.

Changes in whole metabolites after exenatide treatment in overweight/obese polycystic ovary syndrome patients.

Liang Tang, Lei Yuan, Gangyi Yang, Feng Wang, Mao Fu, Min Chen, Dongfang Liu.

   OBJECTIVE: Exenatide is a new agent for diabetes therapy, and its use in polycystic ovary syndrome (PCOS) has gradually increased; however, the clinical benefit and metabolic improvement need further evidence. This research aimed to study the changes in whole metabolites before and after exenatide treatment in overweight/obese PCOS patients to gain a better understanding of exenatide for the treatment of PCOS.
METHODS: Sixty-seven women, including 32 with PCOS and 35 age-matched controls, were recruited. The metabolite changes were detected with non-targeted gas chromatography tandem mass spectrometry (NTGC-MS) before and after exenatide treatment, and changes in clinical biochemical characteristics were also observed.
RESULTS: A total of 62 metabolites were differentially expressed between the healthy and PCOS groups, and 31 differentially expressed metabolites were detected before and after exenatide treatment. Abnormal lipid metabolism and amino acid metabolism were the main metabolic disorders. Exenatide improved lipid and amino acid metabolism, especially amino acid metabolites. Three types of branched-chain amino acids (valine, leucine, and isoleucine), two types of aromatic amino acids (phenylalanine and tyrosine) and lysine are important potential metabolites for the therapeutic efficacy of exenatide. Many abnormal metabolic disorders are related to insulin resistance, oxidative stress, and even ovulatory dysfunction. Moreover, in this small sample clinical study, we also found that exenatide improved insulin sensitivity, reduced body weight and improved glycolipid metabolism in PCOS.
CONCLUSIONS: NTGC-MS-based metabolic pathway analysis revealed that exenatide has a beneficial effect on overweight/obese PCOS patients by regulating metabolic disorders, especially amino acid disorders. This article is protected by copyright. All rights reserved.

Keywords:   PCOS ; Exenatide; Metabolomics

DOI:  https://doi.org/10.1111/cen.14056
Anticancer Res. 2019 Jul;39(7): 3651-3660

Inhibition of Pancreatic Carcinoma Growth Through Enhancing ω-3 Epoxy Polyunsaturated Fatty Acid Profile by Inhibition of Soluble Epoxide Hydrolase.

Rong Xia, Leyu Sun, Jie Liao, Haonan Li, Xiaoming You, Dandan Xu, Jun Yang, Sung Hee Hwang, Ryan D Jones, Bruce Hammock, Guang-Yu Yang.

   BACKGROUND/AIM: Cytochrome P450 epoxygenase is a major enzyme involved in the metabolism of ω-3 polyunsaturated fatty acids (PUFAs) to produce biologically active ω-3 epoxy fatty acids (ω-3 epoxides). In general, all epoxy PUFAs including ω-3 epoxides are quickly metabolized/inactivated by soluble epoxide hydrolase (sEH) to form diol products. The aims of this study were to determine the effect and mechanism of fat-1 transgene, and ω-3 PUFA combined with sEH gene knockout or inhibitor on inhibiting pancreatic cancer and the related mechanisms involved.
MATERIALS AND METHODS: PK03-mutant KrasG12D murine pancreatic carcinoma cells were inoculated into mouse models including fat-1, sEH-/- and C57BL/6J mice. The mice were fed with AIN-76A diet with or without ω-3 PUFA supplementation or treated with sEH inhibitor. In addition to tumor growth (tumor size and weight), cell proliferation, mutant Kras-mediated signaling, inflammatory reaction and angiogenesis were analyzed immunohisto-chemically and by western blot assay. ω-3 PUFA metabolism, particularly focusing on ω-3 epoxy fatty acids (ω-3 epoxides), was measured using a liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach.
RESULTS: Significant decreases of weight and size of the PK03 pancreatic carcinoma were observed in the fat-1 transgenic mice treated with sEH inhibitor compared to those of C57BL/6J control mice fed with AIN-76A diet (weight: 0.28±0.04 g vs. 0.58±0.06 g; size: 187.0±17.5 mm3 vs. 519.3±60.6 mm3). In a separate experiment, sEH-/- mice fed ω-3 PUFA supplement and C57BL/6J mice treated with sEH inhibitor and fed ω-3 PUFA supplement exhibited a significant reduction in the weight and size of the pancreatic carcinoma compared to C57BL/6J control mice (weight: 0.26±.26 g and 0.39±.39 g vs. 0.69±0.11 g, respectively; size: 274.2±36.2 mm3 and 296.4±99.8 mm3 vs. 612.6±117.8 mm3, respectively). Moreover, compared to the pancreatic tumors in C57BL/6J control mice, the tumors in fat-1 transgenic mice treated with sEH inhibitor showed a significant less inflammatory cell infiltrate (62.6±9.2/HPF (high power field) vs. 8.0±1.2/HPF), tumor cell proliferation (48.5±1.7% vs. 16.5±1.6%), and angiogenesis (micro-vessel density (MVD): 35.0±1.0 vs. 11.1±0.5) immunohistochemically, as well as significantly increased caspase-3 labeled apoptosis (0.44±0.06% vs. 0.69±0.06%, respectively). Using western blot approach, significant inhibition of mutant Kras-activated signals including phosphorylated Serine/threonine kinases (cRAF), Mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK) were identified in pancreatic carcinoma of fat-1 transgenic mice treated with sEH inhibitor. Eicosanoic acid metabolic profiling of the serum specimens detected a significant increase of the ratios of epoxides to dihydroxy fatty acid (DiHDPE) for docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and epoxides/dihydroxy octadecenoic acid (DiHOME) for arachidonic acid (ARA) and linoleic acid (LA), as well as a significant increase of epoxy metabolites of DHA, EPA, ARA and LA in fat-1 transgenic mice treated with a sEH inhibitor.
CONCLUSION: ω-3 epoxy products from ω-3 PUFA metabolism play a crucial role in inhibiting pancreatic cancer growth, and use of ω-3 PUFAs combined with sEH inhibition is a strategy with high potential for pancreatic cancer treatment and prevention.

Keywords:  Polyunsaturated fatty acid; epoxy fatty acid; fat-1; pancreatic cancer; soluble epoxide hydrolase

DOI:  https://doi.org/10.21873/anticanres.13513
Nat Commun. 2019 Jul 03. 10(1): 2935

Iron-dependent histone 3 lysine 9 demethylation controls B cell proliferation and humoral immune responses.

Yuhang Jiang, Cuifeng Li, Qian Wu, Peng An, Laiquan Huang, Jia Wang, Chen Chen, Xi Chen, Fan Zhang, Li Ma, Sanhong Liu, Hanqing He, Shuyun Xie, Yangbai Sun, Hanshao Liu, Yu Zhan, Yu Tao, Zhi Liu, Xiaohua Sun, Yiming Hu, Qi Wang, Deji Ye, Jie Zhang, Shanhua Zou, Ying Wang, Gang Wei, Yongzhong Liu, Yufang Shi, Y Eugene Chin, Yongqiang Hao, Fudi Wang, Xiaoren Zhang.

Trace elements play important roles in human health, but little is known about their functions in humoral immunity. Here, we show an important role for iron in inducing cyclin E and B cell proliferation. We find that iron-deficient individuals exhibit a significantly reduced antibody response to the measles vaccine when compared to iron-normal controls. Mice with iron deficiency also exhibit attenuated T-dependent or T-independent antigen-specific antibody responses. We show that iron is essential for B cell proliferation; both iron deficiency and α-ketoglutarate inhibition could suppress cyclin E1 induction and S phase entry of B cells upon activation. Finally, we demonstrate that three demethylases, KDM2B, KDM3B and KDM4C, are responsible for histone 3 lysine 9 (H3K9) demethylation at the cyclin E1 promoter, cyclin E1 induction and B cell proliferation. Thus, our data reveal a crucial role of H3K9 demethylation in B cell proliferation, and the importance of iron in humoral immunity.

DOI: https://doi.org/10.1038/s41467-019-11002-5
Front Neurol. 2019 ;10 642

Yin-Yang Mechanisms Regulating Lipid Peroxidation of Docosahexaenoic Acid and Arachidonic Acid in the Central Nervous System.

Bo Yang, Kevin L Fritsche, David Q Beversdorf, Zezong Gu, James C Lee, William R Folk, C Michael Greenlief, Grace Y Sun.

  Phospholipids in the central nervous system (CNS) are rich in polyunsaturated fatty acids (PUFAs), particularly arachidonic acid (ARA) and docosahexaenoic acid (DHA). Besides providing physical properties to cell membranes, these PUFAs are metabolically active and undergo turnover through the "deacylation-reacylation (Land's) cycle". Recent studies suggest a Yin-Yang mechanism for metabolism of ARA and DHA, largely due to different phospholipases A2 (PLA2s) mediating their release. ARA and DHA are substrates of cyclooxygenases and lipoxygenases resulting in an array of lipid mediators, which are pro-inflammatory and pro-resolving. The PUFAs are susceptible to peroxidation by oxygen free radicals, resulting in the production of 4-hydroxynonenal (4-HNE) from ARA and 4-hydroxyhexenal (4-HHE) from DHA. These alkenal electrophiles are reactive and capable of forming adducts with proteins, phospholipids and nucleic acids. The perceived cytotoxic and hormetic effects of these hydroxyl-alkenals have impacted cell signaling pathways, glucose metabolism and mitochondrial functions in chronic and inflammatory diseases. Due to the high levels of DHA and ARA in brain phospholipids, this review is aimed at providing information on the Yin-Yang mechanisms for regulating these PUFAs and their lipid peroxidation products in the CNS, and implications of their roles in neurological disorders.

Keywords:  4-hydroxyhexenal; 4-hydroxynonenal; arachidonic acid; cPLA2; docosahexaenoic acid; iPLA2; lipid peroxidation; neurodegeneration

DOI:  https://doi.org/10.3389/fneur.2019.00642
J Clin Invest. 2019 Jul 02. pii: 127201. [Epub ahead of print]130

Adipocyte and lipid metabolism in cancer drug resistance.

Yihai Cao.

Development of novel and effective therapeutics for treating various cancers is probably the most congested and challenging enterprise of pharmaceutical companies. Diverse drugs targeting malignant and nonmalignant cells receive clinical approval each year from the FDA. Targeting cancer cells and nonmalignant cells unavoidably changes the tumor microenvironment, and cellular and molecular components relentlessly alter in response to drugs. Cancer cells often reprogram their metabolic pathways to adapt to environmental challenges and facilitate survival, proliferation, and metastasis. While cancer cells' dependence on glycolysis for energy production is well studied, the roles of adipocytes and lipid metabolic reprogramming in supporting cancer growth, metastasis, and drug responses are less understood. This Review focuses on emerging mechanisms involving adipocytes and lipid metabolism in altering the response to cancer treatment. In particular, we discuss mechanisms underlying cancer-associated adipocytes and lipid metabolic reprogramming in cancer drug resistance.

DOI: https://doi.org/10.1172/JCI127201
Exp Ther Med. 2019 Jul;18(1): 188-198

Targeted metabolomics for serum amino acids and acylcarnitines in patients with lung cancer.

Junjun Ni, Li Xu, Wei Li, Chunmei Zheng, Lijun Wu.

  Lung cancer is one of the most prevalent types of cancer, but accurate diagnosis remains a challenge. The aim of the present study was to create a model using amino acids and acylcarnitines for lung cancer screening. Serum samples were obtained from two groups of patients with lung cancer recruited in 2015 (including 40 patients and 100 matched controls) and 2017 (including 17 patients and 30 matched controls). Using a metabolomics method, 21 metabolites (13 types of amino acids and 8 types of acylcarnitines) were measured using liquid chromatography-tandem mass spectrometry. Data (from the 2015 and 2017 data sets) were analysed using a Mann-Whitney U test, Student's t-test, Welch's F test, receiver-operator characteristic curve or logistic regression in order to investigate the potential biomarkers. Six metabolites (glycine, valine, methionine, citrulline, arginine and C16-carnitine) were indicated to be involved in distinguishing patients with lung cancer from healthy controls. The six discriminating metabolites from the 2017 data set were further analysed using Partial least squares-discriminant analysis (PLS-DA). The PLS-DA model was verified using Spearman's correlation analysis and receiver operating characteristic curve analysis. These results demonstrated that the PLS-DA model using the six metabolites (glycine, valine, methionine, citrulline, arginine and C16-carnitine) had a strong ability to identify lung cancer. Therefore, the PLS-DA model using glycine, valine, methionine, citrulline, arginine and C16-carnitine may become a novel screening tool in patients with lung cancer.

Keywords:  acylcarnitines; amino acids; liquid chromatography-tandem mass spectrometry; lung cancer; metabolomics

DOI:  https://doi.org/10.3892/etm.2019.7533
Sci Adv. 2019 Jun;5(6): eaav7769

Nutrient availability shapes methionine metabolism in p16/MTAP-deleted cells.

Sydney M Sanderson, Peter G Mikhael, Vijyendra Ramesh, Ziwei Dai, Jason W Locasale.

Codeletions of gene loci containing tumor suppressors and neighboring metabolic enzymes present an attractive synthetic dependency in cancers. However, the impact that these genetic events have on metabolic processes, which are also dependent on nutrient availability and other environmental factors, is unknown. As a proof of concept, we considered panels of cancer cells with homozygous codeletions in CDKN2a and MTAP, genes respectively encoding the commonly-deleted tumor suppressor p16 and an enzyme involved in methionine metabolism. A comparative metabolomics analysis revealed that while a metabolic signature of MTAP deletion is apparent, it is not preserved upon restriction of nutrients related to methionine metabolism. Furthermore, re-expression of MTAP exerts heterogeneous consequences on metabolism across isogenic cell pairs. Together, this study demonstrates that numerous factors, particularly nutrition, can overwhelm the effects of metabolic gene deletions on metabolism. These findings may also have relevance to drug development efforts aiming to target methionine metabolism.

DOI: https://doi.org/10.1126/sciadv.aav7769
Metabolomics. 2019 Jun 28. 15(7): 100

Metabolomic profiling suggests systemic signatures of premature aging induced by Hutchinson-Gilford progeria syndrome.

Gustavo Monnerat, Geisa Paulino Caprini Evaristo, Joseph Albert Medeiros Evaristo, Caleb Guedes Miranda Dos Santos, Gabriel Carneiro, Leonardo Maciel, Vânia Oliveira Carvalho, Fábio César Sousa Nogueira, Gilberto Barbosa Domont, Antonio Carlos Campos de Carvalho.

   INTRODUCTION: Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder. HGPS children present a high incidence of cardiovascular complications along with altered metabolic processes and an accelerated aging process. No metabolic biomarker is known and the mechanisms underlying premature aging are not fully understood.
OBJECTIVES: The present work aims to evaluate the metabolic alterations in HGPS using high resolution mass spectrometry.
METHODS: The present study analyzed plasma from six HGPS patients of both sexes (7.7 ± 1.4 years old; mean ± SD) and eight controls (8.6 ± 2.3 years old) by LC-MS/MS in high-resolution non-targeted metabolomics (Q-Exactive Plus). Targeted metabolomics was used to validate some of the metabolites identified by the non-targeted method in a triple quadrupole (TSQ-Quantiva).
RESULTS: We found several endogenous metabolites with statistical differences between control and HGPS children. Multivariate statistical analysis showed a clear separation between groups. Potential novel metabolic biomarkers were identified using the multivariate area under ROC curve (AUROC) based analysis, showing an AUC value higher than 0.80 using only two metabolites, and tending to 1.00 when increasing the number of metabolites in the AUROC model. Taken together, changed metabolic pathways involve sphingolipids, amino acids, and oxidation of fatty acids, among others.
CONCLUSION: Our data show significant alterations in cellular energy use and availability, in signal transduction, and lipid metabolites, adding new insights on metabolic alterations associated with premature aging and suggesting novel putative biomarkers.

Keywords:  Biomarkers; HGPS; Lamin; Metabolic profiling; Metabolomics; Premature aging

DOI:  https://doi.org/10.1007/s11306-019-1558-6
Metabolites. 2019 Jun 27. pii: E124. [Epub ahead of print]9(7):

Tissue-Specific Sample Dilution: An Important Parameter to Optimise Prior to Untargeted LC-MS Metabolomics.

Zhanxuan E Wu, Marlena C Kruger, Garth J S Cooper, Sally D Poppitt, Karl Fraser.

  When developing a sample preparation protocol for LC-MS untargeted metabolomics of a new sample matrix unfamiliar to the laboratory, selection of a suitable injection concentration is rarely described. Here we developed a simple workflow to address this issue prior to untargeted LC-MS metabolomics using pig adipose tissue and liver tissue. Bi-phasic extraction was performed to enable simultaneous optimisation of parameters for analysis of both lipids and polar extracts. A series of diluted pooled samples were analysed by LC-MS and used to evaluate signal linearity. Suitable injected concentrations were determined based on both the number of reproducible features and linear features. With our laboratory settings, the optimum concentrations of tissue mass to reconstitution solvent of liver and adipose tissue lipid fractions were found to be 125 mg/mL and 7.81 mg/mL respectively, producing 2811 (ESI+) and 4326 (ESI-) linear features from liver, 698 (ESI+) and 498 (ESI-) linear features from adipose tissue. For analysis of the polar fraction of both tissues, 250 mg/mL was suitable, producing 403 (ESI+) and 235 (ESI-) linear features from liver, 114 (ESI+) and 108 (ESI-) linear features from adipose tissue. Incorrect reconstitution volumes resulted in either severe overloading or poor linearity in our lipid data, while too dilute polar fractions resulted in a low number of reproducible features (<50) compared to hundreds of reproducible features from the optimum concentration used. Our study highlights on multiple matrices and multiple extract and chromatography types, the critical importance of determining a suitable injected concentration prior to untargeted LC-MS metabolomics, with the described workflow applicable to any matrix and LC-MS system.

Keywords:  LC–MS untargeted metabolomics; lipidomics; sample preparation for metabolomics; tissue metabolite profiling

DOI:  https://doi.org/10.3390/metabo9070124
Prostaglandins Other Lipid Mediat. 2019 Jun 28. pii: S1098-8823(18)30161-8. [Epub ahead of print] 106352

Oxylipin profiling in endothelial cells in vitro - Effects of DHA and hydrocortisone upon an inflammatory challenge.

A C Motta, K Strassburg, P Oranje, R J Vreeken, D M Jacobs.

  Omega-3 poly-unsaturated fatty acids have been shown to have beneficial effects on several inflammatory-driven endpoints such as cardiovascular diseases. The anti-inflammatory effects of docosahexaenoic acid (DHA) are largely mediated through various oxylipins. Yet, mechanistic insights are limited. Here, we measured 53 oxylipins using LC-MS/MS in an in vitro model of endothelial cell inflammation, and compared the changes induced by DHA to hydrocortisone, a well-established anti-inflammatory drug. DHA modified several oxylipins derived from different precursors such as DHA, AA, LA and EPA. In response to a TNFα and IL-1-β challenge, DHA clearly reduced many COX-derived pro-inflammatory oxylipins, yet to a minor extent when compared to hydrocortisone. DHA also upregulated metabolites from the CYP and LOX pathways as opposed to hydrocortisone. Thus, DHA reduced pro-inflammation and enhanced pro-resolution, while hydrocortisone blunted both the pro- and anti-inflammatory pathways. Our results may fuel further research on the mitigation of corticosteroids adverse side-effects.

Keywords:  DHA; LC-LC/MS; Oxylipins; hydrocortisone; inflammation; resolution

DOI:  https://doi.org/10.1016/j.prostaglandins.2019.106352
Anal Chem. 2019 Jul 02. 91(13): 8705-8711

AP3: An Advanced Proteotypic Peptide Predictor for Targeted Proteomics by Incorporating Peptide Digestibility.

Zhiqiang Gao, Cheng Chang, Jinghan Yang, Yunping Zhu, Yan Fu.

The selection of proteotypic peptides, that is, detectable unique representatives of proteins of interest, is a key step in targeted proteomics. To date, much effort has been made to understand the mechanisms underlying peptide detection in liquid chromatography-tandem mass spectrometry (LC-MS/MS) based shotgun proteomics and to predict proteotypic peptides in the absence of experimental LC-MS/MS data. However, the prediction accuracy of existing tools is still unsatisfactory. We find that one crucial reason is their neglect of the significant influence of protein proteolytic digestion on peptide detectability in shotgun proteomics. Here, we present an Advanced Proteotypic Peptide Predictor (AP3), which explicitly takes peptide digestibility into account for the prediction of proteotypic peptides. Specifically, peptide digestibility is first predicted for each peptide and then incorporated as a feature into the peptide detectability prediction model. Our results demonstrated that peptide digestibility is the most important feature for the accurate prediction of proteotypic peptides in our model. Compared with the existing available algorithms, AP3 showed 10.3-34.7% higher prediction accuracy. On a targeted proteomics data set, AP3 accurately predicted the proteotypic peptides for proteins of interest, showing great potential for assisting the design of targeted proteomics experiments.

DOI: https://doi.org/10.1021/acs.analchem.9b02520
Anal Chem. 2019 Jul 01.

Small Molecule Isotope Resolved Formula Enumeration: A Methodology for Assigning Isotopologues and Metabolite Formulas in Fourier Transform Mass Spectra.

Joshua M Mitchell, Robert M Flight, Hunter N B Moseley.

Improvements in Fourier transform mass spectrometry (FT-MS) enable increasingly more complex experiments in the field of metabolomics. What is directly detected in FT-MS spectra are spectral features (peaks) that correspond to sets of adducted and charged forms of specific molecules in the sample. The robust assignment of these features is an essential step for MS-based metabolomics experiments, but the sheer complexity of what is detected and a variety of analytically introduced variance, errors, and artifacts has hindered the systematic analysis of complex patterns of observed peaks with respect to isotope content. We have developed a method called SMIRFE that detects small biomolecules and determines their elemental molecular formula (EMF) using detected sets of isotopologue peaks sharing the same EMF. SMIRFE does not use a database of known metabolite formulas; instead a nearly comprehensive search space of all isotopologues within a mass range is constructed and used for assignment. This search space can be tailored for different isotope labeling patterns expected in different stable isotope tracing experiments. Using consumer-level computing equipment, a large search space of 2000 Da was constructed, and assignment performance was evaluated and validated using verified assignments on a pair of peak lists derived from spectra containing unlabeled and 15N-labeled versions of amino acids derivatized using ethylchloroformate. SMIRFE identified 18 of 18 predicted derivatized EMFs, and each assignment was evaluated statistically and assigned an e-value representing the probability to occur by chance.

DOI: https://doi.org/10.1021/acs.analchem.9b00748
J Cancer. 2019 ;10(11): 2425-2433

Advances in the Regulatory Effects of Bioactive Peptides on Metabolic Signaling Pathways in Tumor Cells.

Hongwei Cui, Wenyan Han, Junyao Zhang, Zhihui Zhang, Xiulan Su.

  Changes in cell metabolism are an important feature of tumors that has always been an intense topic of study, particularly in regard to whether metabolic disorders are a cause or an effect of tumorigenesis. Studies have shown that the processes underlying metabolic changes in tumors involve the activation of protooncogenes and the inactivation of cancer suppressor genes, as well as changes in metabolic flux in cells due to the abnormal activation of signaling pathways that modulate metabolic enzymes and/or metabolic regulatory proteins at several levels, including transfer and posttranslational modification. Thus, the repair of abnormal metabolic pathways via intervention in the relevant tumor metabolic pathways that impact specific targets has become a new method of cancer prevention and treatment. Bioactive peptides, which have many biological functions, could specifically target malignant tumors. Their interaction with signal transduction molecules involved in the development and transference of tumors could regulate the relevant cell metabolic pathways and inhibit the development of tumors and/or accelerate apoptosis in tumor cells. In this review, several aspects of tumor suppression using bioactive peptides will be discussed and summarized, including the regulation of the PI3K/AKT/mTOR, AMPK, and STST3 signaling pathways, the modulation of the TRAIL death receptor signaling pathway, the regulation of aerobic glycolysis by PKM2, and the modulation of the NF-кB signaling pathway, to aid in the search for better and more specific antineoplastic drugs in the form of bioactive peptides.

Keywords:  bioactive peptide; cell metabolism; signaling pathway; tumor

DOI:  https://doi.org/10.7150/jca.31359
Clin Chim Acta. 2019 Jul 02. pii: S0009-8981(19)31939-4. [Epub ahead of print]

The urinary bile acid profiling analysis of asymptomatic hypercholanemia of pregnancy: A pseudo-targeted metabolomics study.

Xiao Chen, Xiaoqing Zhang, Biao Xu, Yue Cui, Yifan He, Ting Yang, Yong Shao, Min Ding.

   BACKGROUND: Asymptomatic hypercholanemia of pregnancy (AHP) is a controversial hypercholanemia, which is difficult to distinguish from intrahepatic cholestasis of pregnancy (ICP). Our aim is to elucidate the characteristics of urinary bile acid (BA) profiling of women with AHP and to find potential biomarkers for the diagnosis and differential diagnosis of AHP.
METHODS: We developed a pseudo-targeted approach to perform metabolomics analysis of bile acids (BAs) using ultra-high performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Urinary BAs profiles were compared among AHP women (n = 20), ICP patients (n = 33) and normal controls (n = 35).
RESULTS: The profiling of urinary BAs was significantly different among the AHP, ICP and control groups. Compared to the control group, the AHP group had higher levels of four possible sulfated BAs and trihydroxy BAs, including the species of muricholic acid (MCA), cholic acid (CA) and six possible BAs, whereas, 20 possible sulfated BAs, taurochenodeoxycholic acid (TCDCA), tetrahydrocannabinolic acid (THCA), and seven possible BAs were significantly lower in the AHP group than those in the ICP group. Based on the receiver operating characteristic (ROC) analysis, glycocholic acid (GCA) combined with T-ω-MCA were found to be the potential combination biomarker for the diagnosis (area under the curve was 0.960) of AHP, and mono-S, Gtri-S-2 combined with TLCA-S were found to be the potential combination biomarker for the differential diagnosis (area under the curve was 0.990) of AHP and ICP.
CONCLUSIONS: The metabolisms of urinary Bas were altered in the AHP group compared with the ICP group and the control group. Urinary BA profiling analysis can serve as an effective tool for the diagnosis of AHP and the differential diagnosis of AHP and ICP.

Keywords:  Asymptomatic hypercholanemia of pregnancy; BA profiling; Biomarkers; Differential diagnosis; Intrahepatic cholestasis of pregnancy; Pseudo-targeted method

DOI:  https://doi.org/10.1016/j.cca.2019.07.002
Biomed Chromatogr. 2019 Jun 30. e4633

LC-MS bioanalysis of intact proteins and peptides.

Lijuan Kang, Naidong Weng, Wenying Jian.

  Bioanalysis assays that reliably quantify biotherapeutics and biomarkers in biological samples play pivotal roles in drug discovery and development. Liquid chromatography coupled with mass spectrometry (LC-MS), due to its superior specificity, faster method development, and multiplex capability, has evolved as one of the most important platforms for bioanalysis of biotherapeutics, particularly new scaffolds such as half-life extension platforms for proteins and peptides, as well as antibody drug conjugates. Intact LC-MS analysis is orthogonal to bottom-up surrogate peptide approach by providing whole molecule quantitation and high-level sequence and structure information. Here we review the latest development in LC-MS bioanalysis of intact proteins and peptides by summarizing recent publications and discussing the important topics such as comparison between top-down intact analysis and bottom-up surrogate peptide approach, as well as simultaneous quantitation and catabolite identification. Key bioanalytical issues around intact protein bioanalysis such as sensitivity, data processing strategies, specificity, sample preparation, LC condition are elaborated. For peptides, topics including quantitation of intact peptide versus digested surrogate peptide, metabolites, sensitivity, LC condition, assay performance, internal standard, and sample preparation are discussed.

Keywords:  Bioanalysis; Intact Level; LC-MS; Peptide Bioanalysis; Protein Bioanalysis; Quantitation

DOI:  https://doi.org/10.1002/bmc.4633
Cell Rep. 2019 Jul 02. pii: S2211-1247(19)30768-5. [Epub ahead of print]28(1): 104-118.e8

Reprogramming of Amino Acid Transporters to Support Aspartate and Glutamate Dependency Sustains Endocrine Resistance in Breast Cancer.

Marina Bacci, Nicla Lorito, Luigi Ippolito, Matteo Ramazzotti, Simone Luti, Simone Romagnoli, Matteo Parri, Francesca Bianchini, Federica Cappellesso, Federico Virga, Qiong Gao, Bruno M Simões, Elisabetta Marangoni, Lesley-Ann Martin, Giuseppina Comito, Manuela Ferracin, Elisa Giannoni, Massimiliano Mazzone, Paola Chiarugi, Andrea Morandi.

  Endocrine therapy (ET) is the standard of care for estrogen receptor-positive (ER+) breast cancers. Despite its efficacy, ∼40% of women relapse with ET-resistant (ETR) disease. A global transcription analysis in ETR cells reveals a downregulation of the neutral and basic amino acid transporter SLC6A14 governed by enhanced miR-23b-3p expression, resulting in impaired amino acid metabolism. This altered amino acid metabolism in ETR cells is supported by the activation of autophagy and the enhanced import of acidic amino acids (aspartate and glutamate) mediated by the SLC1A2 transporter. The clinical significance of these findings is validated by multiple orthogonal approaches in a large cohort of ET-treated patients, in patient-derived xenografts, and in in vivo experiments. Targeting these amino acid metabolic dependencies resensitizes ETR cells to therapy and impairs the aggressive features of ETR cells, offering predictive biomarkers and potential targetable pathways to be exploited to combat or delay ETR in ER+ breast cancers.

Keywords:  SLCs; amino acid transporters; aspartate; endocrine therapy; estrogen receptor; glutamate; metabolic reprogramming; miRNA; resistance

DOI:  https://doi.org/10.1016/j.celrep.2019.06.010
Nat Protoc. 2019 Jul 03.

Systems structural biology measurements by in vivo cross-linking with mass spectrometry.

Juan D Chavez, Jared P Mohr, Martin Mathay, Xuefei Zhong, Andrew Keller, James E Bruce.

This protocol describes a workflow for utilizing large-scale cross-linking with mass spectrometry (XL-MS) to make systems-level structural biology measurements in complex biological samples, including cells, isolated organelles, and tissue samples. XL-MS is a structural biology technique that provides information on the molecular structure of proteins and protein complexes using chemical probes that report the proximity of probe-reactive amino acids within proteins, typically lysine residues. Information gained through XL-MS studies is often complementary to more traditional methods, such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy. The use of MS-cleavable cross-linkers, including protein interaction reporter (PIR) technologies, enables XL-MS studies on protein structures and interactions in extremely complex biological samples, including intact living cells. PIR cross-linkers are designed to contain chemical bonds at specific locations within the cross-linker molecule that can be selectively cleaved by collision-induced dissociation or UV light. When broken, these bonds release the intact peptides that were cross-linked, as well as a reporter ion. Conservation of mass dictates that the sum of the two released peptide masses and the reporter mass equals the measured precursor mass. This relationship is used to identify cross-linked peptide pairs. Release of the individual peptides permits accurate measurement of their masses and independent amino acid sequence determination by tandem MS, allowing the use of standard proteomics search engines such as Comet for peptide sequence assignment, greatly simplifying data analysis of cross-linked peptide pairs. Search results are processed with XLinkProphet for validation and can be uploaded into XlinkDB for interaction network and structural analysis.

DOI: https://doi.org/10.1038/s41596-019-0181-3
Front Oncol. 2019 ;9 522

Potential Use of Gluconate in Cancer Therapy.

Maria E Mycielska, Markus T J Mohr, Katharina Schmidt, Konstantin Drexler, Petra Rümmele, Sebastian Haferkamp, Hans J Schlitt, Andreas Gaumann, Jerzy Adamski, Edward K Geissler.

  We have recently discovered that cancer cells take up extracellular citrate through plasma membrane citrate transporter (pmCiC) and advantageously use citrate for their metabolism. Citrate uptake can be blocked with gluconate and this results in decreased tumor growth and altered metabolic characteristics of tumor tissue. Interestingly, gluconate, considered to be physiologically neutral, is incidentally used in medicine as a cation carrier, but not as a therapeutically active substance. In this review we discuss the results of our recent research with available literature and suggest that gluconate may be useful in the treatment of cancer.

Keywords:  cancer metabolism; citrate; gluconate; metabolic targeting; transporter

DOI:  https://doi.org/10.3389/fonc.2019.00522
Anticancer Res. 2019 Jul;39(7): 3385-3394

Acyl-Coenzyme A: Cholesterol Acyltransferase Inhibition in Cancer Treatment.

Judyta Zabielska, Tomasz Sledzinski, Ewa Stelmanska.

  Overexpression of acyl-coenzyme A:cholesterol acyltransferase (ACAT) results in increased cholesteryl ester levels and has been involved in a variety of cancer types. As a consequence, cholesterol metabolism has raised interest as a potential target for cancer treatment. Inhibition of ACAT results in suppression of proliferation in a range of cancer cell types both in vitro and in vivo. The exact mechanism of this phenomenon is being investigated, and the most important findings are presented in this review.

Keywords:  ACAT; Cholesterol metabolism; avasimibe; cancer; review

DOI:  https://doi.org/10.21873/anticanres.13482
Anal Chem. 2019 Jun 27.

Quantification of Drug Molecules in Live Single Cells Using the Single-Probe Mass Spectrometry Technique.

Ning Pan, Shawna J Standke, Naga Rama Kothapalli, Mei Sun, Ryan C Bensen, Anthony W G Burgett, Zhibo Yang.

Analyzing cellular constituents on the single-cell level through mass spectrometry (MS) allows for a wide range of compounds to be studied simultaneously. However, there is a need for quantitative single-cell mass spectrometry (qSCMS) methods to fully characterize drug efficacy from individual cells within cell populations. In this study, qSCMS experiments were carried out using the Single-probe MS technique. The method was successfully used to perform rapid absolute quantifications of the anticancer drug irinotecan in individual mammalian cancer cells under ambient conditions in real time. Traditional liquid chromatography/mass spectrometry (LC/MS) quantifications of irinotecan in cell lysate samples were used to compare the results from Single-probe qSCMS. This technique showcases heterogeneity of drug efficacy on the single-cell level.

DOI: https://doi.org/10.1021/acs.analchem.9b01311
J Am Soc Mass Spectrom. 2019 Jun 27.

Quantitation of Super Basic Peptides in Biological Matrices by a Generic Perfluoropentanoic Acid-Based Liquid Chromatography-Mass Spectrometry Method.

Jianzhong Wen, Weixun Wang, Keun-Joong Lee, Bernard K Choi, Paul Harradine, Gino M Salituro, Lucinda Hittle.

  Peptides represent a promising modality for the design of novel therapeutics that can potentially modulate traditionally non-druggable targets. Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are two large families that are being explored extensively as drug delivery vehicles, imaging reagents, or therapeutic treatments for various diseases. Many CPPs and AMPs are cationic among which a significant portion is extremely basic and hydrophilic (e.g., nona-arginine). Despite their attractive therapeutic potential, it remains challenging to directly analyze and quantify these super cationic peptides from biological matrices due to their poor chromatographic behavior and MS response. Herein, we describe a generic method that combines solid phase extraction and LC-MS/MS for analysis of these peptides. As demonstrated, using a dozen strongly basic peptides, low μM concentration of perfluoropentanoic acid (PFPeA) in the mobile phase enabled excellent compound chromatographic retention, thus avoiding co-elution with solvent front ion suppressants. PFPeA also had a charge reduction effect that allowed the selection of parent/ion fragment pairs in the higher m/z region to further reduce potential low molecular weight interferences. When the method was coupled to the optimized sample extraction process, we routinely achieved low digit ng/ml sensitivity for peptides in plasma/tissue. The method allowed an efficient evaluation of plasma stability of CPPs/AMPs without fluorescence derivatization or other tagging methods. Importantly, using the widely studied HIV-TAT CPP as an example, the method enabled us to directly assess its pharmacokinetics and tissue distribution in preclinical animal models.

Keywords:  Antimicrobial peptides; Cell-penetrating peptides; LC-MS; Peptide quantitation; Perfluoropentanoic acid

DOI:  https://doi.org/10.1007/s13361-019-02257-9
Metabolites. 2019 Jul 02. pii: E127. [Epub ahead of print]9(7):

Human Plasma Metabolomics in Age-Related Macular Degeneration: Meta-Analysis of Two Cohorts.

Inês Laíns, Wonil Chung, Rachel S Kelly, João Gil, Marco Marques, Patrícia Barreto, Joaquim N Murta, Ivana K Kim, Demetrios G Vavvas, John B Miller, Rufino Silva, Jessica Lasky-Su, Liming Liang, Joan W Miller, Deeba Husain.

  The pathogenesis of age-related macular degeneration (AMD), a leading cause of blindness worldwide, remains only partially understood. This has led to the current lack of accessible and reliable biofluid biomarkers for diagnosis and prognosis, and absence of treatments for dry AMD. This study aimed to assess the plasma metabolomic profiles of AMD and its severity stages with the ultimate goal of contributing to addressing these needs. We recruited two cohorts: Boston, United States (n = 196) and Coimbra, Portugal (n = 295). Fasting blood samples were analyzed using ultra-high performance liquid chromatography mass spectrometry. For each cohort, we compared plasma metabolites of AMD patients versus controls (logistic regression), and across disease stages (permutation-based cumulative logistic regression considering both eyes). Meta-analyses were then used to combine results from the two cohorts. Our results revealed that 28 metabolites differed significantly between AMD patients versus controls (false discovery rate (FDR) q-value: 4.1 × 10-2-1.8 × 10-5), and 67 across disease stages (FDR q-value: 4.5 × 10-2-1.7 × 10-4). Pathway analysis showed significant enrichment of glycerophospholipid, purine, taurine and hypotaurine, and nitrogen metabolism (p-value ≤ 0.04). In conclusion, our findings support that AMD patients present distinct plasma metabolomic profiles, which vary with disease severity. This work contributes to the understanding of AMD pathophysiology, and can be the basis of future biomarkers and precision medicine for this blinding condition.

Keywords:  age-related macular degeneration; mass spectrometry; metabolomics

DOI:  https://doi.org/10.3390/metabo9070127