bims-mascan 2019-09-15 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2019–09–15
24 papers selected by
Giovanny Rodriguez Blanco, The Beatson Institute for Cancer Research

Proteomics of Melanoma Response to Immunotherapy Reveals Mitochondrial Dependence.
Methionine metabolism in health and cancer: a nexus of diet and precision medicine.
Proteomic profiling of extracellular vesicles allows for human breast cancer subtyping.
Preclinical studies on metal based anticancer drugs as enabled by integrated metallomics and metabolomics.
Redesigning the T-probe for mass spectrometry analysis of online lysis of non-adherent single cells.
An Isotopic Ratio Outlier Analysis Approach for Global Metabolomics of Biosynthetically Talented Actinomycetes.
Ouabain impairs cancer metabolism and activates AMPK-Src signaling pathway in human cancer cell lines.
Sets of coregulated serum lipids are associated with Alzheimer's disease pathophysiology.
Comparative metabolomics of MCF-7 breast cancer cells using different extraction solvents assessed by mass spectroscopy.
High-throughput proteomic analysis of FFPE tissue samples facilitates tumor stratification.
Unexpected roles for ADH1 and SORD in catalyzing the final step of erythritol biosynthesis.
Fatty acid transport protein 4 is required for incorporation of saturated ultralong-chain fatty acids into epidermal ceramides and monoacylglycerols.
The Hormone KL1: A Regulator of Breast Cancer Cell Metabolism.
Metabolomics-based approach to explore growth phase-dependent markers in cultured diatom Chaetoceros tenuissimus.
Drug Metabolite Cluster-Based Data-Mining Method for Comprehensive Metabolism Study of 5-hydroxy-6,7,3',4'-tetramethoxyflavone in Rats.
Quantitation of Single and Combinatorial Histone Modifications by Integrated Chromatography of Bottom-up Peptides and Middle-down Polypeptide Tails.
Collagen analysis with mass spectrometry.
Urinary metabolites for urological cancer detection: a review on the application of volatile organic compounds for cancers.
Maximizing Tandem MS Acquisition Rates for Shotgun Proteomics.
Association between plasma diacetylspermine and tumor spermine synthase with outcome in triple negative breast cancer.
Mass spectrometry-based forest tree metabolomics.
Lipid Metabolism: a New Player in the Conundrum of Lung Fibrosis.
The ever expanding scope of electrospray mass spectrometry-a 30 year journey.
Analytical separations for lipids in complex, non-polar lipidomes using differential mobility spectrometry.

Cell. 2019 Aug 28. pii: S0092-8674(19)30900-6. [Epub ahead of print]

Proteomics of Melanoma Response to Immunotherapy Reveals Mitochondrial Dependence.

Michal Harel, Rona Ortenberg, Siva Karthik Varanasi, Kailash Chandra Mangalhara, Mariya Mardamshina, Ettai Markovits, Erez N Baruch, Victoria Tripple, May Arama-Chayoth, Eyal Greenberg, Anjana Shenoy, Ruveyda Ayasun, Naama Knafo, Shihao Xu, Liat Anafi, Gali Yanovich-Arad, Georgina D Barnabas, Shira Ashkenazi, Michal J Besser, Jacob Schachter, Marcus Bosenberg, Gerald S Shadel, Iris Barshack, Susan M Kaech, Gal Markel, Tamar Geiger.

  Immunotherapy has revolutionized cancer treatment, yet most patients do not respond. Here, we investigated mechanisms of response by profiling the proteome of clinical samples from advanced stage melanoma patients undergoing either tumor infiltrating lymphocyte (TIL)-based or anti- programmed death 1 (PD1) immunotherapy. Using high-resolution mass spectrometry, we quantified over 10,300 proteins in total and ∼4,500 proteins across most samples in each dataset. Statistical analyses revealed higher oxidative phosphorylation and lipid metabolism in responders than in non-responders in both treatments. To elucidate the effects of the metabolic state on the immune response, we examined melanoma cells upon metabolic perturbations or CRISPR-Cas9 knockouts. These experiments indicated lipid metabolism as a regulatory mechanism that increases melanoma immunogenicity by elevating antigen presentation, thereby increasing sensitivity to T cell mediated killing both in vitro and in vivo. Altogether, our proteomic analyses revealed association between the melanoma metabolic state and the response to immunotherapy, which can be the basis for future improvement of therapeutic response.

Keywords:  anti-PD-1; cancer metabolism; immune checkpoint inhibitors; immunotherapy; lipid metabolism; mass spectrometry; melanoma; mitochondrial metabolism; proteomics; tumor-infiltrating lymphocytes

DOI:  https://doi.org/10.1016/j.cell.2019.08.012
Nat Rev Cancer. 2019 Sep 12.

Methionine metabolism in health and cancer: a nexus of diet and precision medicine.

Sydney M Sanderson, Xia Gao, Ziwei Dai, Jason W Locasale.

Methionine uptake and metabolism is involved in a host of cellular functions including methylation reactions, redox maintenance, polyamine synthesis and coupling to folate metabolism, thus coordinating nucleotide and redox status. Each of these functions has been shown in many contexts to be relevant for cancer pathogenesis. Intriguingly, the levels of methionine obtained from the diet can have a large effect on cellular methionine metabolism. This establishes a link between nutrition and tumour cell metabolism that may allow for tumour-specific metabolic vulnerabilities that can be influenced by diet. Recently, a number of studies have begun to investigate the molecular and cellular mechanisms that underlie the interaction between nutrition, methionine metabolism and effects on health and cancer.

DOI: https://doi.org/10.1038/s41568-019-0187-8
Commun Biol. 2019 ;2 325

Proteomic profiling of extracellular vesicles allows for human breast cancer subtyping.

Stamatia Rontogianni, Eleni Synadaki, Bohui Li, Marte C Liefaard, Esther H Lips, Jelle Wesseling, Wei Wu, Maarten Altelaar.

  Extracellular vesicles (EVs) are a potential source of disease-associated biomarkers for diagnosis. In breast cancer, comprehensive analyses of EVs could yield robust and reliable subtype-specific biomarkers that are still critically needed to improve diagnostic routines and clinical outcome. Here, we show that proteome profiles of EVs secreted by different breast cancer cell lines are highly indicative of their respective molecular subtypes, even more so than the proteome changes within the cancer cells. Moreover, we detected molecular evidence for subtype-specific biological processes and molecular pathways, hyperphosphorylated receptors and kinases in connection with the disease, and compiled a set of protein signatures that closely reflect the associated clinical pathophysiology. These unique features revealed in our work, replicated in clinical material, collectively demonstrate the potential of secreted EVs to differentiate between breast cancer subtypes and show the prospect of their use as non-invasive liquid biopsies for diagnosis and management of breast cancer patients.

Keywords:  Breast cancer; Mass spectrometry; Proteomic analysis

DOI:  https://doi.org/10.1038/s42003-019-0570-8
Metallomics. 2019 Sep 09.

Preclinical studies on metal based anticancer drugs as enabled by integrated metallomics and metabolomics.

Luis Galvez, Mate Rusz, Michaela Schwaiger-Haber, Yasin El Abiead, Gerrit Hermann, Ute Jungwirth, Walter Berger, Bernhard K Keppler, Michael A Jakupec, Gunda Koellensperger.

Resistance development is a major obstacle for platinum-based chemotherapy, with the anticancer drug oxaliplatin being no exception. Acquired resistance is often associated with altered drug accumulation. In this work we introduce a novel -omics workflow enabling the parallel study of platinum drug uptake and its distribution between nucleus/protein and small molecule fraction along with metabolic changes after different treatment time points. This integrated metallomics/metabolomics approach is facilitated by a tailored sample preparation workflow suitable for preclinical studies on adherent cancer cell models. Inductively coupled plasma mass spectrometry monitors the platinum drug, while the metabolomics tool-set is provided by hydrophilic interaction liquid chromatography combined with high-resolution Orbitrap mass spectrometry. The implemented method covers biochemical key pathways of cancer cell metabolism as shown by a panel of >130 metabolite standards. Furthermore, the addition of yeast-based 13C-enriched internal standards upon extraction enabled a novel targeted/untargeted analysis strategy. In this study we used our method to compare an oxaliplatin sensitive human colon cancer cell line (HCT116) and its corresponding resistant model. In the acquired oxaliplatin resistant cells distinct differences in oxaliplatin accumulation correlated with differences in metabolomic rearrangements. Using this multi-omics approach for platinum-treated samples facilitates the generation of novel hypotheses regarding the susceptibility and resistance towards oxaliplatin.

DOI: https://doi.org/10.1039/c9mt00141g
Anal Chim Acta. 2019 Nov 25. pii: S0003-2670(19)30883-9. [Epub ahead of print]1084 53-59

Redesigning the T-probe for mass spectrometry analysis of online lysis of non-adherent single cells.

Yanlin Zhu, Renmeng Liu, Zhibo Yang.

  Single cell mass spectrometry (SCMS) allows for molecular analysis of individual cells while avoiding the inevitable drawbacks of using cell lysate prepared from populations of cells. Based on our previous design of the T-probe, a microscale sampling and ionization device for SCMS analysis, we further developed the device to perform online, and real time lysis of non-adherent live single cells for mass spectrometry (MS) analysis at ambient conditions. This redesigned T-probe includes three parts: a sampling probe with a small tip to withdraw a whole cell, a solvent-providing capillary to deliver lysis solution (i.e., acetonitrile), and a nano-ESI emitter in which rapid cell lysis and ionization occur followed by MS analysis. These three components are embedded between two polycarbonate slides and are jointed through a T-junction to form an integrated device. Colon cancer cells (HCT-116) under control and treatment (using anticancer drug irinotecan) conditions were analyzed. We detected a variety of intracellular species, and structural identification of selected ions was conducted using tandem MS (MS2). We further conducted statistical analysis (e.g., PLS-DA and t-test) to gain biological insights of cellular metabolism. Our results indicate that the influence of anticancer drugs on cellular metabolism of live non-adherent cells can be obtained using the SCMS experiments combined with statistical data analysis.

Keywords:  Non-adherent single cell analysis; Online cell lysis; Single cell mass spectrometry; Single cell metabolomics; T-probe

DOI:  https://doi.org/10.1016/j.aca.2019.07.059
Metabolites. 2019 Sep 10. pii: E181. [Epub ahead of print]9(9):

An Isotopic Ratio Outlier Analysis Approach for Global Metabolomics of Biosynthetically Talented Actinomycetes.

Jordan Carey, Thanh Nguyen, Jennifer Korchak, Christopher Beecher, Felice de Jong, Amy L Lane.

  Actinomycetes are powerhouses of natural product biosynthesis. Full realization of this biosynthetic potential requires approaches for recognizing novel metabolites and determining mediators of metabolite production. Herein, we develop an isotopic ratio outlier analysis (IROA) ultra-high performance liquid chromatography-mass spectrometry (UHPLC/MS) global metabolomics strategy for actinomycetes that facilitates recognition of novel metabolites and evaluation of production mediators. We demonstrate this approach by determining impacts of the iron chelator 2,2'-bipyridyl on the Nocardiopsis dassonvillei metabolome. Experimental and control cultures produced metabolites with isotopic carbon signatures that were distinct from corresponding "standard" culture metabolites, which were used as internal standards for LC/MS. This provided an isotopic MS peak pair for each metabolite, which revealed the number of carbon atoms and relative concentrations of metabolites and distinguished biosynthetic products from artifacts. Principal component analysis (PCA) and random forest (RF) differentiated bipyridyl-treated samples from controls. RF mean decrease accuracy (MDA) values supported perturbation of metabolites from multiple amino acid pathways and novel natural products. Evaluation of bipyridyl impacts on the nocazine/XR334 diketopiperazine (DKP) pathway revealed upregulation of amino acid precursors and downregulation of late stage intermediates and products. These results establish IROA as a tool in the actinomycete natural product chemistry arsenal and support broad metabolic consequences of bipyridyl.

Keywords:  actinomycete; diketopiperazine; global metabolomics; isotopic ratio outlier analysis; liquid chromatography-mass spectrometry; natural product; secondary metabolite; siderophore

DOI:  https://doi.org/10.3390/metabo9090181
Acta Pharmacol Sin. 2019 Sep 12.

Ouabain impairs cancer metabolism and activates AMPK-Src signaling pathway in human cancer cell lines.

Jia-Jia Shen, Yue-Chen Zhan, Hui-Ying Li, Zhen Wang.

  In addition to the well-known cardiotonic effects, cardiac glycosides (CGs) produce potent anticancer effects with various molecular mechanisms. We previously show that ouabain induces autophagic cell death in human lung cancer cells by regulating AMPK-mediated mTOR and Src-mediated ERK1/2 signaling pathways. However, whether and how AMPK and Src signaling interacts in ouabain-treated cancer cells remains unclear. Given the pivotal role of AMPK in metabolism, whether ouabain affects cancer cell metabolism remains elusive. In this study we showed that treatment with ouabain (25 nM) caused simultaneous activation of AMPK and Src signaling pathways in human lung cancer A549 cells and human breast cancer MCF7 cells. Cotreatment with AMPK inhibitor compound C or siRNA greatly abrogates ouabain-induced Src activation, whereas cotreatment with Src inhibitor PP2 has little effect on ouabain-induced AMPK activity, suggesting that AMPK served as an upstream regulator of the Src signaling pathway. On the other hand, ouabain treatment greatly depletes ATP production in A549 and MCF7 cells, and supplement of ATP (100 μM) blocked ouabain-induced AMPK activation. We further demonstrated that ouabain greatly inhibited the mitochondrial oxidative phosphorylation (OXPHOS) in the cancer cells, and exerted differential metabolic effects on glycolysis depending on cancer cell type. Taken together, this study reveals that the altered cancer cell metabolism caused by ouabain may contribute to AMPK activation, as well as its cytotoxicity towards cancer cells.

Keywords:  AMPK; Src; cancer; glycolysis; ouabain; oxidative phosphorylation

DOI:  https://doi.org/10.1038/s41401-019-0290-0
Alzheimers Dement (Amst). 2019 Dec;11 619-627

Sets of coregulated serum lipids are associated with Alzheimer's disease pathophysiology.

Alzheimer Disease Metabolomics Consortium

   Introduction: Comorbidity with metabolic diseases indicates that lipid metabolism plays a role in the etiology of Alzheimer's disease (AD). Comprehensive lipidomic analysis can provide new insights into the altered lipid metabolism in AD.
Method: In this study, a total 349 serum lipids were measured in 806 participants enrolled in the Alzheimer's Disease Neuroimaging Initiative Phase 1 cohort and analyzed using lipid-set enrichment statistics, a data mining method to find coregulated lipid sets.
Results: We found that sets of blood lipids were associated with current AD biomarkers and with AD clinical symptoms. AD diagnosis was associated with 7 of 28 lipid sets of which four also correlated with cognitive decline, including polyunsaturated fatty acids. Cerebrospinal fluid amyloid beta (Aβ1-42) correlated with glucosylceramides, lysophosphatidylcholines and unsaturated triacylglycerides; cerebrospinal fluid total tau and brain atrophy correlated with monounsaturated sphingomyelins and ceramides, in addition to EPA-containing lipids.
Discussion: AD-associated lipid sets indicated that lipid desaturation, elongation, and acyl chain remodeling processes are disturbed in AD subjects. Monounsaturated lipid metabolism was important in early stages of AD, whereas the polyunsaturated lipid metabolism was associated with later stages of AD. Our study provides several new hypotheses for studying the role of lipid metabolism in AD.

Keywords:  Alzheimer's disease; Dyslipidemias; Lipid biochemistry; Lipidomics; Mass spectrometry

DOI:  https://doi.org/10.1016/j.dadm.2019.07.002
Sci Rep. 2019 Sep 11. 9(1): 13126

Comparative metabolomics of MCF-7 breast cancer cells using different extraction solvents assessed by mass spectroscopy.

Mohammad H Semreen, Hasan Y Alniss, Stefan R Grgic, Raafat A El-Awady, Ahmed H Almehdi, Muath K Mousa, Rifat A Hamoudi.

Metabolic profiling of cancer cells can play a vital role in revealing the molecular bases of cancer development and progression. In this study, gas chromatography coupled with mass spectrometry (GC-MS) was employed for the determination of signatures found in ER+/PR+ breast cancer cells derived from MCF-7 using different extraction solvents including: A, formic acid in water; B, ammonium hydroxide in water; C, ethyl acetate; D, methanol: water (1:1, v/v); and E, acetonitrile: water (1:1, v/v). The greatest extraction rate and diversity of metabolites occurs with extraction solvents A and E. Extraction solvent D showed moderate extraction efficiency, whereas extraction solvent B and C showed inferior metabolite diversity. Metabolite set enrichment analysis (MSEA) results showed energy production pathways to be key in MCF-7 cell lines. This study showed that mass spectrometry could identify key metabolites associated with cancers. The highest enriched pathways were related to energy production as well as Warburg effect pathways, which may shed light on how energy metabolism has been hijacked to encourage tumour progression and eventually metastasis in breast cancer.

DOI: https://doi.org/10.1038/s41598-019-49509-y
Mol Oncol. 2019 Sep 08.

High-throughput proteomic analysis of FFPE tissue samples facilitates tumor stratification.

Yi Zhu, Tobias Weiss, Qiushi Zhang, Rui Sun, Bo Wang, Xiao Yi, Zhicheng Wu, Huanhuan Gao, Xue Cai, Guan Ruan, Tiansheng Zhu, Chao Xu, Sai Lou, Xiaoyan Yu, Ludovic Gillet, Peter Blattmann, Karim Saba, Christian D Fankhauser, Michael B Schmid, Dorothea Rutishauser, Jelena Ljubicic, Ailsa Christiansen, Christine Fritz, Niels J Rupp, Cedric Poyet, Elisabeth Rushing, Michael Weller, Patrick Roth, Eugenia Haralambieva, Silvia Hofer, Chen Chen, Wolfram Jochum, Xiaofei Gao, Xiaodong Teng, Lirong Chen, Qing Zhong, Peter J Wild, Ruedi Aebersold, Tiannan Guo.

  Formalin-fixed, paraffin-embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here we developed a pressure cycling technology (PCT)-SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B-cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between one to 15 years in a biobank and show a high degree of the proteome pattern similarity between two types of histological regions in small FFPE samples, i.e. punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of a certain degree of biological variations. Applying the method to two independent DLBCL cohorts we identified myeloperoxidase (MPO), a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered. This article is protected by copyright. All rights reserved.

Keywords:  SWATH; biomarker; formalin-fixed; paraffin-embedded; pressure cycling technology; proteome; tumor

DOI:  https://doi.org/10.1002/1878-0261.12570
J Biol Chem. 2019 Sep 11. pii: jbc.RA119.009049. [Epub ahead of print]

Unexpected roles for ADH1 and SORD in catalyzing the final step of erythritol biosynthesis.

Lisa Schlicker, Doletha M E Szebenyi, Semira R Ortiz, Alexander Heinz, Karsten Hiller, Martha S Field.

  The low-calorie sweetener erythritol is endogenously produced from glucose through the pentose phosphate pathway in humans. Erythritol is of medical interest because elevated plasma levels of this polyol are predictive for visceral adiposity gain and development of type 2 diabetes. However, the mechanisms behind these associations remain unknown because the erythritol biosynthesis pathway, particularly the enzyme catalyzing the final step of erythritol synthesis (reduction of erythrose to erythritol), is not characterized. In this study, we purified two enzymes from rabbit liver capable of catalyzing the conversion of erythrose to erythritol: alcohol dehydrogenase 1 (ADH1) and sorbitol dehydrogenase (SORD). Both recombinant human ADH1 and SORD reduce erythrose to erythritol, using NADPH as a co-factor, and cell culture studies indicate that this activity is primarily NADPH-dependent. We found that ADH1 variants vary markedly in both their affinity for erythrose and their catalytic capacity (turnover number). Interestingly, the recombinant protein produced from the ADH1B2 variant, common in Asian populations, is not active when NADPH is used as a co-factor in vitro. We also confirmed SORD contributes to intracellular erythritol production in human A549 lung cancer cells, where ADH1 is minimally expressed. In summary, human ADH1 and SORD catalyze the conversion of erythrose to erythritol, pointing to novel roles for two dehydrogenase proteins in human glucose metabolism that may contribute to individual responses to diet. Proteomics data are available via ProteomeXchange with identifier PXD015178.

Keywords:  alcohol dehydrogenase (ADH); biomarker; enzyme catalysis; enzyme kinetics; erythritol; glucose metabolism; sorbitol dehydrogenase

DOI:  https://doi.org/10.1074/jbc.RA119.009049
Sci Rep. 2019 Sep 13. 9(1): 13254

Fatty acid transport protein 4 is required for incorporation of saturated ultralong-chain fatty acids into epidermal ceramides and monoacylglycerols.

Meei-Hua Lin, Fong-Fu Hsu, Debra Crumrine, Jason Meyer, Peter M Elias, Jeffrey H Miner.

Fatty acid transport protein 4 (FATP4) is an acyl-CoA synthetase that is required for normal permeability barrier in mammalian skin. FATP4 (SLC27A4) mutations cause ichthyosis prematurity syndrome, a nonlethal disorder. In contrast, Fatp4-/- mice die neonatally from a defective barrier. Here we used electron microscopy and lipidomics to characterize defects in Fatp4-/- mice. Mutants showed lamellar body, corneocyte lipid envelope, and cornified envelope abnormalities. Lipidomics identified two lipids previously speculated to be present in mouse epidermis, sphingosine β-hydroxyceramide and monoacylglycerol; mutants displayed decreased proportions of these and the two ceramide classes that carry ultralong-chain, amide-linked fatty acids (FAs) thought to be critical for barrier function, unbound ω-O-acylceramide and bound ω-hydroxyceramide, the latter constituting the major component of the corneocyte lipid envelope. Other abnormalities included elevated amounts of sphingosine α-hydroxyceramide, phytosphingosine non-hydroxyceramide, and 1-O-acylceramide. Acyl chain length alterations in ceramides also suggested roles for FATP4 in esterifying saturated non-hydroxy and β-hydroxy FAs with at least 25 carbons and saturated or unsaturated ω-hydroxy FAs with at least 30 carbons to CoA. Our lipidomic analysis is the most thorough such study of the Fatp4-/- mouse skin barrier to date, providing information about how FATP4 can contribute to barrier function by regulating fatty acyl moieties in various barrier lipids.

DOI: https://doi.org/10.1038/s41598-019-49684-y
Isr Med Assoc J. 2019 Jul;21(7): 504

The Hormone KL1: A Regulator of Breast Cancer Cell Metabolism.

Bar Davidov, Riva Shmulevich, Ayelet Shabtay, Tami Rubinek, Ido Wolf.

BACKGROUND: Klotho is a transmembrane protein that can be shed and can act as a circulating hormone in three forms: soluble klotho (KL1 + KL2), KL1, and KL2. Klotho was discovered as a gene implicated in aging through inhibition of the IGF-I pathway. Our laboratory discovered the role of klotho as a tumor suppressor in breast cancer and other malignancies. Furthermore, we showed that the KL1 domain mediates this activity. Altered cancer cell metabolism is a hallmark of cancer and our lab demonstrated various effects of klotho on breast cancer cell metabolism. Thus, klotho inhibited glycolysis and activated adenosine monophosphate activating kinase (AMPK), an energy sensor pathway. Moreover, inhibition of AMPK reduced the tumor suppressor activity of klotho.
OBJECTIVES: To assess the effect of KL1 on breast tumor cells metabolism, as KL1 possesses the tumor suppressor activity of klotho.
METHODS: We used MCF-7 breast cancer cells treated with soluble or over-expressed KL1 and klotho. Glycolysis was assessed by measuring mRNA levels of key glycolytic enzymes using reverse transcription polymerase chain reaction and by measuring lactate and glucose levels in media. The AMPK pathway was studied by monitoring AMPK phosphorylation as well as its down-stream target, acetyl-CoA carboxylase, using western blotting. Wound healing assay was used to assess cell migration.
RESULTS: KL1 treatment reduced glycolytic enzymes mRNA levels and the activity of hexokinase, similar to klotho treatment. Furthermore, KL1 reduced glucose uptake and decreased lactate production. KL1 elevated phosphorylated acetyl-CoA carboxylase and phosphorylated AMPK levels. Inhibition AMPK (using a mutant AMPK activator) stopped KL1 from inhibiting cell migration, suggesting AMPK underlies klotho's tumor suppressor activity.
CONCLUSIONS: Our data indicate KL1 as a regulator of metabolic activity in breast cancer and suggest that metabolic alterations underlie KL1 tumor suppressor activities. Furthermore, as KL1 and klotho share a similar effect on cell metabolism, our results further support the central role KL1 domain plays in klotho's tumor suppressor activity.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Aug 27. pii: S1570-0232(19)30549-5. [Epub ahead of print]1128 121779

Metabolomics-based approach to explore growth phase-dependent markers in cultured diatom Chaetoceros tenuissimus.

Takeshi Hano, Yuji Tomaru.

  Chaetoceros tenuissimus is a cosmopolitan marine diatom whose metabolism has been little investigated. In this study, we examined the alterations of the metabolite profile between different growth phases (exponential and stationary phase) in cultured C. tenuissimus and identified growth phase-dependent candidate marker metabolite. First, the preparation methodology was optimized, focusing on extraction solvent. Metabolites of the cultured diatom (exponential phase 2.4 × 106 cells/mL, stationary phase 3.1 × 106 cells/mL) were extracted using two solvents with different constituents (solvent 1, methanol:H2O:chloroform = 5:2:2; solvent 2, methanol:ethanol:chloroform = 1:3:1). Gas chromatography mass spectrometry (GC/MS)-based metabolomics successfully detected 43 water-soluble metabolites in both solvents. The metabolic features were dependent on the growth phase: amino acid levels were higher in the exponential phase, whereas sugars and alcohols were more abundant in the stationary phase. Solvent 1 was superior in the recovery of the candidate metabolite that had a retention time of 18.13 min and predominantly contributed to discrimination between the growth phases; the metabolite level was higher in the stationary phase than in the exponential phase. The candidate metabolite was identified as mannonic acid by using GC/MS and liquid chromatography tandem mass spectrometry. Higher levels of mannonic acid during the stationary phase were also observed in other three diatom species. This study provides further insight into the use of metabolomics in the evaluation of physiological conditions of diatoms and suggests that mannonic acid content is a potential biomarker of the growth phase in cultured diatom cells.

Keywords:  Diatom; Gas chromatography mass spectrometry; Growth phase; Liquid chromatography tandem mass spectrometry; Metabolomics

DOI:  https://doi.org/10.1016/j.jchromb.2019.121779
Molecules. 2019 Sep 09. pii: E3278. [Epub ahead of print]24(18):

Drug Metabolite Cluster-Based Data-Mining Method for Comprehensive Metabolism Study of 5-hydroxy-6,7,3',4'-tetramethoxyflavone in Rats.

Yuqi Wang, Xiaodan Mei, Zihan Liu, Jie Li, Xiaoxin Zhang, Shuang Lang, Long Dai, Jiayu Zhang.

  The screening of drug metabolites in biological matrixes and structural characterization based on product ion spectra is among the most important, but also the most challenging due to the significant interferences from endogenous species. Traditionally, metabolite detection is accomplished primarily on the basis of predicted molecular masses or fragmentation patterns of prototype drug metabolites using ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Although classical techniques are well-suited for achieving the partial characterization of prototype drug metabolites, there is a pressing need for a strategy to enable comprehensive drug metabolism depiction. Therefore, we present drug metabolite clusters (DMCs), different from, but complementary to, traditional approaches for mining the information regarding drugs and their metabolites on the basis of raw, processed, or identified tandem mass spectrometry (MS/MS) data. In this paper, we describe a DMC-based data-mining method for the metabolite identification of 5-hydroxy-6,7,3',4'-tetramethoxyflavone (HTF), a typical hydroxylated-polymethoxyflavonoid (OH-PMF), which addressed the challenge of creating a thorough metabolic profile. Consequently, eight primary metabolism clusters, sixteen secondary metabolism clusters, and five tertiary metabolism clusters were proposed and 106 metabolites (19 potential metabolites included) were detected and identified positively and tentatively. These metabolites were presumed to generate through oxidation (mono-oxidation, di-oxidation), methylation, demethylation, methoxylation, glucuronidation, sulfation, ring cleavage, and their composite reactions. In conclusion, our study expounded drug metabolites in rats and provided a reference for further research on therapeutic material basis and the mechanism of drugs.

Keywords:  5-hydroxy-6,7,3′,4′-tetramethoxyflavone (HTF); data-mining methods; drug metabolite clusters (DMCs); metabolites; ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry

DOI:  https://doi.org/10.3390/molecules24183278
J Am Soc Mass Spectrom. 2019 Sep 11.

Quantitation of Single and Combinatorial Histone Modifications by Integrated Chromatography of Bottom-up Peptides and Middle-down Polypeptide Tails.

Kevin A Janssen, Mariel Coradin, Congcong Lu, Simone Sidoli, Benjamin A Garcia.

  The analysis of histone post-translational modifications (PTMs) by mass spectrometry (MS) has been critical to the advancement of the field of epigenetics. The most sensitive and accurate workflow is similar to the canonical proteomics analysis workflow (bottom-up MS), where histones are digested into short peptides (4-20 aa) and quantitated in extracted ion chromatograms. However, this limits the ability to detect even very common co-occurrences of modifications on histone proteins, preventing biological interpretation of PTM crosstalk. By digesting with GluC rather than trypsin, it is possible to produce long polypeptides corresponding to intact histone N-terminal tails (50-60 aa), where most modifications reside. This middle-down MS approach is used to study distant PTM co-existence. However, the most sensitive middle-down workflow uses weak cation exchange-hydrophilic interaction chromatography (WCX-HILIC), which is less robust than conventional reversed-phase chromatography. Additionally, since the buffer systems for middle-down and bottom-up proteomics differ substantially, it is cumbersome to toggle back and forth between both experimental setups on the same LC system. Here, we present a new workflow using porous graphitic carbon (PGC) as a stationary phase for histone analysis where bottom-up and middle-down sized histone peptides can be analyzed simultaneously using the same reversed-phase buffer setup. By using this protocol for middle-down sized peptides, we identified 406 uniquely modified intact histone tails and achieved a correlation of 0.85 between PGC and WCX-HILIC LC methods. Together, our method facilitates the analysis of single and combinatorial histone PTMs with much simpler applicability for conventional proteomics labs than the state-of-the-art middle-down MS.

Keywords:  Chromatography; Epigenetics; Histones; Middle-down; PTMs; Proteomics

DOI:  https://doi.org/10.1007/s13361-019-02303-6
Mass Spectrom Rev. 2019 Sep 09.

Collagen analysis with mass spectrometry.

Nick A van Huizen, Jan N M Ijzermans, Peter C Burgers, Theo M Luider.

  Mass spectrometry-based techniques can be applied to investigate collagen with respect to identification, quantification, supramolecular organization, and various post-translational modifications. The continuous interest in collagen research has led to a shift from techniques to analyze the physical characteristics of collagen to methods to study collagen abundance and modifications. In this review, we illustrate the potential of mass spectrometry for in-depth analyses of collagen.

Keywords:  collagen; mass spectrometry; post-translational modification; proteomics

DOI:  https://doi.org/10.1002/mas.21600
Am J Clin Exp Urol. 2019 ;7(4): 232-248

Urinary metabolites for urological cancer detection: a review on the application of volatile organic compounds for cancers.

Qin Gao, Wen-Yee Lee.

Cancer is one of the most devastating human diseases that causes a great number of mortalities each year worldwide. Thus, finding and treating cancers early is of increasing interest to the public and presents great opportunity for research. It is well known that the metabolism of cancer cells differs from that of normal tissues. Analysis of volatile organic compounds (VOCs), a group of small molecule metabolites, provides an emerging approach for cancer screening and disease monitoring. VOCs are continuously generated in human body and released through breath, blood, skin, urine and fecal samples, which carry information of the physiological and metabolic status. Furthermore, the development of effective analytical methods for VOCs detection is one of the challenging aspects in cancer research. In this review, the analytical methods such as solid-phase mirco-extraction (SPME) and stir bar sorptive extraction (SBSE) coupled with gas chromatography/mass spectrometry (GC-MS), the application of VOCs in urological cancers diagnosis and potential molecules pathways related to VOCs profile for cancer detection are discussed.

Keywords: Urine; cancer; diagnosis; metabolomics; volatile organic compounds (VOCs)
Anal Chem. 2019 Sep 11.

Maximizing Tandem MS Acquisition Rates for Shotgun Proteomics.

Edna Angelica Trujillo, Alexander S Hebert, Dain R Brademan, Joshua J Coon.

Advances in tandem mass spectrometry (MS/MS) acquisition rate have steadily led to increased performance in shotgun proteomics experiments. To that end, contemporary mass spectrometers are outfitted with multiple analyzers allowing for the simultaneous collection of survey (MS1) and MS/MS spectra. In the latest generation Orbitrap hybrid, MS/MS scans can be acquired at a high rate using the dual cell linear ion trap analyzer, all while the next precursor is being dissociated in a collision cell and a MS1 scan is occurring in the Orbitrap. Often overlooked in these experiments is that the ion trap scan duration is highly variable and dependent upon precursor mass. Here, we examine the use of various static m/z scan ranges for ion trap MS/MS acquisition and determine performance relative to conventional dynamic m/z range scanning. We demonstrate that a fixed m/z scan range generate 12% more MS/MS scans and more unique peptide identifications as compared to the standard dynamic approach, respectively.

DOI: https://doi.org/10.1021/acs.analchem.9b02979
J Natl Cancer Inst. 2019 Sep 10. pii: djz182. [Epub ahead of print]

Association between plasma diacetylspermine and tumor spermine synthase with outcome in triple negative breast cancer.

Johannes F Fahrmann, Jody Vykoukal, Alia Fleury, Satyendra Tripathi, Jennifer B Dennison, Eunice Murage, Peng Wang, Chuan-Yih Yu, Michela Capello, Chad J Creighton, Kim-Anh Do, James P Long, Ehsan Irajizad, Christine Peterson, Hiroyuki Katayama, Mary L Disis, Banu Arun, Samir Hanash.

   BACKGROUND: MYC is an oncogenic driver of development and progression in triple-negative breast cancer (TNBC). Ornithine decarboxylase, the rate-limiting enzyme in polyamine metabolism, is a transcriptional target of MYC. We therefore hypothesized that a plasma polyamine signature may be predictive of TNBC development and progression.
METHODS: Using liquid chromatography mass spectrometry, polyamine levels were determined in plasma samples from newly diagnosed patients with TNBC (n = 87) and cancer-free controls (n = 115). Findings were validated in plasma samples from an independent prospective cohort of 54 TNBC, 55 ER-/PR-/HER2+, and 73 ER+ cases, and 30 cancer-free controls. Gene expression data and clinical data for 921 and 2359 breast cancer tumors were obtained from The Cancer Genome Atlas (TCGA) repository and the Oncomine database, respectively. Relationships between plasma diacetylspermine (DAS) and tumor spermine synthase (SMS) mRNA expression with metastasis free survival and overall survival were determined using Cox proportional hazard models; Fisher's exact tests were used to assess risk of distant metastasis in relation to tumor SMS mRNA expression.
RESULTS: An increase in plasma DAS, a catabolic product of spermine mediated through SMS, was observed in the TNBC subtype of breast cancer. Plasma levels of DAS in TNBC associated with increased risk of metastasis (plasma DAS value ≥ 1.16 HR= 3.06, 95% CI = 1.15-8.13, 2-sided P = .03). SMS mRNA expression in TNBC tumor tissue was also found to be predictive of poor overall survival (top 25th percentile HR = 2.06, 95% CI = 1.04-4.08, 1-sided P= .04) and increased risk of distant metastasis in TNBC (comparison of lowest SMS quartile (reference) to highest SMS quartile Relative Risk= 1.90, 95% CI = 0.97-4.06, 1-sided Fisher's exact test P=.03).
CONCLUSION: Metabolomic profiling identified plasma DAS as a predictive marker for TNBC progression and metastasis.

Keywords:  Biomarker; Metastasis; Prognosis; Triple-Negative Breast Cancer

DOI:  https://doi.org/10.1093/jnci/djz182
Mass Spectrom Rev. 2019 Sep 09.

Mass spectrometry-based forest tree metabolomics.

Ana Margarida Rodrigues, Célia Miguel, Inês Chaves, Carla António.

  Research in forest tree species has advanced slowly when compared with other agricultural crops and model organisms, mainly due to the long-life cycles, large genome sizes, and lack of genomic tools. Additionally, trees are complex matrices, and the presence of interferents (e.g., oleoresins and cellulose) challenges the analysis of tree tissues with mass spectrometry (MS)-based analytical platforms. In this review, advances in MS-based forest tree metabolomics are discussed. Given their economic and ecological significance, particular focus is given to Pinus, Quercus, and Eucalyptus forest tree species to better understand their metabolite responses to abiotic and biotic stresses in the current climate change scenario. Furthermore, MS-based metabolomics technologies produce large and complex datasets that require expertize to adequately manage, process, analyze, and store the data in dedicated repositories. To ensure that the full potential of forest tree metabolomics data are translated into new knowledge, these data should comply with the FAIR principles (i.e., Findable, Accessible, Interoperable, and Re-usable). It is essential that adequate standards are implemented to annotate metadata from forest tree metabolomics studies as is already required by many science and governmental agencies and some major scientific publishers. © 2019 Wiley Periodicals, Inc. Mass Spec Rev 00:1-32, 2019.

Keywords:  FAIR data standards; GC-MS; LC-MS; climate change; omics technologies; pinewood nematode

DOI:  https://doi.org/10.1002/mas.21603
Am J Respir Cell Mol Biol. 2019 Sep 09.

Lipid Metabolism: a New Player in the Conundrum of Lung Fibrosis.

Ross Summer, Ana L Mora.



Keywords:  ER stress; Fatty acids; Lipids; Lung fibrosis,; Metabolism

DOI:  https://doi.org/10.1165/rcmb.2019-0098ED
Nat Commun. 2019 Sep 09. 10(1): 3744

The ever expanding scope of electrospray mass spectrometry-a 30 year journey.

Matthias Mann.

DOI: https://doi.org/10.1038/s41467-019-11747-z
J Lipid Res. 2019 Sep 11. pii: jlr.D094854. [Epub ahead of print]

Analytical separations for lipids in complex, non-polar lipidomes using differential mobility spectrometry.

Sarah E Hancock, Berwyck L J Poad, Mark D P Willcox, Stephen J Blanksby, Todd W Mitchell.

  Secretions from meibomian glands located within the eyelid (commonly known as meibum) are rich in non-polar lipid classes incorporating very-long (22-30 carbons) and ultra-long (>30 carbon) acyl chains. The complex nature of the meibum lipidome and its preponderance of neutral, non-polar lipid classes presents an analytical challenge; with typically poor chromatographic resolution, even between different lipid classes. To address this challenge, we have deployed differential ion mobility-mass spectrometry (DMS-MS) to interrogate the human meibum lipidome and demonstrate near-baseline resolution of the two major non-polar classes contained therein; namely wax esters and cholesteryl esters. Within these two lipid classes, we describe ion-mobility behaviour that is associated with the length of their acyl chains and location of unsaturation. This mobility behaviour was exploited to profile the molecular speciation within each class and thus extend the meibum lipidome coverage. Intriguingly, structure-mobility trends in unsaturated lipids show similar inflections to those previously reported for lipid phase-transition temperatures. Taken together, these data demonstrate that differential ion mobility provides a powerful orthoganol separation technology for the analysis of neutral lipids in complex matrices such as meibum, and may further provide a means to predict physicochemical properties of lipids that could assist in infering their biological function(s).

Keywords:  Cholesterol esters; Eye; Ion-mobility; Lipidomics; Mass spectrometry; Meibum; Physical biochemistry; Sterols; Wax esters

DOI:  https://doi.org/10.1194/jlr.D094854