bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2019‒07‒28
fifty-two papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research


  1. J Proteomics. 2019 Jul 18. pii: S1874-3919(19)30219-2. [Epub ahead of print] 103447
    Xie J, Zhang AH, Qiu S, Zhang TL, Li XN, Yan GL, Sun H, Liu L, Wang XJ.
      Functional metabolomics could bring correlative information about specific cell types under different conditions for exploring cell properties and functions. In this study, we adopt a non-targeted cell metabolomics strategy to reveal the proliferation inhibition mechanism of obacunone on 22RV1 prostate cancer cells. Using high-throughput liquid chromatography-high definition mass spectrometry combined with pattern recognition methods was performed to analyze the cell metabolic profiles and pathway of obacunone on prostate cancer. A total of twenty one proposed metabolites in prostate cancer cell and nine vital metabolic pathways such as nicotinate and nicotinamide metabolism, phenylalanine metabolism as well as tryptophan metabolism were identified from large amounts of data. Then, we have built an overall metabolic description network of obacunone to defense prostate cancer. In addition, morphological observation, cell proliferation and apoptosis analysis of 22RV1 human prostate cancer cells were performed to better understand physiopathologic changes after obacunone treatment. Functional metabolomics is a valuable tool that insight into the natural product mechanisms and contributes to new drug discovery. SIGNIFICANCE: In this study, we probe into the proliferation inhibition effect of obacunone on 22RV1 prostate cancer cells by differentiating metabolic changes of cell sample in control and obacunone administration. Using the non-targeted and targeted cell metabolomics approaches, our findings were manifested that obacunone effectually control proliferation and promote apoptosis in 22RV1 prostate cancer cells, which were related to twenty one proposed metabolites, and nicotinate and nicotinamide metabolism, phenylalanine metabolism, tryptophan metabolism as well as ascorbate metabolism. These data were suggested that functional metabolomics analysis have potential to explore the pharmacodynamic mechanism through resolving metabolic changes in cancer cells that possesses higher clinical application value. The advances in the molecular understanding of the roles of metabolomic pathway concerned with particular metabolites in obacunone administration attract more attention in favor of burgeoning therapeutic measures resisting prostate cancer.
    Keywords:  Metabolic pathway; Metabolites; Metabolomics; Obacunone; Prostate cancer
    DOI:  https://doi.org/10.1016/j.jprot.2019.103447
  2. Int J Mol Sci. 2019 Jul 24. pii: E3624. [Epub ahead of print]20(15):
    Rossi Sebastiano M, Konstantinidou G.
      The deregulation of cancer cell metabolic networks is now recognized as one of the hallmarks of cancer. Abnormal lipid synthesis and extracellular lipid uptake are advantageous modifications fueling the needs of uncontrolled cancer cell proliferation. Fatty acids are placed at the crossroads of anabolic and catabolic pathways, as they are implicated in the synthesis of phospholipids and triacylglycerols, or they can undergo β-oxidation. Key players to these decisions are the long-chain acyl-CoA synthetases, which are enzymes that catalyze the activation of long-chain fatty acids of 12-22 carbons. Importantly, the long-chain acyl-CoA synthetases are deregulated in many types of tumors, providing a rationale for anti-tumor therapeutic opportunities. The purpose of this review is to summarize the last up-to-date findings regarding their role in cancer, and to discuss the related emerging tumor targeting opportunities.
    Keywords:  ACSL1; ACSL3; ACSL4; ACSL5; ACSL6; cancer; cancer therapy; fatty acid; lipid metabolism; therapy target
    DOI:  https://doi.org/10.3390/ijms20153624
  3. Methods Mol Biol. 2019 ;2030 143-151
    Bunk DM, Lowenthal MS.
      The role of amino acid analysis in bioanalysis has changed from a qualitative to a quantitative technique. With the discovery of both electrospray ionization and matrix-assisted laser desorption ionization in the early 1990s, the use of amino acid analysis for qualitative analysis of proteins and peptides has been replaced by mass spectrometry. Accurate measurement of the relative molecular masses of proteins and peptides, peptide mapping, and sequencing by tandem mass spectrometry provide significantly better qualitative information than can be achieved from amino acid analysis. At NIST, amino acid analysis is used to assign concentration values to protein and peptide standard reference materials (SRMs) which, subsequently, will be used in the calibration of a wide variety of protein and peptide assays, such as those used in clinical diagnostics. It is critical that the amino acid analysis method used at NIST for assigning concentration values in SRM deliver the highest accuracy and precision possible. Therefore, we have developed an amino acid analysis method that uses isotope dilution LC-MS/MS-the analytical technique routinely used at NIST to certify analyte concentrations in SRMs for a wide variety of analytes. We present here our most recent method for the quantification of amino acids using isotope dilution LC-MS/MS.
    Keywords:  Amino acid analysis; Isotope dilution; Liquid chromatography; Quantification; Tandem mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_12
  4. Methods Mol Biol. 2019 ;2030 191-206
    Salazar C, Armenta JM, Cortés DF, Shulaev V.
      Amino acid analysis is a powerful tool in life sciences. Current analytical methods used for the detection and quantitation of low abundance amino acids in complex samples face intrinsic challenges such as insufficient sensitivity, selectivity, and throughput. This chapter describes a protocol that makes use of AccQ•Tag chemical derivatization combined with the exceptional chromatographic resolution of ultra-performance liquid chromatography (UPLC) and the sensitivity and selectivity of tandem mass spectrometry (MS/MS). The method has been fully implemented and validated using different tandem quadrupole detectors and thoroughly tested for a variety of samples such as P. falciparum, human red blood cells, and Arabidopsis thaliana extracts. Compared to currently available methods for amino acid analysis, the AccQ•Tag UPLC-MS/MS method presented here provides enhanced sensitivity and reproducibility and offers excellent performance within a short analysis time and a broad dynamic range of analyte concentration. The focus of this chapter is the application of this improved protocol for the compositional amino acid analysis in Arabidopsis thaliana leaf extracts using the Xevo TQ for mass spectrometric detection.
    Keywords:  6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate; AccQ•Tag; Amino acid analysis; UPLC-ESI-MS/MS
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_15
  5. Exp Eye Res. 2019 Jul 17. pii: S0014-4835(19)30252-0. [Epub ahead of print]186 107737
    Dai L, Yang W, Qin X, Li Y, Cao H, Zhou C, Wang Y.
      Myopia is the most common form of refractive eye disease, and the prevalence is increasing rapidly worldwide. However, the key metabolic alterations in individuals with high myopia are not understood clearly, and serum biomarkers remain to be determined. The objectives of this study were to identify serum biomarkers and investigate the metabolic alterations of myopia. The serum metabolomics profiling was investigated on 30 high myopia cases and 30 controls (without myopia) using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS), and an independent additional cohort including 20 cases and 19 controls were investigated to validate potential metabolite candidates for biomarkers. According to the metabolic differences, the myopia patients and controls could be divided into different clusters and nine metabolites were found to be closely correlated with myopia. In the cohort of validation, eight metabolites were confirmed. Metabolic pathway analyses of these metabolites of high myopia involved abnormal phospholipid, diacylglycerol, amino acid, and vitamin metabolism, which were closely correlated with oxidative stress and inflammation. Multiple logistic regression analyses showed that γ-glutamyltyrosine and 12-oxo-20-trihydroxy-leukotriene B4 were potential biomarkers of myopia with a combined high sensitivity (97%), specificity (90%), and area under the curve value (0.983). These findings may contribute to an understanding of the pathophysiological changes and pathogenesis of myopia, and provide novel insight into the early prevention and control of high myopia.
    Keywords:  Biomarkers; LC-QTOF/MS; Metabolite; Metabolomics; Myopia; Serum
    DOI:  https://doi.org/10.1016/j.exer.2019.107737
  6. Methods Mol Biol. 2019 ;2030 207-218
    Nakayama A, Imaizumi A, Yoshida H.
      Plasma free amino acid (PFAA) concentrations in humans are affected by various diseases. However, the variations caused are not dramatic, so a high accurate and precise method for analyzing PFAAs is required. The PFAA analysis protocol described in this chapter covers blood sampling, sample pretreatment, amino acid derivatization, and LC-MS analysis. Each procedure is important for accurate and precise quantification.In the protocol, a human blood sample is collected using an EDTA-2Na or 2K vacuum collection tube and then immediately cooled in water mixed with crushed ice. The sample is then centrifuged on cooling to allow a plasma sample to be removed. A stable-isotope-labeled internal standard solution is added to the plasma, and then the plasma is deproteinized with acetonitrile. The amino acids in the plasma are then derivatized using 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS) reagent which is designed for LC-MS analysis. The derivatized amino acids are separated by reverse-phase HPLC and detected by electrospray ionization mass spectrometry. Using this method, 21 amino acids in human plasma can be analyzed with a 12 min cycle. The accuracy and precision are both better than the required criteria given by the US Food and Drug Administration in guidance of Bioanalytical Method Validation.
    Keywords:  APDS; Amino acid; Human plasma; LC-MS; Precolumn derivatization
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_16
  7. Methods Mol Biol. 2019 ;2030 327-350
    Shanmuganathan M, Britz-McKibbin P.
      Capillary electrophoresis-mass spectrometry (CE-MS) offers a high efficiency microseparation platform for amino acid profiling when analyzing volume-restricted biological samples, such as a dried blood spot punch. Direct analysis of amino acids and their analogs is routinely achieved using strongly acidic buffer conditions under positive-ion mode detection with a coaxial sheath liquid interface for electrospray ionization (ESI). New advances in online sample preconcentration, pre-column chemical derivatization, and/or low flow/sheathless CE-MS interface designs can further improve sensitivity while allowing for resolution of amino acid stereoisomers and labile aminothiols with low nanomolar detection limits. Additionally, multiplexed separations in CE-MS based on serial injection of seven or more samples within a single run greatly boosts sample throughput (<2-3 min/sample) without added infrastructure costs while allowing for stringent quality control and signal batch correction. Accurate prediction of the electromigration behavior of amino acids and their analogs offers a convenient approach for structural elucidation that is complementary to high-resolution MS and MS/MS. Simultaneous analysis of amino acids together with other classes of ionic metabolites by CE-MS allows for comprehensive metabolomic screening as required for new advances in clinical medicine, nutritional sciences, and population health.
    Keywords:  Amino acids; CE-MS; Capillary electrophoresis; Dried blood spots; Mass spectrometry; Plasma; Serum; Urine
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_25
  8. Methods Mol Biol. 2019 ;2030 17-31
    Gogichaeva NV, Alterman MA.
      Here we describe two different AAA protocols based on application of matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS). First protocol describes a MALDI TOF MS-based method for a routine simultaneous qualitative and quantitative analysis of free amino acids and protein hydrolysates. Linear responses between the amino acid concentration and the peak intensity ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300 μM. Limit of quantitation varied from 0.03 μM for arginine to 3.7 μM for histidine and homocysteine. This method has one inherent limitation: the analysis of isomeric and isobaric amino acids. To solve this problem, a second protocol based on the use of MALDI TOF/TOF MS/MS for qualitative analysis of amino and organic acids was developed. This technique is capable of distinguishing isobaric and isomeric compounds. Both methods do not require amino acid derivatization or chromatographic separation, and the data acquisition time is decreased to several seconds for a single sample.
    Keywords:  Absolute protein quantitation; Amino acid analysis; MALDI TOF; MALDI TOF/TOF; Mass spectrometry; Metabolomics; Quantitative mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_3
  9. Methods Mol Biol. 2019 ;2030 57-68
    Yang Y, Fan TW, Lane AN, Higashi RM.
      Stable isotope-resolved metabolomics (SIRM) is increasingly used among researchers for metabolic studies including amino acid metabolism. However, the classical GC- or HPLC-based methods for amino acid quantification do not meet the needs for multiplexed stable isotope-enriched analysis by ultrahigh-resolution Fourier transform mass spectrometry (UHR-FTMS). This is due to insufficient acquisition time during chromatographic separations and large dynamic range in concentrations of analytes, which compromises detection and quantification of the numerous metabolite isotopologues present in crude extracts. This chapter discusses a modified ethyl chloroformate derivatization method to enable rapid quantitative analysis of stable isotope-enriched amino acids using direct infusion ion introduction coupled with UHR-FTMS.
    Keywords:  Amino acids; Chloroformate derivatization; Direct infusion nano-electrospray; Stable isotope-resolved metabolomics; Ultrahigh-resolution Fourier transform mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_6
  10. J Mol Biol. 2019 Jul 17. pii: S0022-2836(19)30450-4. [Epub ahead of print]
    Spégel P, Mulder H.
      The islets of Langerhans harbor multiple endocrine cell-types that continuously respond to circulating nutrient levels in order to adjust their secretion of catabolic and anabolic hormones. Stimulus-secretion coupling in these cells is largely of metabolic nature, i.e. metabolism of nutrient fuels yields signals that trigger and amplify secretion of hormones. Hence, metabolism in this micro-organ is in a major way in control of whole-body metabolism. Therefore, insights into islet metabolism is critical to understand how secretion of insulin is regulated and why it is perturbed in type 2 diabetes. Metabolomics aims at characterizing a wide spectrum of metabolites in cells, tissues and body fluids. For this reason, this technique is well-suited to supply information on stimulus-secretion coupling. Here, we summarize metabolomics studies in islets and β-cells, highlight important discoveries that would have been difficult to make without this technology but also raise awareness of challenges and bottlenecks that curtail its use in metabolic research.
    Keywords:  Diabetes; Mass spectrometry; Metabolite profiling; Pancreatic beta-cell; Stimulus-secretion coupling
    DOI:  https://doi.org/10.1016/j.jmb.2019.07.020
  11. Adv Exp Med Biol. 2019 ;1140 317-326
    Zandkarimi F, Brown LM.
      Lipids play significant roles in biological system, and the study of lipid metabolisms may provide a new insight into the diagnosis and pathophysiology of diseases. Recent developments in high-resolution mass spectrometry techniques combined with high-performance chromatographic methods provide deep insight into lipid analysis. Addition of ion mobility mass spectrometry orthogonal to LC-MS analysis workflow enhances separation of complex lipids, improve isomers resolution, and intensify confidence in lipid identification and characterization. In this chapter, we describe the principle of travelling wave ion mobility mass spectrometry (TWIMS) and its applications in untargeted LC-MS analysis for characterizing the structural diversity and complexity of lipid species in biological samples.
    Keywords:  HDMSE; Ion mobility mass spectrometry; LC-MS; Lipidomics; Lipids; MSE; TWIMS
    DOI:  https://doi.org/10.1007/978-3-030-15950-4_18
  12. Sci Rep. 2019 Jul 19. 9(1): 10473
    Bennett CD, Gill SK, Kohe SE, Wilson MP, Davies NP, Arvanitis TN, Tennant DA, Peet AC.
      Brain tumours are the most common cause of cancer death in children. Molecular studies have greatly improved our understanding of these tumours but tumour metabolism is underexplored. Metabolites measured in vivo have been reported as prognostic biomarkers of these tumours but analysis of surgically resected tumour tissue allows a more extensive set of metabolites to be measured aiding biomarker discovery and providing validation of in vivo findings. In this study, metabolites were quantified across a range of paediatric brain tumours using 1H-High-Resolution Magic Angle Spinning nuclear magnetic resonance spectroscopy (HR-MAS) and their prognostic potential investigated. HR-MAS was performed on pre-treatment frozen tumour tissue from a single centre. Univariate and multivariate Cox regression was used to examine the ability of metabolites to predict survival. The models were cross validated using C-indices and further validated by splitting the cohort into two. Higher concentrations of glutamine were predictive of a longer overall survival, whilst higher concentrations of lipids were predictive of a shorter overall survival. These metabolites were predictive independent of diagnosis, as demonstrated in multivariate Cox regression models. Whilst accurate quantification of metabolites such as glutamine in vivo is challenging, metabolites show promise as prognostic markers due to development of optimised detection methods and increasing use of 3 T clinical scanners.
    DOI:  https://doi.org/10.1038/s41598-019-45900-x
  13. Clin Chim Acta. 2019 Jul 17. pii: S0009-8981(19)31954-0. [Epub ahead of print]
    Huang M, Zhao H, Gao S, Liu Y, Liu Y, Zhang T, Cai X, Li Z, Li L, Li Y, Yu C.
      BACKGROUND: Coronary heart disease (CHD) is the leading cause of death worldwide, and its pathogenesis has attracted much attention. Metabolomics serves as an important tool for diagnosing diseases and exploring their pathogenesis in recent years. In this study, CHD patients were studied by comparing them with normal subjects to elucidate biomarkers that are linearly correlated with the severity of coronary stenosis.METHODS: High-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was used to analyze the urine metabolites of CHD patients and normal subjects. A total of 131 subjects included 23 patients who presented with 50-69% coronary stenosis, 22 with 70-89% stenosis, 29 with 90-99% stenosis, 24 with 100% stenosis, and 29 normal subjects.
    RESULTS: A total of 15 potential biomarkers associated with CHD were identified, and among them 5 biomarkers were linearly correlated with the severity of coronary stenosis in CHD patients. The metabolic pathways involved were amino acid metabolism, fatty acid metabolism, energy metabolism, and nucleotide metabolism.
    CONCLUSION: This study identified the biomarkers and metabolic pathways that may be involved in the occurrence and development of CHD, laying a theoretical foundation for better diagnosis and treatment of CHD in the future.
    Keywords:  Biomarkers; Coronary heart disease; Coronary stenosis; Metabolomics; UPLC-Q-TOF/MS
    DOI:  https://doi.org/10.1016/j.cca.2019.07.017
  14. Cell Commun Signal. 2019 Jul 25. 17(1): 83
    Zhang BK, Moran AM, Bailey CG, Rasko JEJ, Holst J, Wang Q.
      BACKGROUND: Growth factors, such as EGF, activate the PI3K/Akt/mTORC1 signalling pathway, which regulates a distinct program of protein synthesis leading to cell growth. This pathway relies on mTORC1 sensing sufficient levels of intracellular amino acids, such as leucine, which are required for mTORC1 activation. However, it is currently unknown whether there is a direct link between these external growth signals and intracellular amino acid levels. In primary prostate cancer cells, intracellular leucine levels are regulated by L-type amino acid transporter 3 (LAT3/SLC43A1), and we therefore investigated whether LAT3 is regulated by growth factor signalling.METHODS: To investigate how PI3K/Akt signalling regulates leucine transport, prostate cancer cells were treated with different PI3K/Akt inhibitors, or stable knock down of LAT3 by shRNA, followed by analysis of leucine uptake, western blotting, immunofluorescent staining and proximity ligation assay.
    RESULTS: Inhibition of PI3K/Akt signalling significantly reduced leucine transport in LNCaP and PC-3 human prostate cancer cell lines, while growth factor addition significantly increased leucine uptake. These effects appeared to be mediated by LAT3 transport, as LAT3 knockdown blocked leucine uptake, and was not rescued by growth factor activation or further inhibited by signalling pathway inhibition. We further demonstrated that EGF significantly increased LAT3 protein levels when Akt was phosphorylated, and that Akt and LAT3 co-localised on the plasma membrane in EGF-activated LNCaP cells. These effects were likely due to stabilisation of LAT3 protein levels on the plasma membrane, with EGF treatment preventing ubiquitin-mediated LAT3 degradation.
    CONCLUSION: Growth factor-activated PI3K/Akt signalling pathway regulates leucine transport through LAT3 in prostate cancer cell lines. These data support a direct link between growth factor and amino acid uptake, providing a mechanism by which the cells rapidly coordinate amino acid uptake for cell growth.
    Keywords:  EGF; L-type amino acids transporter 3; LAT3; PI3K/Akt signalling pathway; Prostate cancer; SLC43A1
    DOI:  https://doi.org/10.1186/s12964-019-0400-0
  15. Adv Exp Med Biol. 2019 ;1140 635-647
    Wang S, Blair IA, Mesaros C.
      The advancement of mass spectrometry-based analytical platform largely facilitates small-molecule metabolomics studies, which allows simultaneously analysis of a large number of metabolites from bio-samples and give a general picture of metabolic changes related to diseases or environmental alteration. Due to the large diversity of cellular metabolites, globally and precisely examining metabolic profile remains the most challenging part in metabolomic experiment. Mass spectrometry coupled with liquid chromatography enhances sensitivity and resolving power of metabolites identification and quantification, as well as versatility of analyzing a wide array of metabolites. In this chapter, we discussed the technical aspects of each step in the workflow of metabolomics studies we aimed to give technical guidelines for metabolomics investigation design and approach.
    Keywords:  Glycolysis; HILIC; Ion pairing chromatography; Liquid chromatography-mass spectrometry; Metabolomics; Pentose phosphate pathway; Polar metabolites; Tricarboxylic acids cycle
    DOI:  https://doi.org/10.1007/978-3-030-15950-4_38
  16. Adv Exp Med Biol. 2019 ;1140 531-539
    Hoedt E, Zhang G, Neubert TA.
      Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach for high-throughput quantitative proteomics. SILAC allows highly accurate protein quantitation through metabolic encoding of whole cell proteomes using stable isotope labeled amino acids. Since its introduction in 2002, SILAC has become increasingly popular. In this chapter we review the methodology and application of SILAC, with an emphasis on three research areas: dynamics of posttranslational modifications, protein-protein interactions, and protein turnover.
    Keywords:  Mass spectrometry; Metabolic labeling; Quantitative proteomics; Stable isotope labeling by amino acids in cell culture (SILAC)
    DOI:  https://doi.org/10.1007/978-3-030-15950-4_31
  17. JCI Insight. 2019 Jul 25. pii: 129398. [Epub ahead of print]4(14):
    Singh C, Hoppe G, Tran V, McCollum L, Bolok Y, Song W, Sharma A, Brunengraber H, Sears JE.
      We determined which metabolic pathways are activated by hypoxia-inducible factor 1-mediated (HIF-1-mediated) protection against oxygen-induced retinopathy (OIR) in newborn mice, the experimental correlate to retinopathy of prematurity, a leading cause of infant blindness. HIF-1 coordinates the change from oxidative to glycolytic metabolism and mediates flux through serine and 1-carbon metabolism (1CM) in hypoxic and cancer cells. We used untargeted metabolite profiling in vivo to demonstrate that hypoxia mimesis activates serine/1CM. Both [13C6] glucose labeling of metabolites in ex vivo retinal explants as well as in vivo [13C3] serine labeling of metabolites followed in liver lysates strongly suggest that retinal serine is primarily derived from hepatic glycolytic carbon and not from retinal glycolytic carbon in newborn pups. In HIF-1α2lox/2lox albumin-Cre-knockout mice, reduced or near-0 levels of serine/glycine further demonstrate the hepatic origin of retinal serine. Furthermore, inhibition of 1CM by methotrexate blocked HIF-mediated protection against OIR. This demonstrated that 1CM participates in protection induced by HIF-1 stabilization. The urea cycle also dominated pathway enrichment analyses of plasma samples. The dependence of retinal serine on hepatic HIF-1 and the upregulation of the urea cycle emphasize the importance of the liver to remote protection of the retina.
    Keywords:  Angiogenesis; Metabolism; Retinopathy
    DOI:  https://doi.org/10.1172/jci.insight.129398
  18. J Biol Chem. 2019 Jul 23. pii: jbc.RA119.008743. [Epub ahead of print]
    Fan TWM, Bruntz RC, Yang Y, Song H, Chernyavskaya Y, Deng P, Zhang Y, Shah PP, Beverly LJ, Qi Z, Mahan AL, Higashi RM, Dang CV, Lane AN.
      Nucleotide synthesis is essential to proliferating cells but the preferred precursors for de novo biosynthesis are not defined in human cancer tissues. We have employed multiplexed Stable Isotope Resolved Metabolomics (mSIRM) to track the metabolism of 13C6-glucose, D2-glycine, 13C2-glycine, and D3-serine into purine nucleotides in freshly resected cancerous and matched non-cancerous lung tissues from non-small cell lung cancer (NSCLC) patients and compared the metabolism with established NSCLC PC9 and A549 cell lines in vitro.  Surprisingly, 13C6-glucose was the best carbon source for purine synthesis in human NSCLC tissues, in contrast to the non-cancerous lung tissues from the same patient, which showed lower mitotic indices and MYC expression. We also observed that D3-Ser was preferentially incorporated into purine rings over D2-glycine in both tissues and cell lines. MYC suppression attenuated 13C6-glucose, D3-serine, and 13C2-glycine incorporation into purines and reduced proliferation in PC9 but not in A549 cells. Using detailed kinetic modeling, we showed that the preferred use of glucose as a carbon source for purine ring synthesis in NSCLC tissues involves cytoplasmic activation/compartmentation of the glucose-to-serine pathway and enhanced reversed one-carbon fluxes that attenuate exogenous serine incorporation into purines. Our findings also indicate that the substrate for de novo nucleotide synthesis differs profoundly between cancer cell lines and fresh human lung cancer tissues; the latter preferred glucose to exogenous serine or glycine but not the former. This distinction in substrate utilization in purine synthesis in human cancer tissues should be considered when targeting one-carbon metabolism for cancer therapy.
    Keywords:  dynamic compartmentation; ex vivo human lung tissue slice cultures; lung cancer; metabolic tracer; metabolism; multiplexed Stable Isotope-Resolved Metabolomics (mSIRM); nucleoside/nucleotide biosynthesis; one-carbon metabolism
    DOI:  https://doi.org/10.1074/jbc.RA119.008743
  19. Methods Mol Biol. 2019 ;2030 153-172
    Giordano G, Gucciardi A, Pirillo P, Naturale M.
      Enzyme deficiencies in amino acid metabolism may increase the levels of a single or several compounds in physiological fluids becoming diagnostically significant biomarkers for one or a group of metabolic disorders. Therefore, it is important to monitor a wide range of free amino acids simultaneously and to quantify them. This is time consuming if we use the classical methods and, especially now that many laboratories have introduced Newborn Screening Programs for the semiquantitative analysis, the detection and quantification of some amino acids need to be performed in a short time to reduce the rate of false positives.We have modified the stable isotope dilution HPLC-ESI-MS/MS method previously described by Qu (Qu et al., 2002) for a more rapid, robust, sensitive, and specific detection and quantification of underivatized amino acids. The modified method reduces the time of analysis to 10 min with very good reproducibility of retention times and a better separation of the metabolites and their isomers.The omission of the derivatization step, enabled to achieve some important advantages: fast and simple sample preparation, exclusion of artifacts, and interferences. The use of this technique is highly sensitive and specific and allowed to monitor 40 underivatized amino acids including the key isomers and quantification of some of them, in order to cover many diagnostically important intermediates of metabolic pathways.We propose this HPLC-ESI-MS/MS method for underivatized amino acids as a support for the newborn screening as secondary test using the same dried blood spots for a more accurate and specific examination in case of suspected metabolic diseases. In this way we avoid plasma collection from the patient as it normally occurs, reducing anxiety for the parents and further costs for analysis.The same method was validated and applied also to plasma and urine samples with good reproducibility, accuracy, and precision. The fast run time, the feasibility of high sample throughput, and the small amount of sample required make this method very suitable for routine analysis in the clinical setting.
    Keywords:  Blood spot; HPLC-ESI-MS/MS; Newborn screening; Underivatized amino acids
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_13
  20. Methods Mol Biol. 2019 ;2030 85-109
    Mak J, Cowan TM, Le A.
      Quantitative amino acid analysis has diverse applications in clinical diagnostics, biomedical research, and agriculture. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables more rapid and specific detection of amino acids in comparison to traditional, gold-standard ninhydrin-based methods. However, triple quadrupole mass spectrometers are unable to definitively differentiate isomers and are susceptible to ion suppression, both of which prevent accurate quantitation. Therefore, appropriate chromatography must be applied before ionization.We have shown that two-dimensional LC enables rapid and specific amino acid quantitation without derivatization by resolving isomers, such as alloisoleucine, isoleucine, and leucine, and reducing matrix effects (Le et al., J Chromatogr B Analyt Technol Biomed Life Sci 944:166-174, 2014). In this clinically validated protocol, we provide an updated description of the chromatographic setup and selected reaction monitoring (SRM) transitions. Then, we describe sample processing for serum, plasma, urine, cerebral spinal fluid, and dried blood spots. Most importantly, we outline a singular quantitative design for efficient data analysis of the listed sample types and quality assurance strategies to ensure test fidelity. Lastly, we share extensive knowledge critical to the success of this method. A liquid sample can be processed and be ready for injection within 5 min, and each sample is analyzed by the MS in 14.5 min.
    Keywords:  2D-LC-MS/MS; Alloisoleucine; Amino acid analysis; Blood spots; Guanidinoacetic acid; Liquid chromatography; Mass spectrometry; Targeted metabolomics; Trap and reverse elute; Underivatized
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_8
  21. Metabolites. 2019 Jul 25. pii: E156. [Epub ahead of print]9(8):
    Stevens VL, Hoover E, Wang Y, Zanetti KA.
      Metabolomics provides a comprehensive assessment of numerous small molecules in biological samples. As it integrates the effects of exogenous exposures, endogenous metabolism, and genetic variation, metabolomics is well-suited for studies examining metabolic profiles associated with a variety of chronic diseases. In this review, we summarize the studies that have characterized the effects of various pre-analytical factors on both targeted and untargeted metabolomic studies involving human plasma, serum, and urine and were published through 14 January 2019. A standardized protocol was used for extracting data from full-text articles identified by searching PubMed and EMBASE. For plasma and serum samples, metabolomic profiles were affected by fasting status, hemolysis, collection time, processing delays, particularly at room temperature, and repeated freeze/thaw cycles. For urine samples, collection time and fasting, centrifugation conditions, filtration and the use of additives, normalization procedures and multiple freeze/thaw cycles were found to alter metabolomic findings. Consideration of the effects of pre-analytical factors is a particularly important issue for epidemiological studies where samples are often collected in nonclinical settings and various locations and are subjected to time and temperature delays prior being to processed and frozen for storage.
    Keywords:  metabolite; metabolomics; pre-analytical factors
    DOI:  https://doi.org/10.3390/metabo9080156
  22. Methods Mol Biol. 2019 ;2030 11-16
    Mirgorodskaya OA, Körner R, Kozmin YP, Roepstorff P.
      Amino acid analysis is among the most accurate methods for absolute quantification of proteins and peptides. Here we combine acid hydrolysis with the addition of isotopically labeled standard amino acids and analysis by mass spectrometry for accurate and sensitive protein quantitation. Quantitation of less than 10 fmol of protein standards with errors below 10% has been demonstrated using this method.
    Keywords:  Absolute protein quantitation; Amino acid analysis; Isotope-labeled amino acids; MALDI; Mass spectrometry; Quantitative mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_2
  23. Methods Mol Biol. 2019 ;2030 173-190
    Dettmer K, Stevens AP, Fagerer SR, Kaspar H, Oefner PJ.
      Two mass spectrometry-based methods for the quantitative analysis of free amino acids are described. The first method uses propyl chloroformate/propanol derivatization and gas chromatography-quadrupole mass spectrometry (GC-qMS) analysis in a single-ion monitoring mode. Derivatization is carried out directly in the aqueous samples, thereby allowing automation of the entire procedure, including addition of reagents, extraction, and injection into the GC-MS. The method delivers the quantification of 26 amino acids. The iTRAQ method employs the labeling of amino acids with isobaric iTRAQ tags. The tags contain two different cleavable reporter ions, one for the sample and one for the standard, which are detected by fragmentation in a tandem mass spectrometer (MS/MS). Reversed-phase liquid chromatography (RP-LC) of the labeled amino acids is performed prior to mass spectrometric analysis to separate isobaric amino acids. The commercial iTRAQ kit allows for the analysis of 42 physiological amino acids with a respective isotope-labeled standard for each of these 42 amino acids.
    Keywords:  Amino acid analysis; Amino acids; Derivatization; Gas chromatography; Liquid chromatography-tandem mass spectrometry; Mass spectrometry; Propyl chloroformate; Stable isotope dilution; iTRAQ
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_14
  24. Methods Mol Biol. 2019 ;2030 403-414
    Thiele B, Hupert M, Santiago-Schübel B, Oldiges M, Hofmann D.
      In this chapter we describe a method for quantification of 20 proteinogenic amino acids by liquid chromatography-mass spectrometry which affords neither derivatization nor the use of organic solvents. Analysis of the underivatized amino acids is performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in the positive ESI mode. Separation is achieved on a strong cation exchange (SCX) column (Luna 5 μ SCX 100 Å) with 5% acetic acid in water (A) and 75 mM ammonium acetate in water (B). Quantification is accomplished by use of d2-phenylalanine as internal standard achieving limits of detection of 5-50 nM. The method was successfully applied for the determination of proteinogenic amino acids in plant extracts.
    Keywords:  Amino acids; LC-MS/MS; Plant metabolism; Strong cation exchange column
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_30
  25. Adv Exp Med Biol. 2019 ;1140 327-358
    Chen X, Lee J, Wu H, Tsang AW, Furdui CM.
      Redox (portmanteau of reduction-oxidation) reactions involve the transfer of electrons between chemical species in biological processes fundamental to life. It is of outmost importance that cells maintain a healthy redox state by balancing the action of oxidants and antioxidants; failure to do so leads to a multitude of diseases including cancer, diabetes, fibrosis, autoimmune diseases, and cardiovascular and neurodegenerative diseases. From the perspective of precision medicine, it is therefore beneficial to interrogate the redox phenotype of the individual-similar to the use of genomic sequencing-in order to design tailored strategies for disease prevention and treatment. This chapter provides an overview of redox metabolism and focuses on how mass spectrometry (MS) can be applied to advance our knowledge in redox biology and precision medicine.
    Keywords:  DNA oxidation; Lipidomics; Mass spectrometry; Metabolomics; Oxidative stress; Precision medicine; Proteomics; Redox biomarkers
    DOI:  https://doi.org/10.1007/978-3-030-15950-4_19
  26. Methods Mol Biol. 2019 ;2030 439-450
    van Helmond W, de Puit M.
      UPLC-MS is a commonly used technique to first separate complex samples and subsequently quantify molecules of interest. Herein we describe the use of UPLC-MS using an amide stationary phase to quantify non-derivatized amino acids extracted from fingerprints. As detector either a triple-quadrupole MS/MS or a TOF-MS detector was used. This method allows for a simple and fast sample preparation, which facilitates the analysis of large amounts of samples.
    Keywords:  Amino acid analysis; Amino acid profiling; Fingerprint; HILIC; Mass spectrometry; UPLC-MS
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_33
  27. Invest Ophthalmol Vis Sci. 2019 Jul 01. 60(8): 3119-3126
    Sumarriva K, Uppal K, Ma C, Herren DJ, Wang Y, Chocron IM, Warden C, Mitchell SL, Burgess LG, Goodale MP, Osborn MP, Ferreira AJ, Law JC, Cherney EF, Jones DP, Brantley MA.
      Purpose: To determine plasma metabolite and metabolic pathway differences between patients with type 2 diabetes with diabetic retinopathy (DR) and without retinopathy (diabetic controls), and between patients with proliferative DR (PDR) and nonproliferative DR (NPDR).Methods: Using high-resolution mass spectrometry with liquid chromatography, untargeted metabolomics was performed on plasma samples from 83 DR patients and 90 diabetic controls. Discriminatory metabolic features were identified through partial least squares discriminant analysis, and linear regression was used to adjust for age, sex, diabetes duration, and hemoglobin A1c. Pathway analysis was performed using Mummichog 2.0.
    Results: In the adjusted analysis, 126 metabolic features differed significantly between DR patients and diabetic controls. Pathway analysis revealed alterations in the metabolism of amino acids, leukotrienes, niacin, pyrimidine, and purine. Arginine, citrulline, glutamic γ-semialdehyde, and dehydroxycarnitine were key contributors to these pathway differences. A total of 151 features distinguished PDR patients from NPDR patients, and pathway analysis revealed alterations in the β-oxidation of saturated fatty acids, fatty acid metabolism, and vitamin D3 metabolism. Carnitine was a major contributor to the pathway differences.
    Conclusions: This study demonstrates that arginine and citrulline-related pathways are dysregulated in DR, and fatty acid metabolism is altered in PDR patients compared with NPDR patients.
    DOI:  https://doi.org/10.1167/iovs.19-27321
  28. Oncogene. 2019 Jul 19.
    Cordero A, Kanojia D, Miska J, Panek WK, Xiao A, Han Y, Bonamici N, Zhou W, Xiao T, Wu M, Ahmed AU, Lesniak MS.
      Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer patients is associated with increased incidence of breast cancer brain metastases (BCBM), but the mechanisms underlying this phenomenon remain unclear. Here, to identify brain-predominant genes critical for the establishment of BCBM, we conducted an in silico screening analysis and identified that increased levels of fatty acid-binding protein 7 (FABP7) correlate with a lower survival and higher incidence of brain metastases in breast cancer patients. We validated these findings using HER2+ BCBM cells compared with parental breast cancer cells. Importantly, through knockdown and overexpression assays, we characterized the role of FABP7 in the BCBM process in vitro and in vivo. Our results uncover a key role of FABP7 in metabolic reprogramming of HER2 + breast cancer cells, supporting a glycolytic phenotype and storage of lipid droplets that enable their adaptation and survival in the brain microenvironment. In addition, FABP7 is shown to be required for upregulation of key metastatic genes and pathways, such as integrins-Src and VEGFA, and for the growth of HER2+ breast cancer cells in the brain microenvironment in vivo. Together, our results support FABP7 as a potential target for the treatment of HER2+ BCBM.
    DOI:  https://doi.org/10.1038/s41388-019-0893-4
  29. J Am Soc Mass Spectrom. 2019 Jul 25.
    Poad BLJ, Marshall DL, Harazim E, Gupta R, Narreddula VR, Young RSE, Duchoslav E, Campbell JL, Broadbent JA, Cvačka J, Mitchell TW, Blanksby SJ.
      The specific positions of carbon-carbon double bond(s) within an unsaturated fatty acid exert a significant effect on the physical and chemical properties of the lipid that ultimately inform its biological function(s). Contemporary liquid chromatography-mass spectrometry (MS) strategies based on electrospray ionization coupled to tandem MS can easily detect fatty acyl lipids but generally cannot reveal those specific site(s) of unsaturation. Herein, we describe a novel and versatile workflow whereby fatty acids are first converted to fixed charge N-(4-aminomethylphenyl)pyridinium (AMPP) derivatives and subsequently subjected to ozone-induced dissociation (OzID) on a modified triple quadrupole mass spectrometer. The AMPP modification enhances the detection of fatty acids introduced by direct infusion. Fragmentation of the derivatized fatty acids also provides diagnostic fragment ions upon collision-induced dissociation that can be targeted in precursor ion scans to subsequently trigger OzID analyses in an automated data-dependent workflow. It is these OzID analyses that provide unambiguous assignment of carbon-carbon double bond locations in the AMPP-derivatized fatty acids. The performance of this analysis pipeline is assessed in profiling the patterns of unsaturation in fatty acids within the complex biological secretion vernix caseosa. This analysis uncovers significant isomeric diversity within the fatty acid pool of this sample, including a number of hitherto unreported double bond positional isomers that hint at the activity of potentially new metabolic pathways.
    Keywords:  Fatty Acids; Lipids; ozone-induced dissociation; vernix caseosa
    DOI:  https://doi.org/10.1007/s13361-019-02285-5
  30. Adv Exp Med Biol. 2019 ;1140 575-583
    Snider J, Wang D, Bogenhagen DF, Haley JD.
      The global measurement of assembly and turnover of protein containing complexes within cells has advanced with the development of pulse stable isotope labelled amino acid approaches. Stable isotope labeling with amino acids in cell culture (SILAC) allows the incorporation of "light" 12-carbon amino acids or "heavy" 13-carbon amino acids into cells or organisms and the quantitation of proteins and peptides containing these amino acid tags using mass spectrometry. The use of these labels in pulse or pulse-chase scenarios has enabled measurements of macromolecular dynamics in cells, on time scales of several hours. Here we review advances with this approach and alternative or parallel strategies. We also examine the statistical considerations impacting datasets detailing mitochondrial assembly, to highlight key parameters in establishing significance and reproducibility.
    Keywords:  Protein complex assembly; Protein synthesis; Pulse SILAC
    DOI:  https://doi.org/10.1007/978-3-030-15950-4_34
  31. Pharmacol Res. 2019 Jul 16. pii: S1043-6618(19)30855-2. [Epub ahead of print] 104352
    Zeng JD, Wu WKK, Wang HY, Li XX.
      Mammalian / mechanistic target of rapamycin (mTOR) is a critical sensor of environmental cues that regulates cellular macromolecule synthesis and metabolism in eukaryotes. DNA methylation is the most well-studied epigenetic modification that is capable of regulating gene transcription and affecting genome stability. Both dysregulation of mTOR signaling and DNA methylation patterns have been shown to be closely linked to tumor progression and serve as promising targets for cancer therapy. Although their respective roles in tumorigenesis have been extensively studied, whether molecular interplay exists between them is still largely unknown. In this review, we provide a brief overview of mTOR signaling, DNA methylation as well as related serine and one-carbon metabolism, one of the most critical aspects of metabolic reprogramming in cancer. Based on the latest understanding regarding the regulation of metabolic processes by mTOR signaling as well as interaction between metabolism and epigenetics, we further discuss how serine and one-carbon metabolism may serve as a bridge that links mTOR signaling and DNA methylation to promote tumor growth. Elucidating their relationship may provide novel insight for cancer therapy in the future.
    Keywords:  5-azacytidine (9444); Cancer therapy; DNA methylation; Methionine (PubChem CID 6137); One-carbon metabolism; S-adenosylmethionine (34755); Serine; Tumorigenesis; decitabine (451668); everolimus (6442177); glutathione (124886); mTOR; rapamycin (5284616); temsirolimus (6918289); tetrahydrofolate (135444742); vitamin B12 (5311498)
    DOI:  https://doi.org/10.1016/j.phrs.2019.104352
  32. Int J Mol Sci. 2019 Jul 09. pii: E3374. [Epub ahead of print]20(13):
    Zambrano A, Molt M, Uribe E, Salas M.
      An important hallmark in cancer cells is the increase in glucose uptake. GLUT1 is an important target in cancer treatment because cancer cells upregulate GLUT1, a membrane protein that facilitates the basal uptake of glucose in most cell types, to ensure the flux of sugar into metabolic pathways. The dysregulation of GLUT1 is associated with numerous disorders, including cancer and metabolic diseases. There are natural products emerging as a source for inhibitors of glucose uptake, and resveratrol is a molecule of natural origin with many properties that acts as antioxidant and antiproliferative in malignant cells. In the present review, we discuss how GLUT1 is involved in the general scheme of cancer cell metabolism, the mechanism of glucose transport, and the importance of GLUT1 structure to understand the inhibition process. Then, we review the current state-of-the-art of resveratrol and other natural products as GLUT1 inhibitors, focusing on those directed at treating different types of cancer. Targeting GLUT1 activity is a promising strategy for the development of drugs aimed at treating neoplastic growth.
    Keywords:  GLUT1; cancer metabolism; cancer therapy; glucose uptake inhibition; resveratrol
    DOI:  https://doi.org/10.3390/ijms20133374
  33. Methods Mol Biol. 2019 ;2030 307-313
    Hirayama A, Ikeda S, Sato A, Soga T.
      Capillary electrophoresis-mass spectrometry (CE-MS) has been developed as a powerful tool in the analysis of charged compounds. To simultaneously analyze free amino acids, an electrolyte with low pH was used to positively charge all of the amino acids. In this condition, all protonated amino acids migrated toward the cathode in CE and then were sensitively and selectively detected by MS. This method is simple, rapid, and selective and could readily be applied to the analysis of free amino acids in various samples. In this chapter, the detailed procedure to analyze amino acids using CE-tandem mass spectrometry (MS/MS) is described.
    Keywords:  Capillary electrophoresis; Mass spectrometry; Tandem mass spectrometry; Tumor tissue
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_23
  34. Methods Mol Biol. 2019 ;2030 395-402
    Werneth M, Koellensperger G, Hann S.
      Analysis of underivatized amino acids is challenging regarding both separation and detection of this small, polar, and largely UV-inactive compounds. Additives for reversed phase chromatography such as ion pairing reagents can hamper mass spectrometric detection. Zwitterionic hydrophilic interaction chromatography using MS compatible eluents together with tandem mass spectrometry in multiple reaction monitoring mode for selective detection is an attractive approach to overcome the abovementioned issues.
    Keywords:  Amino acid analysis; HILIC; MRM; Qualifier transition; Quantifier transition; Root exudation; Tandem mass spectrometry; Triple quadrupole mass spectrometry; Zwitterionic phase
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_29
  35. Mol Genet Metab. 2019 Jul 18. pii: S1096-7192(19)30292-6. [Epub ahead of print]
    Brown M, Ashcraft P, Arning E, Bottiglieri T, Roullet JB, Gibson KM.
      Increased gamma-hydroxybutyric acid in urine and blood are metabolic hallmarks of succinic semialdehyde dehydrogenase deficiency, a defect of 4-aminobutyric acid metabolism. Here, we examined the hypothesis that succinic semialdehyde dehydrogenase deficiency could be identified via measurement of gamma-hydroxybutyric acid in newborn and post-newborn dried bloodspots. Quantitation of gamma-hydroxybutyric acid using liquid chromatography-tandem mass spectrometry in twelve archival newborn patient dried bloodspots was 360 ± 57 μM (mean, standard error; range 111-767), all values exceeding the previously established cutoff for newborn detection of 78 μΜ established from 2831 dried bloodspots derived from newborns, neonates and children. Gamma-hydroxybutyric acid in post-newborn dried bloodspots (n = 19; ages 0.8-38 years) was 191 ± 65 μM (mean, standard error; range 20-1218), exceeding the aforementioned GHB cutoff for patients approximately 10 years of age or younger. Further, gamma-hydroxybutyric acid in post-newborn dried bloodspots displayed a significant (p < .0001) inverse correlation with age. This preliminary study suggests that succinic semialdehyde dehydrogenase deficiency may be identified in newborn and post-newborn dried bloodspots via quantitation of gamma-hydroxybutyric acid, while forming the platform for more extensive studies in affected and unaffected dried bloodspots.
    Keywords:  Dried bloodspots; GABA metabolism; Gamma-hydroxybutyric acid; Newborn screening
    DOI:  https://doi.org/10.1016/j.ymgme.2019.07.010
  36. Methods Mol Biol. 2019 ;2030 1-10
    Louwagie M, Kieffer-Jaquinod S, Brun V.
      Recombinant proteins are essential components of therapeutic, biotechnological, food, and household products. In some cases, recombinant proteins must be purified and their quantity and/or concentration precisely determined. In this chapter, we describe a protocol for the quantification of purified recombinant proteins. The protocol is based on a microwave-assisted acidic hydrolysis of the target protein followed by high-resolution mass spectrometry (HRMS) analysis of the hydrolytic products. Absolute quantification is obtained by adding controlled amounts of labeled amino acids that serve as standards.
    Keywords:  Amino acid analysis; Mass spectrometry; Protein; Quantification
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_1
  37. Sci Rep. 2019 Jul 26. 9(1): 10873
    Song J, Wang X, Guo Y, Yang Y, Xu K, Wang T, Sa Y, Yuan L, Jiang H, Guo J, Sun Z.
      The complexity of follicular fluid metabolome presents a significant challenge for qualitative and quantitative metabolite profiling, and for discovering the comprehensive biomarkers. In order to address this challenge, a novel SWATHtoMRM metabolomics method was used for providing broad coverage and excellent quantitative capability to discover the human follicular fluid metabolites related to recurrent spontaneous abortion (RSA) after in vitro fertilization and embryo transfer, and to evaluate their relationship with pregnancy outcome. The follicular fluid samples from the spontaneous abortion group (n = 22) and the control group (n = 22) were analyzed using ultra-performance liquid chromatography high-resolution mass spectrometry. A novel, high-coverage, targeted metabolomics method (SWATH to MRM) and a targeted metabolomics method were used to find and validate the differential metabolites between the two groups. A total of 18 follicular fluid metabolites, including amino acids, cholesterol, vitamins, fatty acids, cholic acid, lysophosphatidylcholine and other metabolites, were identified. In the RSA group, 8 metabolites, namely dehydroepiandrosterone, lysoPC(16:0), lysoPC(18:2), lysoPC(18:1), lysoPC(18:0), lysoPC(20:5), lysoPC(20:4), and lysoPC(20:3), were up-regulated, and 10 metabolites, namely phenylalanine, linoleate, oleic acid, docosahexaenoic acid, lithocholic acid, 25-hydroxyvitamin D3, hydroxycholesterol, 13-hydroxy-alpha-tocopherol, leucine, and tryptophan, were down-regulated. These differential metabolites related to RSA may provide a possible diagnostic basis and therapeutic target for RSA, as well as a scientific basis for elucidating the mechanism of RSA.
    DOI:  https://doi.org/10.1038/s41598-019-47370-7
  38. Lipids. 2019 Jul 24.
    Picklo MJ, Hanson BK, Bukowski MR.
      Ceramides (CER) are biologically active sphingolipid precursors that are mechanistically linked to several pathogenic states including cancer, insulin resistance, and neurodegeneration. CER are commonly quantified through mass spectrometry-based methods founded upon a product ion scan (PIS) in positive mode to produce a characteristic m/z 264 ion. The ionization efficiency (IE) of CER species decreases with an increase in CER mass, thus quantitation of CER typically involves application of mass-dependent response factors (RF) for each CER species. In this work, we observed that the RF were systematically dependent on the number of fatty acid acyl carbons and the collision energy (CE) used to generate the m/z 264 ion. Using these complimentary trends, we determined an "isosbestic" CE where the RF for all CER species were equivalent, allowing for CER quantitation without postcollection correction factors. A comparison of this common CE/common RF method to the multiple RF method demonstrated good agreement between the two methods. Use of the common CE/common RF method will reduce data processing and reduce the use of multiple CER species standards.
    Keywords:  Ceramide; Collision energy; Ionization efficiency; Mass spectrometry
    DOI:  https://doi.org/10.1002/lipd.12179
  39. Methods Mol Biol. 2019 ;2030 69-83
    Tripp JA, Devièse T, McCullagh JSO.
      Single-compound analysis of stable or radioactive isotopes has found application in a number of fields ranging from archaeology to forensics. Often, the most difficult part of these analyses is the development of a method for isolating the compound(s) of interest, which can derive from a wide range of sample types including the hair, nails, and bone.Here we describe three complementary preparative HPLC techniques suitable for separating and isolating amino acids from bone collagen and hair keratin. Using preparative reversed-phase, ion-pair, or mixed-mode chromatography in aqueous carbon-free mobile phases, or those from which carbon can easily be removed, underivatized single amino acids can be isolated and further analyzed using mass spectrometric techniques.
    Keywords:  Accelerator mass spectrometry (AMS); Amino acid (AA); Compound-specific isotope analysis (CSIA); Compound-specific radiocarbon analysis (CSRA); High-performance liquid chromatography (HPLC); Isotope ratio mass spectrometry (IRMS); Liquid chromatography-isotope ratio mass spectrometry (LC-IRMS); Mixed-mode high-performance liquid chromatography (MM-HPLC); Preparative chromatography; Radiocarbon dating; Stable isotopes; ion-pair high-performance liquid chromatography (IP-HPLC); Δ14C; δ13C
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_7
  40. Methods Mol Biol. 2019 ;2030 253-261
    Nakano Y, Taniguchi M, Umakoshi Y, Watai D, Fukusaki E.
      D-Amino acids have recently attracted much attention in various research fields including medical, clinical, and food industry due to their important biological functions that differ from L-amino acid. Most chiral amino acid separation techniques require complicated derivatization procedures in order to achieve the desirable chromatographic behavior and detectability. This chapter describes a highly sensitive analytical method for the enantioseparation of chiral amino acids without any derivatization process using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method allows the simultaneous analysis of 18 D-amino acids with high sensitivity and reproducibility. Additionally, this chapter also focuses on the application of the method to real samples for the quantification of targeted amino acids.
    Keywords:  Chiral; D-Amino acid; Enantioseparation; LC-MS/MS; Quantitative analysis; Underivatized
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_19
  41. Methods Mol Biol. 2019 ;2030 365-379
    Ziegler J, Hussain H, Neubert RHH, Abel S.
      A method for selective and sensitive quantification of amino acids is described. The combination of established derivatization procedures of secondary and primary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) and subsequent detection of derivatized amino acids by LC-ESI-MS/MS using multiple reaction monitoring provides high selectivity. The attachment of an apolar moiety enables purification of derivatized amino acids from matrix by a single solid-phase extraction step, which increases sensitivity by reduced ion suppression during LC-ESI-MS/MS detection. Additionally, chromatography of all amino acids can be performed on reversed-phase HPLC columns using eluents without additives, which are known to cause significant decreases in signal to noise ratios. The method has been routinely applied for amino acid profiling of low amounts of liquids and tissues of various origins with a sample throughput of about 50-100 samples a day. In addition to a detailed description of the method, some representative examples are presented.
    Keywords:  Amino acid profiling; Derivatization; Fmoc-Cl; Mass spectrometry; Multiple reaction monitoring; Reversed-phase HPLC; Solid-phase extraction
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_27
  42. Adv Exp Med Biol. 2019 ;1140 1-26
    Woods AG, Sokolowska I, Ngounou Wetie AG, Channaveerappa D, Dupree EJ, Jayathirtha M, Aslebagh R, Wormwood KL, Darie CC.
      Within the past years, we have witnessed a great improvement is mass spectrometry (MS) and proteomics approaches in terms of instrumentation, protein fractionation, and bioinformatics. With the current technology, protein identification alone is no longer sufficient. Both scientists and clinicians want not only to identify the proteins, but also to identify the protein's post-translational modifications (PTMs), protein isoforms, protein truncation, protein-protein interactions (PPI), and protein quantitation. Here, we describe the principle of MS and proteomics, and strategies to identify proteins, protein's PTMs, protein isoforms, protein truncation, PPIs, and protein quantitation. We also discuss the strengths and weaknesses within this field. Finally, in our concluding remarks we assess the role of mass spectrometry and proteomics in the scientific and clinical settings, in the near future. This chapter provides an introduction and overview for subsequent chapters that will discuss specific MS proteomic methodologies and their application to specific medical conditions. Other chapters will also touch upon areas that expand beyond proteomics, such as lipidomics and metabolomics.
    Keywords:  LC-MS/MS; MALDI-MS; Mass spectrometry; Proteomics
    DOI:  https://doi.org/10.1007/978-3-030-15950-4_1
  43. Cancer Metab. 2019 ;7 7
    Thomas LW, Esposito C, Stephen JM, Costa ASH, Frezza C, Blacker TS, Szabadkai G, Ashcroft M.
      Background: Mitochondrial oxidative phosphorylation (OXPHOS) via the respiratory chain is required for the maintenance of tumour cell proliferation and regulation of epithelial to mesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study, we interrogate the importance of CHCHD4-regulated mitochondrial metabolism for tumour cell proliferation and EMT-related phenotypes, and elucidate key pathways involved.Results: Using in silico analyses of 967 tumour cell lines, and tumours from different cancer patient cohorts, we show that CHCHD4 expression positively correlates with OXPHOS and proliferative pathways including the mTORC1 signalling pathway. We show that CHCHD4 expression significantly correlates with the doubling time of a range of tumour cell lines, and that CHCHD4-mediated tumour cell growth and mTORC1 signalling is coupled to respiratory chain complex I (CI) activity. Using global metabolomics analysis, we show that CHCHD4 regulates amino acid metabolism, and that CHCHD4-mediated tumour cell growth is dependent on glutamine. We show that CHCHD4-mediated tumour cell growth is linked to CI-regulated mTORC1 signalling and amino acid metabolism. Finally, we show that CHCHD4 expression in tumours is inversely correlated with EMT-related gene expression, and that increased CHCHD4 expression in tumour cells modulates EMT-related phenotypes.
    Conclusions: CHCHD4 drives tumour cell growth and activates mTORC1 signalling through its control of respiratory chain mediated metabolism and complex I biology, and also regulates EMT-related phenotypes of tumour cells.
    Keywords:  Coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4); HIF-1α; complex I; disulfide relay system; hypoxia; mitochondria; respiratory chain; tumour growth; tumour metabolism
    DOI:  https://doi.org/10.1186/s40170-019-0200-4
  44. Biomed Pharmacother. 2019 Jul 18. pii: S0753-3322(19)32555-7. [Epub ahead of print]118 109210
    Zou S, Wang X, Liu P, Ke C, Xu S.
      Certain cancer cells with nutrient auxotrophy and have a much higher nutrient demand compared with normal human cells. Arginine as a versatile amino acid, has multiple biological functions in metabolic and signaling pathways. Depletion of this amino acid by arginine depletor is generally well tolerated and has become a targeted therapy for arginine auxotrophic cancers. However, the modulatory eff ;ect of arginine on cancer cells is very complicated and still controversial. Therefore, this article focuses on arginine metabolism and depletion therapy in cancer treatment to provide systemical review on this issue.
    Keywords:  Arginine; Cancer; Deprivation; Metabolism
    DOI:  https://doi.org/10.1016/j.biopha.2019.109210
  45. J Proteomics. 2019 Jul 16. pii: S1874-3919(19)30218-0. [Epub ahead of print]206 103446
    Goh WWB, Zhao Y, Sue AC, Guo T, Wong L.
      Cancer is a heterogeneous disease, confounding the identification of relevant markers and drug targets. Network-based analysis is robust against noise, potentially offering a promising approach towards biomarker identification. We describe here the application of two network-based methods, qPSP (Quantitative Proteomics Signature Profiling) and PFSNet (Paired Fuzzy SubNetworks), in an intra-tissue proteome data set of prostate tissue samples. Despite high basal variation, we find that traditional statistical analysis may exaggerate the extent of heterogeneity. We also report that network-based analysis outperforms protein-based feature selection with concomitantly higher cross-validation accuracy. Overall, network-based analysis provides emergent signal that boosts sensitivity while retaining good precision. It is a potential means of circumventing heterogeneity for stable biomarker discovery.
    Keywords:  Bioinformatics; Biomarker; Networks; Proteomics; Systems biology
    DOI:  https://doi.org/10.1016/j.jprot.2019.103446
  46. Proc Natl Acad Sci U S A. 2019 Jul 24. pii: 201902346. [Epub ahead of print]
    Bockwoldt M, Houry D, Niere M, Gossmann TI, Reinartz I, Schug A, Ziegler M, Heiland I.
      Nicotinamide adenine dinucleotide (NAD) provides an important link between metabolism and signal transduction and has emerged as central hub between bioenergetics and all major cellular events. NAD-dependent signaling (e.g., by sirtuins and poly-adenosine diphosphate [ADP] ribose polymerases [PARPs]) consumes considerable amounts of NAD. To maintain physiological functions, NAD consumption and biosynthesis need to be carefully balanced. Using extensive phylogenetic analyses, mathematical modeling of NAD metabolism, and experimental verification, we show that the diversification of NAD-dependent signaling in vertebrates depended on 3 critical evolutionary events: 1) the transition of NAD biosynthesis to exclusive usage of nicotinamide phosphoribosyltransferase (NamPT); 2) the occurrence of nicotinamide N-methyltransferase (NNMT), which diverts nicotinamide (Nam) from recycling into NAD, preventing Nam accumulation and inhibition of NAD-dependent signaling reactions; and 3) structural adaptation of NamPT, providing an unusually high affinity toward Nam, necessary to maintain NAD levels. Our results reveal an unexpected coevolution and kinetic interplay between NNMT and NamPT that enables extensive NAD signaling. This has implications for therapeutic strategies of NAD supplementation and the use of NNMT or NamPT inhibitors in disease treatment.
    Keywords:  NAD pathway dynamics and evolution; NAD-dependent signaling and biosynthesis; mathematical modeling; nicotinamide N-methyltransferase (NNMT); nicotinamide phosphoribosyltransferase (NamPT)
    DOI:  https://doi.org/10.1073/pnas.1902346116
  47. Methods Mol Biol. 2019 ;2030 111-118
    Kato M, Takatsu A.
      Here, we describe an amino acid analysis that is based on the use of hydrophilic interaction liquid chromatography coupled with isotope dilution mass spectrometry for the accurate quantification of underivatized amino acids from hydrolyzed peptide/protein. Twelve underivatized amino acids were separated and detected during an 88-min runtime. The absolute limits of detection and limits of quantification (on column) of the four amino acids (isoleucine, phenylalanine, proline, and valine) were in the range of 6-80 and 20-200 fmol, respectively. As little as 25 pmol of peptide or protein hydrolysate is sufficient for determining absolute content.
    Keywords:  Accuracy; Amino acid analysis; Hydrophilic interaction liquid chromatography; Isotope dilution mass spectrometry; Peptide/protein quantification
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_9
  48. Prostaglandins Other Lipid Mediat. 2019 Jul 16. pii: S1098-8823(19)30049-8. [Epub ahead of print] 106350
    Liening S, Romp E, Werz O, Scriba G, Garscha U.
      Glutathione (GSH) conjugates of oxygenated polyunsaturated fatty acids comprise a group of pro-inflammatory and pro-resolving lipid mediators formed in immunocompetent cells. While the pro-inflammatory conjugates as the cysteinyl leukotrienes (cys-LTs), eoxins (EXs) and five-oxo-GSH conjugate (FOG7) derive from arachidonic acid (AA), the group of conjugates in tissue regeneration (CTRs) such as maresin CTRs (MCTRs), protectin CTRs (PCTRs) and resolvin CTRs (RCTRs) are biosynthesized from docosahexaenoic acid (DHA). Here, we present a gradient UPLC-MS/MS method for the analysis of pro-inflammatory and pro-resolving GSH conjugates using positive electrospray ionization (ESI(+)) and collision-induced fragmentation for unambiguous identification and structural information, and a negative ionization (ESI(-)) mode for quantification of the GSH conjugates. The method was employed to detect GSH conjugates in human platelets and macrophages. MCTRs were detected in platelets upon addition of exogenous docosahexaenoic acid (DHA) and the biosynthesis was independent on leukotriene C4 (LTC4) synthase activity. Pathogenic bacteria stimulated the formation of EXs and PCTRs in M2 macrophages, whereas Ca2+-ionophore activated the biosynthesis of LTC4 in M1 and M2 macrophage phenotypes. Together, our methodology covers the qualitative and quantitative analysis of GSH conjugates and gives an analytical basis for the detection and structural elucidation of cysteinyl-containing lipid mediators.
    DOI:  https://doi.org/10.1016/j.prostaglandins.2019.106350
  49. Methods Mol Biol. 2019 ;2030 293-306
    Toyo'oka T.
      L-Pyroglutamic acid succinimidyl ester (L-PGA-OSu) and its isotopic variant (L-PGA[d5]-OSu) were synthesized and used as the chiral labeling reagents for the enantioseparation of amino acids by reversed-phase UPLC-ESI-MS/MS. The enantiomers of amino acids were labeled with the reagents at 60 °C for 10 min in an alkaline medium. The resulting diastereomers were well separated by the reversed-phase chromatography using an ODS column, packed with small particles (1.7 μm) (Rs = 1.95-8.05). A highly sensitive detection at a low-fmol level (0.5-3.2 fmol) was obtained from the selected reaction monitoring (SRM) chromatograms. An isotope labeling strategy using light and heavy variants for the differential analysis of the DL-amino acids in different sample groups is also presented in this paper. The ratios of D/L-alanine in different yogurt products were successfully determined by the proposed method. The D/L ratios were almost comparable to those obtained from only using light reagent (i.e., L-PGA-OSu). Therefore, the proposed strategy seems to be useful for the differential analysis of DL-amino acids, not only in food products but also in biological samples.
    Keywords:  Amino acid; Enantioseparation; Isotope-coded derivatization; L-pyroglutamic acid succinimidyl ester; Mass spectrometry; Reversed-phase chromatography
    DOI:  https://doi.org/10.1007/978-1-4939-9639-1_22
  50. Cancers (Basel). 2019 Jul 11. pii: E972. [Epub ahead of print]11(7):
    Kuciauskas D, Dreize N, Ger M, Kaupinis A, Zemaitis K, Stankevicius V, Suziedelis K, Cicenas J, Graves LM, Valius M.
      Antitumor drug resistance remains a major challenge in cancer chemotherapy. Here we investigated the mechanism of acquired resistance to a novel anticancer agent RH1 designed to be activated in cancer cells by the NQO1 enzyme. Data show that in some cancer cells RH1 may act in an NQO1-independent way. Differential proteomic analysis of breast cancer cells with acquired resistance to RH1 revealed changes in cell energy, amino acid metabolism and G2/M cell cycle transition regulation. Analysis of phosphoproteomics and protein kinase activity by multiplexed kinase inhibitor beads showed an increase in the activity of protein kinases involved in the cell cycle and stemness regulation and downregulation of proapoptotic kinases such as JNK in RH1-resistant cells. Suppression of JNK leads to the increase of cancer cell resistance to RH1. Moreover, resistant cells have enhanced expression of stem cell factor (SCF) and stem cell markers. Inhibition of SCF receptor c-KIT resulted in the attenuation of cancer stem cell enrichment and decreased amounts of tumor-initiating cells. RH1-resistant cells also acquire resistance to conventional therapeutics while remaining susceptible to c-KIT-targeted therapy. Data show that RH1 can be useful to treat cancers in the NQO1-independent way, and targeting of the cancer stem cells might be an effective approach for combating resistance to RH1 therapy.
    Keywords:  JNK; MIBs; RH1; c-KIT; cancer drug resistance; cancer stem cells; chemotherapy; label-free proteomics; phosphoproteome; protein kinases
    DOI:  https://doi.org/10.3390/cancers11070972
  51. Int J Mol Sci. 2019 Jul 14. pii: E3461. [Epub ahead of print]20(14):
    Zebrowska A, Skowronek A, Wojakowska A, Widlak P, Pietrowska M.
      Exosomes and other classes of extracellular vesicles (EVs) have gained interest due to their role in cell-to-cell communication. Knowledge of the molecular content of EVs may provide important information on features of parental cells and mechanisms of cross-talk between cells. To study functions of EVs it is essential to know their composition, that includes proteins, nucleic acids, and other classes biomolecules. The metabolome, set of molecules the most directly related to the cell phenotype, is the least researched component of EVs. However, the metabolome of EVs circulating in human blood and other bio-fluids is of particular interest because of its potential diagnostic value in cancer and other health conditions. On the other hand, the metabolome of EVs released to culture media in controlled conditions in vitro could shed light on important aspects of communication between cells in model systems. This paper summarizes the most common approaches implemented in EV metabolomics and integrates currently available data on the composition of the metabolome of EVs obtained in different models with particular focus on human body fluids and cancer cells.
    Keywords:  exosomes; extracellular vesicles; lipids; liquid biopsy; mass spectrometry; metabolites; metabolomics
    DOI:  https://doi.org/10.3390/ijms20143461
  52. Front Plant Sci. 2019 ;10 850
    Lei Z, Watson BS, Huhman D, Yang DS, Sumner LW.
      A total of 1,622 samples representing 201 Medicago truncatula ecotypes were analyzed using ultrahigh pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS) to ascertain saponin profiles in different M. truncatula ecotypes and to provide data for a genome-wide association study and subsequent line selection for saponin biosynthesis. These ecotypes originated from 14 different Mediterranean countries, i.e., Algeria, Cyprus, France, Greece, Israel, Italy, Jordan, Libya, Morocco, Portugal, Spain, Syria, Tunisia, and Turkey. The results revealed significant differences in the saponin content among the ecotypes. European ecotypes generally contained higher saponin content than African ecotypes (p < 0.0001). This suggests that M. truncatula ecotypes modulate their secondary metabolism to adapt to their environments. Significant differences in saponin accumulation were also observed between the aerial and the root tissues of the same ecotypes (p < 0.0001). While some saponins were found to be present in both the aerial and root tissues, zanhic acid glycosides were found predominantly in the aerial tissues. Bayogenin and hederagenin glycosides were found mostly in roots. The differential spatially resolved accumulation of saponins suggests that saponins in the aerial and root tissues play different roles in plant fitness. Aerial saponins such as zanhic glycosides may act as animal feeding deterrent and root saponins may protect against soil microbes.
    Keywords:  LC-MS/MS; Medicago truncatula; ecotypes; metabolomics; triterpene saponin
    DOI:  https://doi.org/10.3389/fpls.2019.00850