bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2019‒05‒12
fifty-nine papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research


  1. Expert Opin Ther Targets. 2019 May 10. 1-11
    Pope ED, Kimbrough EO, Vemireddy LP, Surapaneni PK, Copland JA, Mody K.
      INTRODUCTION: Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers. Progress has been made in treatment of HCC; however, improved outcomes are much needed. The increased metabolic needs of cancer cells underscore the importance of metabolic pathways in cancer cell survival. Lipid metabolism has a role in HCC development; aberrant overexpression of several key enzymes is seen in many solid human tumors. Areas covered: We discuss aberrant lipid metabolism and the promise of multiple targets, in particular related to HCC treatment. We searched PubMed and clinicaltrials.gov for published and unpublished studies from 2000 to 2019. These terms were used: lipids, fatty acid metabolism, lipid metabolism, liver cancer, HCC, de novo fatty acid synthesis, ATP citrate lyase, stearoyl CoA denaturase, fatty acid synthase, acetyl coenzyme A carboxylase, CD147, KLF4, monoglyceride lipase, AMP activated protein kinase. Expert opinion: The importance of dysregulation of fatty acid synthesis in cancer is a growing area of research. HCC demonstrates significant alteration in lipid metabolism, representing great potential as a target for novel therapeutics. Various agents have demonstrated promising anti-neoplastic activity. This strategy deserves further development for improved outcomes.
    Keywords:  Fatty acid; SCD1; hepatocellular carcinoma; lipid metabolism; lipids; liver cancer; stearoyl co-A desaturase
    DOI:  https://doi.org/10.1080/14728222.2019.1615883
  2. Cell Metab. 2019 Apr 28. pii: S1550-4131(19)30188-3. [Epub ahead of print]
    Duman C, Yaqubi K, Hoffmann A, Acikgöz AA, Korshunov A, Bendszus M, Herold-Mende C, Liu HK, Alfonso J.
      Glioblastoma multiforme (GBM) undergoes metabolic reprogramming to meet the high ATP and anabolic demands of the tumor cells. However, the role of fatty acid oxidation (FAO) and its regulators in the GBM context has been largely unknown. Here, we show that the neural stem cell pro-proliferative factor acyl-CoA-binding protein (ACBP, also known as DBI) is highly expressed in GBM, and by binding to acyl-CoAs, it cell-autonomously maintains high proliferation rates, promoting tumor growth and poor survival in several preclinical models. Mechanistic experiments using ACBP-acyl-CoA binding affinity variants and pharmacological FAO modulators suggest that ACBP supports tumor growth by controlling the availability of long-chain fatty acyl-CoAs to mitochondria, promoting FAO in GBM. Thus, our findings uncover a critical link between lipid metabolism and GBM progression established by ACBP and offer a potential therapeutic strategy for an effective anti-proliferative metabolic management of GBM.
    Keywords:  ACBP; DBI; GABA(A) receptor; acyl-CoA; brain cancer; glioblastoma; lipid metabolism; mitochondrial respiration; senescence; β-oxidation
    DOI:  https://doi.org/10.1016/j.cmet.2019.04.004
  3. Animals (Basel). 2019 May 07. pii: E222. [Epub ahead of print]9(5):
    Belghit I, Lock EJ, Fumière O, Lecrenier MC, Renard P, Dieu M, Berntssen MHG, Palmblad M, Rasinger JD.
      Insect protein has the potential to become a sustainable feed ingredient for the rapidly growing aquaculture industry. In the European Union, insect derived protein is placed under the same legislation as processed animal proteins (PAP). It is therefore of interest to develop methods for regulatory use, which unambiguously identify the species origin of insect-based ingredients. We performed (i) total protein quantification of insect samples using the traditional nitrogen-to-protein conversion factor of 6.25 and the sum of anhydrous amino acids, (ii) quantitative amino acid profiling and (iii) high-throughput tandem mass spectrometry to describe and differentiate 18 different commercial-grade insect meal samples derived from Hermetia illucens (8), Tenebrio molitor (5), Alphitobius diaperinus (3) and Acheta domesticus (2). In addition, we investigated and compared different protein extraction and digestion protocols for proteomic analysis. We found that irrespective of sample preparation, shotgun proteomics in combination with direct spectral comparison were able to differentiate insect meal according to their taxonomic classification. The insect specific spectral libraries created in the present work can in future be used to develop more sensitive targeted methods of insect PAP identification and quantification in commercial feed mixtures.
    Keywords:  insect meal; protein quantification; shotgun proteomics; species differentiation
    DOI:  https://doi.org/10.3390/ani9050222
  4. Nat Med. 2019 May;25(5): 850-860
    Li H, Ning S, Ghandi M, Kryukov GV, Gopal S, Deik A, Souza A, Pierce K, Keskula P, Hernandez D, Ann J, Shkoza D, Apfel V, Zou Y, Vazquez F, Barretina J, Pagliarini RA, Galli GG, Root DE, Hahn WC, Tsherniak A, Giannakis M, Schreiber SL, Clish CB, Garraway LA, Sellers WR.
      Despite considerable efforts to identify cancer metabolic alterations that might unveil druggable vulnerabilities, systematic characterizations of metabolism as it relates to functional genomic features and associated dependencies remain uncommon. To further understand the metabolic diversity of cancer, we profiled 225 metabolites in 928 cell lines from more than 20 cancer types in the Cancer Cell Line Encyclopedia (CCLE) using liquid chromatography-mass spectrometry (LC-MS). This resource enables unbiased association analysis linking the cancer metabolome to genetic alterations, epigenetic features and gene dependencies. Additionally, by screening barcoded cell lines, we demonstrated that aberrant ASNS hypermethylation sensitizes subsets of gastric and hepatic cancers to asparaginase therapy. Finally, our analysis revealed distinct synthesis and secretion patterns of kynurenine, an immune-suppressive metabolite, in model cancer cell lines. Together, these findings and related methodology provide comprehensive resources that will help clarify the landscape of cancer metabolism.
    DOI:  https://doi.org/10.1038/s41591-019-0404-8
  5. Cancer Res. 2019 May 07. pii: canres.2043.2018. [Epub ahead of print]
    Kaminski L, Torrino S, Dufies M, Djabari Z, Haider R, Roustan FR, Jaune E, Laurent K, Nottet N, Michiels JF, Gesson M, Rocchi S, Mazure NM, Durand M, Tanti JF, Ambrosetti D, Clavel S, Ben-Sahra I, Bost F.
      Although tumorigenesis is dependent on the reprogramming of cellular metabolism, the metabolic pathways engaged in the formation of metastases remain largely unknown. The transcriptional co-activator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) plays a pleiotropic role in the control of cancer cell metabolism and has been associated with a good prognosis in prostate cancer (PCa). Here we show that PGC-1α represses the metastatic properties of PCa cells via modulation of the polyamine biosynthesis pathway. Mechanistically, PGC-1α inhibits the expression of c-MYC and ornithine decarboxylase 1 (ODC1), the rate limiting enzyme for polyamine synthesis. Analysis of in vivo metastases and clinical data from prostate cancer patients support the proposition that the PGC-1α/c-MYC/ODC1 axis regulates polyamine biosynthesis and prostate cancer aggressiveness. In conclusion, downregulation of PGC-1α renders PCa cells dependent on polyamine to promote metastasis.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-18-2043
  6. Biomed Pharmacother. 2019 May 06. pii: S0753-3322(19)30029-0. [Epub ahead of print]115 108907
    Sun X, Lan J, Tong R, Zhang H, Sun S, Xiong A, Wang Z, Yang L.
      Plantaginis semen, the dried mature seed of Plantago asiatica L. or Plantago deprdssa Willd., has a prominent effect on the treatment of obesity, type 2 diabetes and lipid disorders, however, its clinical application is limited due to inadequate in-depth mechanism exploration and incomplete discussion of action targets of its in vivo. Therefore, an untargeted metabolomics approach was firstly applied to study the serum metabolic differences in mice. Metabolomics analysis was performed using ultra performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) together with multivariate statistical data analysis. The results showed that Plantaginis semen can mainly improve blood lipids, some degree in blood glucose and insulin levels in high-fat mice, in addition, the phenotype of liver and fat stained sections demonstrated remarkable results. A total of 22 metabolites involved in arachidonic acid, glycerophospholipid, glycosphingolipid, linoleate, Omega-3 fatty acid, phosphatidylinositol phosphate and tyrosine metabolisms were identified. In further, it was found that the possible mechanisms of Plantaginis semen on hyperlipidemic mice lied in the biosynthesis of thyroxine, biological effects of enzymes of phospholipase A2 activity, glucosylceramide synthase and inositol essential enzyme 1α, genes expressions of fatty acid metabolism and inflammation. Serum metabolomics revealed that Plantaginis semen could cure the organism disease via regulating multiple metabolic pathways which will be helpful for understanding the mechanism of this herb and providing references for better applications of it in clinic, even researches on other TCMs.
    Keywords:  Hyperlipidemic mice; Metabolomics; Plantaginis semen; UPLC-QTOF-MS
    DOI:  https://doi.org/10.1016/j.biopha.2019.108907
  7. Nat Med. 2019 May;25(5): 825-837
    Wang Z, Yip LY, Lee JHJ, Wu Z, Chew HY, Chong PKW, Teo CC, Ang HY, Peh KLE, Yuan J, Ma S, Choo LSK, Basri N, Jiang X, Yu Q, Hillmer AM, Lim WT, Lim TKH, Takano A, Tan EH, Tan DSW, Ho YS, Lim B, Tam WL.
      Understanding cellular metabolism holds immense potential for developing new classes of therapeutics that target metabolic pathways in cancer. Metabolic pathways are altered in bulk neoplastic cells in comparison to normal tissues. However, carcinoma cells within tumors are heterogeneous, and tumor-initiating cells (TICs) are important therapeutic targets that have remained metabolically uncharacterized. To understand their metabolic alterations, we performed metabolomics and metabolite tracing analyses, which revealed that TICs have highly elevated methionine cycle activity and transmethylation rates that are driven by MAT2A. High methionine cycle activity causes methionine consumption to far outstrip its regeneration, leading to addiction to exogenous methionine. Pharmacological inhibition of the methionine cycle, even transiently, is sufficient to cripple the tumor-initiating capability of these cells. Methionine cycle flux specifically influences the epigenetic state of cancer cells and drives tumor initiation. Methionine cycle enzymes are also enriched in other tumor types, and MAT2A expression impinges upon the sensitivity of certain cancer cells to therapeutic inhibition.
    DOI:  https://doi.org/10.1038/s41591-019-0423-5
  8. Hepatology. 2019 May 07.
    Ma F, Zhu Y, Liu X, Zhou Q, Hong X, Qu C, Feng X, Zhang Y, Ding Q, Zhao J, Hou J, Zhong M, Zhuo H, Zhong L, Ye Z, Xie W, Liu Y, Xiong Y, Chen H, Piao D, Sun B, Gao Z, Li Q, Zhang Z, Qiu X, Zhang Z.
      Cancer cells metabolize different energy sources to generate biomass rapidly. The purine biosynthetic pathway was recently identified as an important source of metabolic intermediates for these processes. However, very little was known about the regulatory mechanisms of the purine metabolism in hepatocellular carcinoma (HCC). Herein, we explored the role of Dual-specificity tyrosine- (Y) -phosphorylation-regulated kinase 3 (Dyrk3) in HCC metabolism. Dyrk3 was significantly downregulated in HCC compared with normal controls. Its introduction in HCC cells markedly suppressed tumor growth and metastasis in xenograft tumor models. Mass spectrometry analysis of metabolites suggests that the effect of Dyrk3 on HCC occurred at least partially through downregulating purine metabolism, as evidenced by the fact that inhibiting purine synthesis reverted the HCC progression mediated by the loss of Dyrk3. We further provide evidence that this action of Dyrk3 knockdown requires nuclear receptor coactivator 3 (NCOA3), which previously has been shown as a coactivator of activating transcription factor 4 (ATF4) to target purine pathway genes for transcriptional activation. Mechanistically, Dyrk3 directly phosphorylated NCOA3 at Ser-1330, disrupting its binding to ATF4 and thereby causing the inhibition of ATF4 transcription activity. However, phosphorylation-resistant NCOA3-S1330A mutant has the opposite effect. Interestingly, the promoter activity of Dyrk3 was negatively regulated by ATF4, indicating a double-negative feedback loop. Importantly, levels of Dyrk3 and phospho-NCOA3-S1330 inversely correlate with the expression of ATF4 in human HCC specimens. CONCLUSIONS: Our findings not only illustrate a previously unsuspected function of Dyrk3 in reprograming HCC metabolism by negatively regulating NCOA3/ATF4 transcription factor complex, but also identify NCOA3 as a novel phosphorylation substrate of Dyrk3, suggesting Dyrk3/NCOA3/ATF4 axis as a potential new candidate for HCC therapy. This article is protected by copyright. All rights reserved.
    Keywords:  ATF4; Dyrk3; Hepatocellular carcinoma; NCOA3; Purine metabolism
    DOI:  https://doi.org/10.1002/hep.30703
  9. Metabolites. 2019 May 07. pii: E91. [Epub ahead of print]9(5):
    Schlicker L, Boers HM, Dudek CA, Zhao G, Barua A, Trezzi JP, Meyer-Hermann M, Jacobs DM, Hiller K.
      Food supplementation with a fiber mix of guar gum and chickpea flour represents a promising approach to reduce the risk of type 2 diabetes mellitus (T2DM) by attenuating postprandial glycemia. To investigate the effects on postprandial metabolic fluxes of glucose-derived metabolites in response to this fiber mix, a randomized, cross-over study was designed. Twelve healthy, male subjects consumed three different flatbreads either supplemented with 2% guar gum or 4% guar gum and 15% chickpea flour or without supplementation (control). The flatbreads were enriched with ~2% of 13C-labeled wheat flour. Blood was collected at 16 intervals over a period of 360 min after bread intake and plasma samples were analyzed by GC-MS based metabolite profiling combined with stable isotope-assisted metabolomics. Although metabolite levels of the downstream metabolites of glucose, specifically lactate and alanine, were not altered in response to the fiber mix, supplementation of 4% guar gum was shown to significantly delay and reduce the exogenous formation of these metabolites. Metabolic modeling and computation of appearance rates revealed that the effects induced by the fiber mix were strongest for glucose and attenuated downstream of glucose. Further investigations to explore the potential of fiber mix supplementation to counteract the development of metabolic diseases are warranted.
    Keywords:  13C-enrichment; GC-MS; chickpea flour; guar gum; metabolism; starch; wheat flour
    DOI:  https://doi.org/10.3390/metabo9050091
  10. Semin Cell Dev Biol. 2019 May 03. pii: S1084-9521(18)30203-9. [Epub ahead of print]
    Audrito V, Managò A, Gaudino F, Deaglio S.
      Cancer cells rewire their metabolism to support proliferation, growth and survival. In metastatic melanoma the BRAF oncogenic pathway is a master regulator of this process, highlighting the importance of metabolic reprogramming in the pathogenesis of this tumor and offering potential therapeutic approaches. Metabolic adaptation of melanoma cells generally requires increased amounts of NAD+, an essential redox cofactor in cellular metabolism and a signaling molecule. Nicotinamide phosphoribosyltransferase (NAMPT) is the most important NAD+ biosynthetic enzyme in mammalian cells and a direct target of the BRAF oncogenic signaling pathway. These findings suggest that NAMPT is an attractive new therapeutic target, particularly in combination strategies with BRAF or MEK inhibitors. Here we review current knowledge on how oncogenic signaling reprograms metabolism in BRAF-mutated melanoma, and discuss how NAMPT/NAD+ axis contributes to these processes. Lastly, we present evidence supporting a role of NAMPT as a novel therapeutic target in metastatic melanoma.
    Keywords:  BRAF; Metabolic reprogramming; NAD(+)metabolism; NAMPT; combination therapy; melanoma
    DOI:  https://doi.org/10.1016/j.semcdb.2019.05.001
  11. Lipids Health Dis. 2019 May 10. 18(1): 112
    Yang F, Liu M, Qin N, Li S, Yu M, Wang C, Ma Q.
      BACKGROUND: Hyperuricemia as a metabolic disease is usually associated with lipid metabolic disorder. The purpose of this study is to identify potential lipid biomarkers and provide the evidence for the relationship between hyperuricemia and lipid-related diseases.METHODS: Lipidomics-a specialized study of lipid metabolites-has become a highly sensitive and powerful tool for biomarker discovery. In this work, an ultra-performance liquid chromatography-quadruole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS)-based on Lipidomics approach was employed to investigate serum samples from potassium oxonate-treated rats to find potential biomarkers. Principal component analysis (PCA) was used to analyze the MS data to assess the establishment of hyperuricemia model. Orthogonal partial least-squares discriminant analysis (OPLS-DA) in combination with independent samples t-test was performed for biomarker selection and identification.
    RESULTS: Thirteen potential biomarkers in rat serum were identified in the screen, and two abnormal metabolism pathways were found, namely glycerolphospholipid metabolism and glycosylphosphatidylinositol-anchored protein biosynthesis.
    CONCLUSIONS: In this work, the Lipidomics approach based on UPLC-Q-TOF/MS was employed to investigate serum metabolic changes in the rat model, 13 potential biomarkers related to hyperuricemia were identified, primarily involved in glycerolphospholipid metabolism and glycosylphosphatidylinositol-anchored protein biosynthesis. Abnormal glycerophospholipid metabolism pathway may be associated with lipid metabolism disorder caused by hyperuricemia, while the relationship between hyperuricemia and glycosylphosphatidylinositol-anchored protein biosynthesis needs further study.
    Keywords:  Hyperuricemia; Lipidomics; Potassium oxonate; UPLS-Q-TOF/MS
    DOI:  https://doi.org/10.1186/s12944-019-1054-z
  12. Anal Chem. 2019 May 10.
    Hinz C, Liggi S, Mocciaro G, Jung SM, Induruwa I, Pereira MCDA, Bryant CE, Meckelmann SW, O'Donnell VB, Farndale RW, Fjeldsted JC, Griffin JL.
      Analysis of oxylipins by liquid chromatography mass spectrometry (LC/MS) is challenging because of the small mass range occupied by this diverse lipid class, the presence of numerous structural isomers, and their low abundance in biological samples. Although highly sensitive LC/MS/MS methods are commonly used, further separation is achievable by using drift tube ion mobility coupled with high-resolution mass spectrometry (DTIM-MS). Herein, we present a combined analytical and computational method for the identification of oxylipins and fatty acids. We use a reversed-phase LC/DTIM-MS workflow able to profile and quantify the oxylipin and fatty acid content of biological samples while simultaneously acquiring full scan and product ion spectra. The information regarding accurate mass, collision-cross section values in nitrogen (DTCCSN2) and retention times of the species found are compared to an internal library of lipid standards as well as the LIPID MAPS Structure Database by using specifically developed processing tools. Features detected within the DTCCSN2 and m/z ranges of the analyzed standards are flagged as oxylipin-like species, which can be further characterized using drift time alignment of product and precursor ions distinctive of DTIM-MS. This not only helps identification by reducing the number of annotations from LIPID MAPS, but also guides discovery studies of potentially novel species. Testing the methodology on Salmonella enterica serovar Typhimurium infected murine bone-marrow derived macrophages and thrombin activated human platelets yields results in agreement with literature. This workflow has also annotated features as potentially novel oxylipins, confirming its ability in providing further insights into lipid analysis of biological samples.
    DOI:  https://doi.org/10.1021/acs.analchem.8b04615
  13. J Biol Chem. 2019 May 06. pii: jbc.RA119.008915. [Epub ahead of print]
    Kornilov A, Kennedy PD, Aldrovandi M, Watson AJA, Hinz C, Harless B, Colombo J, Maxey KM, Tyrrell VJ, Simon M, Aggarwal VK, Boeglin WE, Brash AR, Murphy RC, O'Donnell VB.
      Eicosanoids are critical mediators of fever, pain and inflammation generated by immune and tissue cells.  We recently described a new bioactive eicosanoid generated by cyclooxygenase-1 (COX-1) turnover during platelet activation that can stimulate human neutrophil integrin expression. On the basis of mass spectrometry (MS/MS and MS3), stable isotope labeling and GC/MS analysis, we previously proposed a structure of 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (DXA3). Here, we achieved enzymatic synthesis and 1H-NMR characterization of this compound with results in conflict with the previously proposed structural assignment. Accordingly, by using LC-MS, we screened autoxidation reactions of 11-HpETE and thereby identified a candidate sharing the precise reverse phase chromatographic and MS characteristics of the platelet product. We optimized these methods to increase yield, allowing full structural analysis by 1H-NMR. The revised assignment is presented here as 8,9-11,12-diepoxy-13-hydroxy-eicosadienoic acid, abbreviated to 8,9-11,12-DiEp-13-HEDE or DiEpHEDE, substituted for the previous name DXA3 We found that in platelets, the lipid likely forms via dioxolane ring opening with rearrangement to the diepoxy moieties, followed by oxygen insertion at C13.  We present its enzymatic biosynthetic pathway and MS/MS fragmentation pattern, and using the synthetic compound, demonstrate that it has bioactivity. For the platelet lipid, we estimate 16 isomers based on our current knowledge (and 4 isomers for the synthetic lipid). Determining the exact isomeric structure of the platelet lipid remains to be undertaken.
    Keywords:  8,9-11,12-diepoxy-13-hydroxy-eicosadienoic acid (8,9-11,12-DiEp-13-HEDE); 8-hydroxy-9,11- dioxolane eicosatetraenoic acid (DXA3); DiEpHEDE; Immunity; cyclooxygenase (COX); eicosanoid; leukocyte-regulating lipid; lipid; lipid metabolism; platelet
    DOI:  https://doi.org/10.1074/jbc.RA119.008915
  14. J Leukoc Biol. 2019 May 09.
    Tyurina YY, Croix CSM, Watkins SC, Watson AM, Epperly MW, Anthonymuthu TS, Kisin ER, Vlasova II, Krysko O, Krysko DV, Kapralov AA, Dar HH, Tyurin VA, Amoscato AA, Popova EN, Bolevich SB, Timashev PS, Kellum JA, Wenzel SE, Mallampalli RK, Greenberger JS, Bayir H, Shvedova AA, Kagan VE.
      In addition to the known prominent role of polyunsaturated (phospho)lipids as structural blocks of biomembranes, there is an emerging understanding of another important function of these molecules as a highly diversified signaling language utilized for intra- and extracellular communications. Technological developments in high-resolution mass spectrometry facilitated the development of a new branch of metabolomics, redox lipidomics. Analysis of lipid peroxidation reactions has already identified specific enzymatic mechanisms responsible for the biosynthesis of several unique signals in response to inflammation and regulated cell death programs. Obtaining comprehensive information about millions of signals encoded by oxidized phospholipids, represented by thousands of interactive reactions and pleiotropic (patho)physiological effects, is a daunting task. However, there is still reasonable hope that significant discoveries, of at least some of the important contributors to the overall overwhelmingly complex network of interactions triggered by inflammation, will lead to the discovery of new small molecule regulators and therapeutic modalities. For example, suppression of the production of AA-derived pro-inflammatory mediators, HXA3 and LTB4, by an iPLA2 γ inhibitor, R-BEL, mitigated injury associated with the activation of pro-inflammatory processes in animals exposed to whole-body irradiation. Further, technological developments promise to make redox lipidomics a powerful approach in the arsenal of diagnostic and therapeutic instruments for personalized medicine of inflammatory diseases and conditions.
    Keywords:  Oxidized phospholipids; eicosanoids; lipid mediators; lipoxygenase; peroxidation; phospholipase A2; phospholipid hydrolysis
    DOI:  https://doi.org/10.1002/JLB.3MIR0119-004RR
  15. Nat Metab. 2019 Mar;1 404-415
    Chen L, Zhang Z, Hoshino A, Zheng HD, Morley M, Arany Z, Rabinowitz JD.
      NADPH donates high energy electrons for antioxidant defense and reductive biosynthesis. Cytosolic NADP is recycled to NADPH by the oxidative pentose phosphate pathway (oxPPP), malic enzyme 1 (ME1) and isocitrate dehydrogenase 1 (IDH1). Here we show that any one of these routes can support cell growth, but the oxPPP is uniquely required to maintain a normal NADPH/NADP ratio, mammalian dihydrofolate reductase (DHFR) activity and folate metabolism. These findings are based on CRISPR deletions of glucose-6-phosphate dehydrogenase (G6PD, the committed oxPPP enzyme), ME1, IDH1, and combinations thereof in HCT116 colon cancer cells. Loss of G6PD results in high NADP, which induces compensatory increases in ME1 and IDH1 flux. But the high NADP inhibits dihydrofolate reductase (DHFR), resulting in impaired folate-mediated biosynthesis, which is reversed by recombinant expression of E. coli DHFR. Across different cancer cell lines, G6PD deletion produced consistent changes in folate-related metabolites, suggesting a general requirement for the oxPPP to support folate metabolism.
  16. J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Apr 24. pii: S1570-0232(19)30369-1. [Epub ahead of print]1118-1119 157-163
    Kim SY, Kim BK, Gwon MR, Seong SJ, Ohk B, Kang WY, Lee HW, Jung HY, Cho JH, Chung BH, Lee SH, Kim YH, Yoon YR, Kim CD, Cho S.
      To improve early renal allograft function, it is important to develop a noninvasive diagnostic method for acute T cell-mediated rejection (TCMR). This study aims to explore potential noninvasive urinary biomarkers to screen for acute TCMR in kidney transplant recipients (KTRs) using untargeted metabolomic profiling. Urinary metabolites, collected from KTRs with stable graft function (STA) or acute TCMR episodes, were analyzed using liquid chromatography-mass spectrometry (LC-MS). Multivariate statistical analyses were performed to discriminate differences in urinary metabolites between the two groups. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic performance of potential urinary biomarkers. Statistical analysis revealed the differences in urinary metabolites between the two groups and indicated several statistically significant metabolic features suitable for potential biomarkers. By comparing the retention times and mass fragmentation patterns of the chemicals in metabolite databases, samples, and standards, six of these features were clearly identified. ROC curve analysis showed the best performance of the training set (area under the curve value, 0.926; sensitivity, 90.0%; specificity, 84.6%) using a panel of five potential biomarkers: guanidoacetic acid, methylimidazoleacetic acid, dopamine, 4-guanidinobutyric acid, and L-tryptophan. The diagnostic accuracy of this model was 62.5% for an independent test dataset. LC-MS-based untargeted metabolomic profiling is a promising method to discriminate between acute TCMR and STA groups. Our model, based on a panel of five potential biomarkers, needs to be further validated in larger scale studies.
    Keywords:  Acute T cell-mediated rejection; Kidney transplantation; Liquid chromatography-tandem mass spectrometry; Metabolomic profiling; Metabolomics; Urine
    DOI:  https://doi.org/10.1016/j.jchromb.2019.04.047
  17. BMC Bioinformatics. 2019 May 01. 20(Suppl 7): 195
    Hao Y, Li D, Xu Y, Ouyang J, Wang Y, Zhang Y, Li B, Xie L, Qin G.
      BACKGROUND: Lipid metabolism reprogramming is a hallmark for tumor which contributes to tumorigenesis and progression, but the commonality and difference of lipid metabolism among pan-cancer is not fully investigated. Increasing evidences suggest that the alterations in tumor metabolism, including metabolite abundance and accumulation of metabolic products, lead to local immunosuppression in the tumor microenvironment. An integrated analysis of lipid metabolism in cancers from different tissues using multiple omics data may provide novel insight into the understanding of tumorigenesis and progression.RESULTS: Through systematic analysis of the multiple omics data from TCGA, we found that the most-widely altered lipid metabolism pathways in pan-cancer are fatty acid metabolism, arachidonic acid metabolism, cholesterol metabolism and PPAR signaling. Gene expression profiles of fatty acid metabolism show commonalities across pan-cancer, while the alteration in cholesterol metabolism and arachidonic acid metabolism differ with tissue origin, suggesting tissue specific lipid metabolism features in different tumor types. An integrated analysis of gene expression, DNA methylation and mutations revealed factors that regulate gene expression, including the differentially methylated sites and mutations of the lipid genes, as well as mutation and differential expression of the up-stream transcription factors for the lipid metabolism pathways. Correlation analysis of the proportion of immune cells in the tumor microenvironment and the expression of lipid metabolism genes revealed immune-related differentially expressed lipid metabolic genes, indicating the potential crosstalk between lipid metabolism and immune response. Genes related to lipid metabolism and immune response that are associated with poor prognosis were discovered including HMGCS2, GPX2 and CD36, which may provide clues for tumor biomarkers or therapeutic targets.
    CONCLUSIONS: Our study provides an integrated analysis of lipid metabolism in pan-cancer, highlights the perturbation of key metabolism processes in tumorigenesis and clarificates the regulation mechanism of abnormal lipid metabolism and effects of lipid metabolism on tumor immune microenvironment. This study also provides new clues for biomarkers or therapeutic targets of lipid metabolism in tumors.
    Keywords:  Lipid metabolism; Multiple omics analysis; Pan-cancer; Tumor immune micro-environment
    DOI:  https://doi.org/10.1186/s12859-019-2734-4
  18. Methods Mol Biol. 2019 ;1985 57-79
    Toyo'oka T.
      The determination of enantiomers of biological molecules is an important issue because a significant difference in the activity of the enantiomers is generally observed in biological systems. Chiral separations can be carried out by direct resolution using a chiral stationary column or by indirect resolution based on the derivatization with a chiral reagent. Many chiral-labeling reagents for ultraviolet-visible and fluorescence detections have been developed for various functional groups, such as amine and carboxylic acid. However, there are hardly any labeling reagents for LC-MS-specific detection. Based on this observation, we have developed several chiral-labeling reagents for LC-MS/MS analysis.This chapter describes methodologies and applications for the indirect LC-MS/MS determination of biological chiral molecules using triazine-based chiral-labeling reagents, i.e., (S and R)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3(S and R)-Apy) for carboxylic acids and (S and R)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate (DMT-(S and R)-Pro-OSu) for amines and amino acids. A reliable method for the non-targeted chiral metabolomics is also described in this chapter.
    Keywords:  Chiral-labeling reagent; Enantioseparation; Indirect resolution; Mass spectrometry; Triazine-based reagent; UHPLC separation
    DOI:  https://doi.org/10.1007/978-1-4939-9438-0_4
  19. J Am Soc Mass Spectrom. 2019 May 09.
    McCool EN, Lodge JM, Basharat AR, Liu X, Coon JJ, Sun L.
      Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as an efficient approach for top-down proteomics recently for its high-capacity separation and highly sensitive detection of proteoforms. However, the commonly used collision-based dissociation methods often cannot provide extensive fragmentation of proteoforms for thorough characterization. Activated ion electron transfer dissociation (AI-ETD), that combines infrared photoactivation concurrent with ETD, has shown better performance for proteoform fragmentation than higher energy-collisional dissociation (HCD) and standard ETD. Here, we present the first application of CZE-AI-ETD on an Orbitrap Fusion Lumos mass spectrometer for large-scale top-down proteomics of Escherichia coli (E. coli) cells. CZE-AI-ETD outperformed CZE-ETD regarding proteoform and protein identifications (IDs). CZE-AI-ETD reached comparable proteoform and protein IDs with CZE-HCD. CZE-AI-ETD tended to generate better expectation values (E values) of proteoforms than CZE-HCD and CZE-ETD, indicating a higher quality of MS/MS spectra from AI-ETD respecting the number of sequence-informative fragment ions generated. CZE-AI-ETD showed great reproducibility regarding the proteoform and protein IDs with relative standard deviations less than 4% and 2% (n = 3). Coupling size exclusion chromatography (SEC) to CZE-AI-ETD identified 3028 proteoforms and 387 proteins from E. coli cells with 1% spectrum level and 5% proteoform-level false discovery rates. The data represents the largest top-down proteomics dataset using the AI-ETD method so far. Single-shot CZE-AI-ETD of one SEC fraction identified 957 proteoforms and 253 proteins. N-terminal truncations, signal peptide cleavage, N-terminal methionine removal, and various post-translational modifications including protein N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation were detected.
    Keywords:  Activated ion electron transfer dissociation; Capillary zone electrophoresis-tandem mass spectrometry; Disulfide bonds; Escherichia coli; Lysine succinylation; S-thiolation; Top-down proteomics
    DOI:  https://doi.org/10.1007/s13361-019-02206-6
  20. Metabolites. 2019 May 07. pii: E90. [Epub ahead of print]9(5):
    Considine EC, Khashan AS, Kenny LC.
      The aim of this preliminary study was to investigate the potential of maternal serum to provide metabolomic biomarker candidates for the prediction of spontaneous preterm birth (SPTB) in asymptomatic pregnant women at 15 and/or 20 weeks' gestation. Metabolomics LC-MS datasets from serum samples at 15- and 20-weeks' gestation from a cohort of approximately 50 cases (GA < 37 weeks) and 55 controls (GA > 41weeks) were analysed for candidate biomarkers predictive of SPTB. Lists of the top ranked candidate biomarkers from both multivariate and univariate analyses were produced. At the 20 weeks' GA time-point these lists had high concordance with each other (85%). A subset of 4 of these features produce a biomarker panel that predicts SPTB with a partial Area Under the Curve (pAUC) of 12.2, a sensitivity of 87.8%, a specificity of 57.7% and a p-value of 0.0013 upon 10-fold cross validation using PanelomiX software. This biomarker panel contained mostly features from groups already associated in the literature with preterm birth and consisted of 4 features from the biological groups of "Bile Acids", "Prostaglandins", "Vitamin D and derivatives" and "Fatty Acids and Conjugates".
    Keywords:  biomarkers; complex disease; heterogeneity; metabolomics; panel; prediction; preterm birth
    DOI:  https://doi.org/10.3390/metabo9050090
  21. Clin Chim Acta. 2019 May 03. pii: S0009-8981(19)31843-1. [Epub ahead of print]
    Yang L, Li Z, Song Y, Liu Y, Zhao H, Liu Y, Zhang T, Yuan Y, Cai X, Wang S, Wang P, Gao S, Li L, Li Y, Yu C.
      BACKGROUND: As a recognized risk factor for cardiovascular disease (CVD), hyperlipidemia (HLP) has developed into a high incidence disease that seriously threatens human health. Finding a new target for effective treatment of HLP will be a powerful way to reduce the incidence of CVD. The purpose of this study was to find potential biomarkers in urine of HLP patients and analyze their metabolic pathways to study the pathogenesis of HLP.METHODS: An UPLC-Q-TOF/MS technology was used to detect the metabolites in urine of 60 HLP patients and 60 normal controls. Based on PLS-DA pattern recognition, potential biomarkers related to HLP were screened out.
    RESULTS: 22 potential biomarkers related to HLP were identified, which involved amino acid metabolism, fatty acid metabolism, nucleotide metabolism, steroid hormone metabolism and intestinal flora metabolism, and their possible pathogenesis was found to be related to inflammatory reaction and oxidative stress.
    CONCLUSION: The non-targeted metabolomic method based on UPLC-Q-TOF/MS technology can effectively identify potential biomarkers in the urine of HLP patients and explore the possible pathogenesis. Our research will lay a foundation for finding new targets for the treatment of HLP and provide a basis for clinical research on the treatment of HLP.
    Keywords:  Biomarkers; Hyperlipidemia; Metabolomics; Pathogenesis; UPLC-Q-TOF/MS
    DOI:  https://doi.org/10.1016/j.cca.2019.05.001
  22. Mol Omics. 2019 May 09.
    Zheng H, Wu J, Huang H, Meng C, Li W, Wei T, Su Z.
      Diabetes is one of the most severe chronic diseases worldwide. It is widely accepted that apoptosis of the pancreatic beta cell is an important cause for the induction of hyperglycemia and high levels of free fatty acids (FFAs), also called lipotoxicity associated with pancreatic beta cell dysfunction. Lipotoxicity has been proven to be an important pathogenic factor of diabetes. However, until now, the mechanism of FFA-induced lipotoxicity in INS-1 cells has not been fully understood. Current anti-diabetic drugs that protect islet cells are often toxic to healthy cells, resulting in negative side effects. Thus, there is an urgent need to identify more effective and safer anti-diabetic agents to protect pancreatic islet cells. Rubusoside (RUB) is a major ingredient in the leaves of Rubus suavissimus S. Lee, which decreases blood glucose levels by protecting pancreatic islet cells. However, the exact mechanism of this effect is unknown. In this study, metabolomics experiments based on UPLC-Q/TOF MS characterized a total of 15 metabolites as potential biomarkers associated with lipotoxicity induced by palmitic acid in INS-1 cells. According to the metabolic pathway analysis, pentose and glucuronate interconversions, and glycerophospholipid metabolism were recognized as the most influenced metabolic pathways associated with lipotoxicity. Unexpectedly, deviations of 14 metabolites in lipotoxic INS-1 cells were regulated by RUB, suggesting synergistic mediation of the abnormal metabolic pathways. The metabolomics strategy based on UPLC/Q-TOF MS analysis provides a new insight into the mechanisms of lipotoxicity induced by palmitic acid and the anti-lipotoxic activity of RUB in INS-1 cells.
    DOI:  https://doi.org/10.1039/c9mo00029a
  23. Sci Transl Med. 2019 May 08. pii: eaau1167. [Epub ahead of print]11(491):
    Zhang L, Yao Y, Zhang S, Liu Y, Guo H, Ahmed M, Bell T, Zhang H, Han G, Lorence E, Badillo M, Zhou S, Sun Y, Di Francesco ME, Feng N, Haun R, Lan R, Mackintosh SG, Mao X, Song X, Zhang J, Pham LV, Lorenzi PL, Marszalek J, Heffernan T, Draetta G, Jones P, Futreal A, Nomie K, Wang L, Wang M.
      Metabolic reprogramming is linked to cancer cell growth and proliferation, metastasis, and therapeutic resistance in a multitude of cancers. Targeting dysregulated metabolic pathways to overcome resistance, an urgent clinical need in all relapsed/refractory cancers, remains difficult. Through genomic analyses of clinical specimens, we show that metabolic reprogramming toward oxidative phosphorylation (OXPHOS) and glutaminolysis is associated with therapeutic resistance to the Bruton's tyrosine kinase inhibitor ibrutinib in mantle cell lymphoma (MCL), a B cell lymphoma subtype with poor clinical outcomes. Inhibition of OXPHOS with a clinically applicable small molecule, IACS-010759, which targets complex I of the mitochondrial electron transport chain, results in marked growth inhibition in vitro and in vivo in ibrutinib-resistant patient-derived cancer models. This work suggests that targeting metabolic pathways to subvert therapeutic resistance is a clinically viable approach to treat highly refractory malignancies.
    DOI:  https://doi.org/10.1126/scitranslmed.aau1167
  24. Proteomics. 2019 May 09. e1800394
    Han S, Huo Z, Nguyen K, Zhu F, Underwood PW, Basso KBG, George TJ, Hughes SJ.
      Exosomes are membrane-bound vesicles that traffic small molecular cargos. These cargos participate in cell-cell communication and contribute to the pathogenesis of many disease states, including cancer. How these mechanisms contribute to communication within the pancreatic adenocarcinoma (PDAC) microenvironment and how they contribute to PDAC biology are poorly understood. In this study, we performed comprehensive, quantitative comparisons of the proteomes of three PDAC cell lines to those of the exosomes they produce. Approximately 35% of whole cell proteins sort into exosomes. ANalysis of Composition Of Microbiomes (ANCOM) analysis determined a cluster of 98 enriched Pancreatic Cancer Exosome Proteins (ePC-ECPs). Further, these proteins are predicted by Ingenuity Pathway Analysis (IPA) as actively involved in signaling pathways regulating cell death and survival, cellular movement, and cell-to-cell signaling and interaction in particular (the top three p-value significant pathways). Significant enrichment of canonical pathways of Acute Phase Response Signaling (inflammatory response signaling pathways) and FXR and RXR activation in metabolic pathways are also predicted. Ninety-seven ePC-ECPs are associated with cancer and among them, 34 of those proteins are specifically associated with PDAC. In conclusion, exosomes from PDAC are enriched with cancer-associated signaling proteins. Further assessment of these proteins as PDAC biomarkers or therapeutic targets is warranted. This article is protected by copyright. All rights reserved.
    Keywords:  ANCOM; exosomes; pancreatic adenocarcinoma; proteome; signaling pathway
    DOI:  https://doi.org/10.1002/pmic.201800394
  25. Methods Mol Biol. 2019 ;1967 21-43
    Metcalfe C.
      Reduction of labile disulphide bonds on leukocyte cell surface proteins plays a regulatory role in immune cell activation. Here I describe a method for the fast, efficient, and unbiased purification of cell-surface proteins containing such labile disulphide bonds. Free thiols liberated from the reduction of labile disulphide bonds are labeled with biotin, purified, enriched, and subsequently identified using liquid chromatography coupled to tandem mass spectrometry. Both the proteins containing the labile disulphide bonds and the position of bonds within the protein are revealed, thus providing a valuable addition to the immunology or biochemistry toolkit.
    Keywords:  Differential labeling; Functional disulphide bonds; Immune regulation; Proteomics; Redox labile
    DOI:  https://doi.org/10.1007/978-1-4939-9187-7_3
  26. Mol Cancer Res. 2019 May 08. pii: molcanres.0020.2019. [Epub ahead of print]
    Gonthier K, Poluri RTK, Weidmann C, Tadros M, Audet-Walsh E.
      Mutations of the isocitrate dehydrogenase genes IDH1 and IDH2, key enzymes involved in citrate metabolism, are important oncogenic events in several cancer types, including in 1-3% of all prostate cancer (PCa) cases. However, if IDH1 and other IDH isoforms are associated with PCa progression, as well as the regulatory factors controlling their expression and activity, remain mostly unknown. Using publicly available datasets, we showed that PCa harbors the highest IDH1 expression across the human cancer spectrum and that IDH1 expression is altered during PCa progression. We showed that the androgen receptor (AR), a key oncogene in PCa, controls multiple IDH isoforms in both in vitro and in vivo models, predominantly positively regulating IDH1. Chromatin immunoprecipitation experiments confirmed the recruitment of AR at several regulatory regions of IDH1 and enzymatic assays demonstrated that AR significantly induces IDH activity. Genetic blockade of IDH1 significantly impaired PCa cell proliferation, consistent with IDH1 having a key function in these cancer cells. Importantly, knockdown of IDH1 blocked the AR-mediated induction in IDH activity, indicating that AR promotes a mitochondrial to cytoplasmic reprogramming of IDH activity. Overall, our study demonstrates that IDH1 expression is associated with PCa progression, that AR signaling integrates one of the first transcriptional mechanisms shown to regulate IDH1, and that AR reprograms PCa cell metabolism by selectively inducing extra-mitochondrial IDH activity. Implications: The discovery that AR reprograms IDH activity highlights a novel metabolic reprogramming necessary for PCa growth and suggests targeting IDH activity as a new therapeutic approach for PCa treatment.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-19-0020
  27. Methods Mol Biol. 2019 ;1985 391-405
    Castro-Puyana M, Marina ML.
      Capillary electrophoresis (CE) is a well-established and one of the most powerful separation techniques in the field of chiral separations. Its hyphenation with mass spectrometry (MS) combines both the high separation efficiency and low sample consumption of CE and the high sensitivity and structural information of MS. Thus, the outstanding chiral resolution power of CE along with the MS advantages makes CE-MS a perfect combination to achieve sensitive enantioseparations. This chapter describes three representative examples of different approaches used in the chiral analysis of amino acids in biological fluids by CE-MS. The first methodology uses the partial filling technique to avoid the entry of cyclodextrins in the MS source. The second method shows the possibility to carry out the direct coupling EKC-MS even when a relative high concentration of a native cyclodextrin is used as chiral selector. The last example illustrates an alternative strategy based on the formation of stable diastereomers between an enantiomerically pure chiral reagent and the amino acids enantiomers which can be separated in an achiral environment.
    Keywords:  Capillary electrophoresis; Chiral; Cyclodextrins; Diastereomeric separation; Enantiomeric separation; Mass spectrometry; Partial filling technique
    DOI:  https://doi.org/10.1007/978-1-4939-9438-0_23
  28. Nat Struct Mol Biol. 2019 May;26(5): 361-371
    Metzger E, Wang S, Urban S, Willmann D, Schmidt A, Offermann A, Allen A, Sum M, Obier N, Cottard F, Ulferts S, Preca BT, Hermann B, Maurer J, Greschik H, Hornung V, Einsle O, Perner S, Imhof A, Jung M, Schüle R.
      Histone lysine methylation is generally performed by SET domain methyltransferases and regulates chromatin structure and gene expression. Here, we identify human C21orf127 (HEMK2, N6AMT1, PrmC), a member of the seven-β-strand family of putative methyltransferases, as a novel histone lysine methyltransferase. C21orf127 functions as an obligate heterodimer with TRMT112, writing the methylation mark on lysine 12 of histone H4 (H4K12) in vitro and in vivo. We characterized H4K12 recognition by solving the crystal structure of human C21orf127-TRMT112, hereafter termed 'lysine methyltransferase 9' (KMT9), in complex with S-adenosyl-homocysteine and H4K12me1 peptide. Additional analyses revealed enrichment for KMT9 and H4K12me1 at the promoters of numerous genes encoding cell cycle regulators and control of cell cycle progression by KMT9. Importantly, KMT9 depletion severely affects the proliferation of androgen receptor-dependent, as well as that of castration- and enzalutamide-resistant prostate cancer cells and xenograft tumors. Our data link H4K12 methylation with KMT9-dependent regulation of androgen-independent prostate tumor cell proliferation, thereby providing a promising paradigm for the treatment of castration-resistant prostate cancer.
    DOI:  https://doi.org/10.1038/s41594-019-0219-9
  29. Expert Rev Proteomics. 2019 May 04.
    Bardanzellu F, Fanos V.
      INTRODUCTION: In recent years, several studies have highlighted the promising role of metabolomics in the analysis of amniotic fluid (AF), to describe and characterize the interactions occurring between the mother and the fetus during prenatal development. Among the available biological fluids, AF represents an ideal substrate to provide dynamic information regarding fetal organogenesis and metabolism through pregnancy, since it originates from both maternal and fetal tissues and contains substances derived from placenta, fetal skin, lungs, gastric fluid and fetal urine. Areas covered: In this paper, we provide an update reporting the most recent results on AF metabolomics in the assessment of feto-maternal health, regarding physiological pregnancies but even fields such as prematurity, bronchopulmonary dysplasia, fetal malformations, chromosomopathies, maternal diseases, placental inflammation or infections, maternal diet or exposure to exogenous substances, according to the literature found on MEDLINE since 2015. Expert opinion: Metabolomics shows a promising role in describing both physiology and disease; the goal would be the identification of biomarkers able to precociously and efficaciously detect pathological conditions, allowing the identification of complicated pregnancy and improving their management. However, this field is under development and its reliability still needs to be clarified, especially through more numerous and accurate studies.
    Keywords:  chorioamnionitis; fetal malformations; gas chromatography; gestational age; gestational diabetes mellitus; lipidomics; liquid chromatography; mass spectrometry; pregnancy; prematurity
    DOI:  https://doi.org/10.1080/14789450.2019.1615892
  30. J Pharm Sci. 2019 May 02. pii: S0022-3549(19)30297-7. [Epub ahead of print]
    Cheng Y, Hu M, Zamiri C, Carcelen T, Demeule B, Tomlinson A, Gu J, Yigzaw Y, Kalo M, Yu XC.
      Polysorbate 20 (PS20), a widely used surfactant in protein therapeutics, has been reported to undergo hydrolytic degradation during product storage, causing the release of free fatty acids. The accumulation of free fatty acids in protein therapeutics was found to result in the formation of particles due to their limited aqueous solubility at 2-8°C. Quantitation of free fatty acids originating from PS20 degradation is thus important during bio-process optimization and stability testing in formulation development to ensure optimum PS20 stability as well as product and process consistency in final drug products. This work reports the development of a simple and robust, high-throughput, reversed-phase UHPLC-MS method for high sensitivity quantitation of lauric acid and myristic acid by using isotope labeled fatty acid internal standards. The high sensitivity (< 100 ng/mL for lauric acid) and suitable precision (intermediate precision RSD of 11%) of this method enables accurate detection of lauric acid produced from the degradation of less than 1% of PS20 in a 0.2 mg/mL formulation. Using accelerated thermal stability testing, this method identifies processes that exhibit fast PS20 degradation within only days, and consequently allows faster iterative optimization of the process.
    Keywords:  LPLA2; Polysorbate 20; enzymatic degradation; free fatty acids; hydrolytic degradation; lauric acid; myristic acid; reversed-phase liquid chromatography mass spectrometer (RPLC-MS)
    DOI:  https://doi.org/10.1016/j.xphs.2019.04.029
  31. Lipids Health Dis. 2019 May 10. 18(1): 108
    Sun Y, Zhang N, Ding YL, Yu LJ, Cai J, Ma , Yang W, Lu WK, Niu JL.
      BACKGROUND: This study aims to investigate the effect of lipid metabolism disorder on liver function in patients with malignant tumors after chemotherapy.METHOD: A total of 428 patients with malignant tumors with normal liver function in our hospital between May 2013 to June 2018 were divided into an observation group (lipid metabolism disorder, n = 265) and control group (normal lipid metabolism, n = 163). The lipid metabolism levels and liver damage of the two groups were compared before and after chemotherapy.
    RESULTS: No significant differences in age, gender, body mass index, tumor types, history of surgery, levels of alanine aminotransferase (ALT; an indicator of liver function), and chemotherapy regimen were observed between the two groups. However, the observation group showed increased levels of total cholesterol (P = 0.000), triglycerides (P = 0.000), and low-density lipoprotein (P = 0.01), as well as decreased levels of high-density lipoprotein (P = 0.000) before chemotherapy compared with the control group. Furthermore, patients with lipid metabolism disorders were more likely to develop abnormal liver function after chemotherapy. Moreover, mixed lipid metabolism disorder was more likely to cause severe liver damage after chemotherapy. Additionally, the number of patients with lipid metabolism disorders after chemotherapy (n = 367) was significantly increased compared with before chemotherapy (n = 265) (P < 0.01), indicating that chemotherapy might induce or aggravate an abnormal lipid metabolism.
    CONCLUSIONS: After receiving chemotherapy, patients with malignant tumors presenting lipid metabolism disorders are more prone to liver damage and lipid metabolism disorders than patients with a normal lipid metabolism.
    Keywords:  Chemotherapy; Lipid metabolism; Liver function; Tumor
    DOI:  https://doi.org/10.1186/s12944-019-1063-y
  32. Methods Mol Biol. 2019 ;1985 453-478
    Klein-Júnior LC, Mangelings D, Vander Heyden Y.
      In this chapter, the application of design of experiments (DoE) for chiral separation optimization using supercritical fluid chromatography (SFC), liquid chromatography (LC), capillary electrophoresis (CE), and capillary electrochromatography (CEC) methods is reviewed. Both screening and optimization steps are covered, including a discussion of each aspect, such as factor-, level-, and response selection. Different designs are also presented, highlighting their applications.
    Keywords:  Capillary electrochromatography; Capillary electrophoresis; Chiral separation; Design of experiments; Liquid chromatography; Method development; Supercritical fluid chromatography
    DOI:  https://doi.org/10.1007/978-1-4939-9438-0_27
  33. J Agric Food Chem. 2019 May 09.
    Dhar Dubey KK, Sharma G, Kumar A.
      Conjugated fatty acids (CFAs) including both conjugated linoleic acids (CLAs) and conjugated linolenic acids (CLNAs) have various health promoting effects. These beneficial effects are comprised by their anti-oxidant, anti-atherogenecity, anti-carcinogenic activities, etc. Several reports indicate that CLNAs such as eleostearic acid, punicic acid, jacaric acid and calendic acid possess anti-cancer properties. These CLNAs are produced and accumulated in seeds of certain commonly available plants. This review discusses their role in chemoprevention of cancer. Using in vitro as well as in vivo models of cancer, bioactivities of these CLNAs have been explored in detail. CLNAs have been shown to have potent anti-cancer activity as compared to the CLAs. Although the molecular basis of these effects has been summarized here, more detailed studies are needed to explore the underlying mechanisms. Further clinical trials are obligatory for assessing the safety and efficacy of CLNAs as an anti-cancer agent.
    DOI:  https://doi.org/10.1021/acs.jafc.9b01379
  34. J Proteome Res. 2019 May 10.
    Aggarwal S, Talukdar NC, Yadav AK.
      Proteomics by mass spectrometry (MS) allows the large-scale identification and quantitation of the cellular proteins in a given biological context. Systems biology studies from proteomics data are largely limited by accuracy and coverage of quantitative proteomics along with missing values. Towards this end, statistically robust biological observations are required, comprising multiple replicates, preferably with little technical variations. Multiplexed labeling techniques in proteomics allow quantitative comparisons of several biological samples or conditions. In this focused review, we discuss an emerging technique called higher order multiplexing or enhanced multiplexing, a unique combination of traditional MS1 and MS2 based quantitative proteomics methods that allows for expanding the multiplexing capability of MS methods to save valuable instrument time, achieve statistical robustness, enhancing coverage, quantitation accuracy and reduce the run-to-run variability. We discuss the various innovative studies and experimental designs that exploit the power of this technique and its variants, in order to provide an overview of a rapidly growing area and also to highlight the advantages and challenges that lie ahead in the widespread adoption of this technique.
    DOI:  https://doi.org/10.1021/acs.jproteome.9b00228
  35. Oncol Rep. 2019 May 02.
    Ando N, Hara M, Shiga K, Yanagita T, Takasu K, Nakai N, Maeda Y, Hirokawa T, Takahashi H, Ishiguro H, Matsuo Y, Takiguchi S.
      Eicosapentaenoic acid (EPA) improves interleukin (IL)‑6 hypercytokinemia in patients with advanced cancer due to its anti‑inflammatory effects. This EPA mechanism has been revealed to lead to several anticancer effects. While the effects of EPA on cancer cells have been investigated, particularly in terms of angiogenesis, its effects on the tumor stroma remain unclear. In the present study, the authors clarified the role of EPA in cancer angiogenesis against colon cancer‑associated fibroblasts (CAFs) from the colon stroma. With established human CAFs and normal fibroblasts from colon stroma (NFs), the authors evaluated IL‑6 and vascular endothelial growth factor (VEGF) secretion with or without EPA treatment using ELISA. The signal inhibition of mitogen‑activated protein kinase (ERK) in CAFs by EPA was evaluated using western blotting. In vitro anti‑angiogenesis effects were evaluated by the angiogenesis assay on Matrigel using human umbilical vein endothelial cells (HUVECs) cultured with the supernatant obtained from CAF cultures with or without EPA. IL‑6 secretion was greater from CAFs compared with that from NFs and stimulation with lipopolysaccharide (LPS) resulted in greater IL‑6 secretion from the two fibroblast types compared with that from fibroblasts without LPS stimulation. While LPS stimulation increased VEGF secretion from the two fibroblast types, EPA decreased IL‑6 and VEGF secretion from CAFs. Western blotting revealed that the addition of 30 µM EPA inhibited the ERK phosphorylation signal in CAFs. Furthermore, the angiogenesis assay with Matrigel revealed that the CAF culture supernatants treated with EPA suppressed tubular formation in HUVECs. These reductions may have been caused by the inhibition of ERK phosphorylation by EPA. Thus, EPA reduces cancer angiogenesis associated with CAFs. Additional studies will be needed to clarify the continuous anti‑angiogenetic effect of chemotherapy using angiogenesis inhibitors (e.g. bevacizumab and aflibercept) in conjunction with or without EPA, and the clinical usage of EPA in conjunction with chemotherapy in vivo.
    DOI:  https://doi.org/10.3892/or.2019.7141
  36. Amino Acids. 2019 May 09.
    Oppermann H, Heinrich M, Birkemeyer C, Meixensberger J, Gaunitz F.
      The previous studies demonstrated that carnosine (β-alanyl-L-histidine) inhibits the growth of tumor cells in vitro and in vivo. Considering carnosine for the treatment of glioblastoma, we investigated which proton-coupled oligopeptide transporters (POTs) are present in glioblastoma cells and how they contribute to the uptake of carnosine. Therefore, mRNA expression of the four known POTs (PEPT1, PEPT2, PHT1, and PHT2) was examined in three glioblastoma cell lines, ten primary tumor cell cultures, in freshly isolated tumor tissue and in healthy brain. Using high-performance liquid chromatography coupled to mass spectrometry, the uptake of carnosine was investigated in the presence of competitive inhibitors and after siRNA-mediated knockdown of POTs. Whereas PEPT1 mRNA was not detected in any sample, expression of the three other transporters was significantly increased in tumor tissue compared to healthy brain. In cell culture, PHT1 expression was comparable to expression in tumor tissue, PHT2 exhibited a slightly reduced expression, and PEPT2 expression was reduced to normal brain tissue levels. In the cell line LN405, the competitive inhibitors β-alanyl-L-alanine (inhibits all transporters) and L-histidine (inhibitor of PHT1/2) both inhibited the uptake of carnosine. SiRNA-mediated knockdown of PHT1 and PHT2 revealed a significantly reduced uptake of carnosine. Interestingly, despite its low expression at the level of mRNA, knockdown of PEPT2 also resulted in decreased uptake. In conclusion, our results demonstrate that the transporters PEPT2, PHT1, and PHT2 are responsible for the uptake of carnosine into glioblastoma cells and full function of all three transporters is required for maximum uptake.
    Keywords:  Carnosine; Glioblastoma; Peptide Transport; siRNA
    DOI:  https://doi.org/10.1007/s00726-019-02739-w
  37. Mol Cell Proteomics. 2019 May 06. pii: mcp.RA119.001496. [Epub ahead of print]
    Nguyen EV, Pereira BA, Lawrence MG, Ma X, Rebello RJ, Chan H, Niranjan B, Wu Y, Ellem S, Guan X, Wu J, Skhinas JN, Cox TR, Risbridger GP, Taylor RA, Lister NL, Daly R.
      In prostate cancer, cancer-associated fibroblasts (CAF) exhibit contrasting biological properties to non-malignant prostate fibroblasts (NPF) and promote tumorigenesis. Resolving intercellular signaling pathways between CAF and prostate tumor epithelium may offer novel opportunities for research translation. To this end, the proteome and phosphoproteome of four pairs of patient-matched CAF and NPF were characterized to identify discriminating proteomic signatures. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a hyper reaction monitoring data-independent acquisition (HRM-DIA) workflow. Proteins that exhibited a significant increase in CAF versus NPF were enriched for the functional categories 'cell adhesion' and the 'extracellular matrix'. The CAF phosphoproteome exhibited enhanced phosphorylation of proteins associated with the 'spliceosome' and 'actin binding'. STRING analysis of the CAF proteome revealed a prominent interaction hub associated with collagen synthesis, modification, and signaling. It contained multiple collagens, including the fibrillar types COL1A1/2 and COL5A1; the receptor tyrosine kinase discoidin domaincontaining receptor 2 (DDR2), a receptor for fibrillar collagens; and lysyl oxidase-like 2 (LOXL2), an enzyme which promotes collagen crosslinking. Increased activity and/or expression of LOXL2 and DDR2 in CAF were confirmed by enzymatic assays and Western blot analyses. Pharmacological inhibition of CAF-derived LOXL2 perturbed extracellular matrix (ECM) organization and decreased CAF migration in a wound healing assay. Furthermore, it significantly impaired the motility of co-cultured RWPE-2 prostate tumor epithelial cells. These results indicate that CAF-derived LOXL2 is an important mediator of intercellular communication within the prostate tumor microenvironment and is a potential therapeutic target.
    Keywords:  Cancer biomarker(s); Cancer-associated fibroblasts; Fibroblasts; LOXL2; Non-malignant prostate fibroblasts; Phosphoproteome; Prostate cancer; Prostate cancer biomarkers; Tumor microenvironment*
    DOI:  https://doi.org/10.1074/mcp.RA119.001496
  38. Anal Chem. 2019 May 10.
    Zhang W, Ren Y, Lin Z, Ouyang Z.
      The transition of Mass spectrometry (MS) for clinical analysis is highly desirable and a major progress has been made with direct sampling ionization for operation simplification. High-precision quantitation, however, remains as a major challenge in this transition. Herein, a novel method was developed for direct quantitation of biofluid samples, using extremely simplified procedure for incorporation of internal standards selected against the traditional rules. Slug flow microextraction was used for the development, with conditions predicted by a theoretical model, viz. using internal standards of partition coefficients very different from the analytes and large sample-to-extraction solvent volume ratios. Direct quantitation of drug compounds in urine and blood samples was demonstrated. This development enabled an extremely simplified protocol that is expected to have a significant impact on on-site or clinical analysis.
    DOI:  https://doi.org/10.1021/acs.analchem.9b01694
  39. Dig Dis Sci. 2019 May 10.
    Gao B, Lang S, Duan Y, Wang Y, Shawcross DL, Louvet A, Mathurin P, Ho SB, Stärkel P, Schnabl B.
      BACKGROUND: Alcohol-related liver disease is one of the most prevalent chronic liver diseases worldwide. Mechanisms involved in the pathogenesis of alcohol-related liver disease are not well understood. Oxylipins play a crucial role in numerous biological processes and pathological conditions. Nevertheless, oxylipins are not well studied in alcohol-related liver disease.AIMS: (1) To characterize the patterns of bioactive ω-3 and ω-6 polyunsaturated fatty acid metabolites in alcohol use disorder and alcoholic hepatitis patients and (2) to identify associations of serum oxylipins with clinical parameters in patients with alcohol-related liver disease.
    METHODS: We performed a comprehensive liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis of serum and fecal oxylipins derived from ω-6 arachidonic acid, ω-3 eicosapentaenoic acid, and docosahexaenoic acid in a patient cohort with alcohol-related liver disease.
    RESULTS: Our results show profound alterations in the serum oxylipin profile of patients with alcohol use disorder and alcoholic hepatitis compared to nonalcoholic controls. Spearman correlation of the oxylipins with clinical parameters shows a link between different serum oxylipins and intestinal permeability, aspartate aminotransferase, bilirubin, albumin, international normalized ratio, platelet count, steatosis, fibrosis and model for end-stage liver disease score. Especially, higher level of serum 20-HETE was significantly associated with decreased albumin, increased hepatic steatosis, polymorphonuclear infiltration, and 90-day mortality.
    CONCLUSIONS: Patients with alcohol-related liver disease have different oxylipin profiles. Future studies are required to confirm oxylipins as disease biomarker or to connect oxylipins to disease pathogenesis.
    Keywords:  AA; DHA; EPA; Lipid mediator; Metabolomics; PUFA
    DOI:  https://doi.org/10.1007/s10620-019-05638-y
  40. Cancer Metastasis Rev. 2019 May 08.
    Ji K, Mayernik L, Moin K, Sloane BF.
      The glycolytic phenotype of the Warburg effect is associated with acidification of the tumor microenvironment. In this review, we describe how acidification of the tumor microenvironment may increase the invasive and degradative phenotype of cancer cells. As a template of an extracellular acidic microenvironment that is linked to proteolysis, we use the resorptive pit formed between osteoclasts and bone. We describe similar changes that have been observed in cancer cells in response to an acidic microenvironment and that are associated with proteolysis and invasive and metastatic phenotypes. This includes consideration of changes observed in the intracellular trafficking of vesicles, i.e., lysosomes and exosomes, and in specialized regions of the membrane, i.e., invadopodia and caveolae. Cancer-associated cells are known to affect what is generally referred to as tumor proteolysis but little direct evidence for this being regulated by acidosis; we describe potential links that should be verified.
    Keywords:  Acidosis; Caveolae; Exosomes; Invadopodia; Lysosomes
    DOI:  https://doi.org/10.1007/s10555-019-09796-3
  41. Exp Cell Res. 2019 May 06. pii: S0014-4827(19)30232-0. [Epub ahead of print]
    Wang A, Han TL, Chen Z, Zhou X, Yu X, Qi H, Baker PN, Zhang H.
      Maternal obesity is associated with adverse effects on the health of offsprings. Consumption of a high-carbohydrate (HC) diet has been found to promote abnormal fatty acid metabolism in adipose tissue. Therefore, we hypothesised that maternal obesity combined with an offspring HC diet would alter the fatty acid metabolism of adipose tissue and subsequently contribute to offspring obesity. Leprdb/+ mice were used to model pre-pregnancy maternal obesity and the C57BL/6 wildtype were used as a control group. Offspring were fed either HC diet or a normal-carbohydrate (NC) diet after weaning. Brown adipose tissue (BAT) and white adipose tissue (WAT) were collected from offspring at 20 weeks of age and their fatty acid metabolome was characterized using gas chromatography-mass spectrometry. We found that HC diet increased the body weight of offspring (males increased by 14.70% and females increased by 1.05%) compared to control mothers. However, maternal obesity alone caused a 7.9% body weight increase in female offspring. Maternal obesity combined with an offspring HC diet resulted in dynamic alterations of the fatty acid profiles of adipose tissue in male offspring. Under the impact of a HC diet, the fatty acid metabolome was solely elevated in female WAT, whereas, the fatty acid metabolites in BAT showed a similar trend in the male and female offsprings. 6,9-octadecadienoic acid and 12,15-cis-octadecatrienoic acid were significantly affected in female WAT, in response to offspring consumption of a HC diet. Our study demonstrated that maternal obesity and offspring HC diet have different metabolic effects on adipose tissue in male and female offsprings.
    Keywords:  Adipose tissue; Fatty acid; High-carbohydrate diet; Maternal obesity; Metabolomics; Mice offsprings
    DOI:  https://doi.org/10.1016/j.yexcr.2019.05.001
  42. Sci Rep. 2019 May 10. 9(1): 7247
    León M, Ferreira CR, Eberlin LS, Jarmusch AK, Pirro V, Rodrigues ACB, Favaron PO, Miglino MA, Cooks RG.
      Chemical imaging by mass spectrometry (MS) has been largely used to study diseases in animals and humans, especially cancer; however, this technology has been minimally explored to study the complex chemical changes associated with fetal development. In this work, we report the histologically-compatible chemical imaging of small molecules by desorption electrospray ionization (DESI) - MS of a complete swine fetus at 50 days of gestation. Tissue morphology was unperturbed by morphologically-friendly DESI-MS analysis while allowing detection of a wide range of small molecules. We observed organ-dependent localization of lipids, e.g. a large diversity of phosphatidylserine lipids in brain compared to other organs, as well as metabolites such as N-acetyl-aspartic acid in the developing nervous system and N-acetyl-L-glutamine in the heart. Some lipids abundant in the lungs, such as PC(32:0) and PS(40:6), were  similar to surfactant composition reported previously. Sulfatides were highly concentrated in the fetus liver, while hexoses were barely detected at this organ but were abundant in lung and heart. The chemical information on small molecules recorded via DESI-MS imaging coupled with traditional anatomical evaluation is a powerful source of bioanalytical information which reveals the chemical changes associated with embryonic and fetal development that, when disturbed, causes congenital diseases such as spina bifida and cleft palate.
    DOI:  https://doi.org/10.1038/s41598-019-43698-2
  43. Am J Physiol Cell Physiol. 2019 May 08.
    Venturini G, Malagrino PA, Padilha K, Tanaka LY, Laurindo FR, Dariolli R, Carvalho VM, Cardozo KHM, Krieger JE, Pereira ADC.
      OBJECTIVE: Atherosclerotic plaque development is closely associated with the hemodynamic forces applied to endothelial cells (EC). Among these, shear stress (SS) plays a key role in disease development since changes in flow intensity and direction could stimulate an atheroprone or atheroprotective phenotype. EC under low or oscillatory SS (LSS) shows upregulation of inflammatory, adhesion and cellular permeability molecules. On the contrary, cells under high or laminar SS (HSS) increase their expression of protective and anti-inflammatory factors. The mechanism behind SS regulation of an atheroprotective phenotype is not completely elucidated. Approach and Results: Here we used proteomics and metabolomics to better understand the changes in endothelial cells (HUVECs) under in vitro LSS and HSS that promote an atheroprone or atheroprotective profile and how these modifications can be connected to atherosclerosis development. Our data showed that lipid metabolism, in special cholesterol metabolism, was downregulated in cells under LSS. The LDLR showed significant alterations both at the quantitative expression level, as well as regarding post-translational modifications. Under LSS, LDLR was seen at lower concentrations and with a different glycosylation profile. Finally, modulating LDLR with atorvastatin led to the recapitulation of a HSS metabolic phenotype in EC under LSS.CONCLUSIONS: Altogether, our data suggest that there is significant modulation of lipid metabolism in endothelial cells under different SS intensities and that this could contribute to the atheroprone phenotype of LSS. Statin treatment was able to partially recover the protective profile of these cells.
    Keywords:  LDLR; Shear Stress; cholesterol; lipids; lipids metabolism
    DOI:  https://doi.org/10.1152/ajpcell.00128.2018
  44. Inflamm Bowel Dis. 2019 May 11. pii: izz098. [Epub ahead of print]
    Diab J, Hansen T, Goll R, Stenlund H, Ahnlund M, Jensen E, Moritz T, Florholmen J, Forsdahl G.
      BACKGROUND: The onset of ulcerative colitis (UC) is associated with alterations in lipid metabolism and a disruption of the balance between pro- and anti-inflammatory molecules. Only a few studies describe the mucosal lipid biosignatures during active UC. Moreover, the dynamics of lipid metabolism in the remission state is poorly defined. Therefore, this study aims to characterize mucosal lipid profiles in treatment-naïve UC patients and deep remission UC patients compared with healthy subjects.METHODS: Treatment-naïve UC patients (n = 21), UC patients in deep remission (n = 12), and healthy volunteers (n = 14) were recruited. The state of deep remission was defined by histological and immunological remission defined by a normalized TNF-α gene expression. Mucosa biopsies were collected by colonoscopy. Lipid analysis was performed by means of ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS-MS). In total, 220 lipids from 11 lipid classes were identified.
    RESULTS: The relative concentration of 122 and 36 lipids was altered in UC treatment-naïve patients and UC remission patients, respectively, compared with healthy controls. The highest number of significant variations was in the phosphatidylcholine (PC), ceramide (Cer), and sphingomyelin (SM) composition. Multivariate analysis revealed discrimination among the study groups based on the lipid profile. Furthermore, changes in phosphatidylethanolamine(38:3), Cer(d18:1/24:0), and Cer(d18:1/24:2) were most distinctive between the groups.
    CONCLUSION: This study revealed a discriminant mucosal lipid composition pattern between treatment-naïve UC patients, deep remission UC patients, and healthy controls. We report several distinctive lipids, which might be involved in the inflammatory response in UC, and could reflect the disease state.
    Keywords:  inflammatory bowel disease; lipidomics; phospholipids; sphingolipids; ulcerative colitis
    DOI:  https://doi.org/10.1093/ibd/izz098
  45. Cancer Res. 2019 May 07. pii: canres.2529.2018. [Epub ahead of print]
    Gordon JA, Noble JW, Midha A, Derakhshan F, Wang G, Adomat HH, Tomlinson Guns ES, Lin YY, Ren S, Collins CC, Nelson PS, Morrisey C, Wasan KM, Cox ME.
      Aberrant cholesterol metabolism is increasingly appreciated to be essential for prostate cancer (PCa) initiation and progression. Transcript expression of the high-density lipoprotein-cholesterol receptor scavenger receptor B1 (SR-B1) is elevated in primary PCa. Hypothesizing that SR-B1 expression may help facilitate malignant transformation, we document increased SR-B1 protein and transcript expression in PCa relative to normal prostate epithelium that persists in lethal castration-resistant prostate cancer (CRPC) metastasis. As intratumoral steroid synthesis from the precursor cholesterol can drive androgen receptor (AR) pathway activity in CRPC, we screened androgenic benign and cancer cell lines for sensitivity to SR-B1 antagonism. Benign cells were insensitive to SR-B1 antagonism, and cancer line sensitivity inversely correlated with expression levels of full-length and splice-variant AR. In androgen-responsive CRPC cell model C4-2, SR-B1 antagonism suppressed cholesterol uptake, de novo steroidogenesis, and AR activity. SR-B1 antagonism also suppressed growth and viability and induced endoplasmic reticulum stress and autophagy. The inability of exogenous steroids to reverse these effects indicates that AR pathway activation is insufficient to overcome cytotoxic stress caused by a decrease in the availability of cholesterol. Furthermore, SR-B1 antagonism decreased cholesterol uptake, growth, and viability of the AR-null CRPC cell model PC-3, and the small molecule SR-BI antagonist Block Lipid Transport-1 decreased xenograft growth rate despite poor pharmacologic properties. Overall, our findings show that SR-B1 is upregulated in primary and castration-resistant disease and is essential for cholesterol uptake needed to drive both steroidogenic and non-steroidogenic biogenic pathways, thus implicating SR-B1 as a novel and potentially actionable target in CRPC.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-18-2529
  46. Sci Rep. 2019 May 10. 9(1): 7217
    Trilla-Fuertes L, Gámez-Pozo A, Prado-Vázquez G, Zapater-Moros A, Díaz-Almirón M, Fortes C, Ferrer-Gómez M, López-Vacas R, Parra Blanco V, Márquez-Rodas I, Soria A, Fresno Vara JÁ, Espinosa E.
      Melanoma is the most lethal cutaneous cancer. New drugs have recently appeared; however, not all patients obtain a benefit of these new drugs. For this reason, it is still necessary to characterize melanoma at molecular level. The aim of this study was to explore the molecular differences between melanoma tumor subtypes, based on BRAF and NRAS mutational status. Fourteen formalin-fixed, paraffin-embedded melanoma samples were analyzed using a high-throughput proteomics approach, combined with probabilistic graphical models and Flux Balance Analysis, to characterize these differences. Proteomics analyses showed differences in expression of proteins related with fatty acid metabolism, melanogenesis and extracellular space between BRAF mutated and BRAF non-mutated melanoma tumors. Additionally, probabilistic graphical models showed differences between melanoma subgroups at biological processes such as melanogenesis or metabolism. On the other hand, Flux Balance Analysis predicts a higher tumor growth rate in BRAF mutated melanoma samples. In conclusion, differential biological processes between melanomas showing a specific mutational status can be detected using combined proteomics and computational approaches.
    DOI:  https://doi.org/10.1038/s41598-019-43512-z
  47. Postgrad Med. 2019 May 07. 1-10
    Nelson JR, Raskin S.
      Eicosapentaenoic acid (EPA) is a key anti-inflammatory/anti-aggregatory long-chain polyunsaturated omega-3 fatty acid. Conversely, the omega-6 fatty acid, arachidonic acid (AA) is a precursor to a number of pro-inflammatory/pro-aggregatory mediators. EPA acts competitively with AA for the key cyclooxygenase and lipoxygenase enzymes to form less inflammatory products. As a result, the EPA:AA ratio may be a marker of chronic inflammation, with a lower ratio corresponding to higher levels of inflammation. It is now well established that inflammation plays an important role in cardiovascular disease. This review examines the role of the EPA:AA ratio as a marker of cardiovascular disease and the relationship between changes in the ratio (mediated by EPA intake) and changes in cardiovascular risk. Epidemiological studies have shown that a lower EPA:AA ratio is associated with an increased risk of coronary artery disease, acute coronary syndrome, myocardial infarction, stroke, chronic heart failure, peripheral artery disease, and vascular disease. Increasing the EPA:AA ratio through treatment with purified EPA has been shown in clinical studies to be effective in primary and secondary prevention of coronary artery disease and reduces the risk of cardiovascular events following percutaneous coronary intervention. The EPA:AA ratio is a valuable predictor of cardiovascular risk. Results from ongoing clinical trials will help to define thresholds for EPA treatment associated with better clinical outcomes.
    Keywords:  Eicosapentaenoic acid; arachidonic acid; cardiovascular disease; coronary artery disease; inflammation; long-chain polyunsaturated omega-3 fatty acids; omega 3 fatty acids
  48. Methods Mol Biol. 2019 ;1967 197-210
    Bukowski MR, Picklo MJ.
      Protein-glutathione mixed disulphides (PSSG) are an important redox-sensitive posttranslational modification. Quantitation of protein-glutathione mixed disulphides (PSSG) is achieved by the reduction of the disulphide bond to liberate glutathione (GSH); however, this method leaves the assay susceptible to contamination by cytosolic GSH and glutathione disulphide (GSSG) captured during protein precipitation. The method herein describes a workflow in which protein from mouse liver is precipitated and adventitious GSH contamination is removed by reaction with N-ethylmaleimide. The sample is divided into two equal aliquots, a control aliquot that allows for direct quantitation of adventitious GSSG and a chemically reduced aliquot that contains GSH from both the GSSG and PSSG disulphides. Determining the concentration of adventitious GSSG allows for correction of the latter value to provide an accurate assay of PSSG. This assay also provides quantitation of cytosolic GSH and GSSG.
    Keywords:  Glutathione; Glutathione disulphide; LC-MS/MS; Mixed disulphide; S-glutathionylation
    DOI:  https://doi.org/10.1007/978-1-4939-9187-7_12
  49. Genomics Proteomics Bioinformatics. 2019 May 06. pii: S1672-0229(18)30171-2. [Epub ahead of print]
    Sun H, Chen L, Cao S, Liang Y, Xu Y.
      It has been observed that both cancer tissue cells and normal proliferating cells (NPCs) have the Warburg effect. Our goal here is to demonstrate that they do this for different reasons. To accomplish this, we have analyzed the transcriptomic data of over 7000 cancer and control tissues of 14 cancer types in TCGA and data of five NPC types in GEO. Our analyses reveal that NPCs accumulate large quantities of ATPs produced by the respiration process before starting the Warburg effect, to raise the intracellular pH from ∼6.8 to ∼7.2 and to prepare for cell division energetically. Once cell cycle starts, the cells start to rely on glycolysis for ATP generation followed by ATP hydrolysis and lactic acid release, to maintain the elevated intracellular pH as needed by cell division since together the three processes are pH neutral. The cells go back to the normal respiration-based ATP production once the cell division phase ends. In comparison, cancer cells have reached their intracellular pH at ∼7.4 from top down as multiple acid-loading transporters are up-regulated and most acid-extruding ones except for lactic acid exporters are repressed. Cancer cells use continuous glycolysis for ATP production as way to acidify the intracellular space since the lactic acid secretion is decoupled from glycolysis-based ATP generation and is pH balanced by increased expressions of acid-loading transporters. Co-expression analyses suggest that lactic acid secretion is regulated by external, non-pH related signals. Overall, our data strongly suggest that the two cell types have the Warburg effect for very different reasons.
    Keywords:  Cancer; Cell proliferation; Fenton reaction; Warburg effect; pH homeostasis
    DOI:  https://doi.org/10.1016/j.gpb.2018.12.006
  50. Analyst. 2019 May 08.
    Ashwood C, Pratt B, MacLean BX, Gundry RL, Packer NH.
      Porous graphitized carbon (PGC) based chromatography achieves high-resolution separation of glycan structures released from glycoproteins. This approach is especially valuable when resolving structurally similar isomers and for discovery of novel and/or sample-specific glycan structures. However, the implementation of PGC-based separations in glycomics studies has been limited because system-independent retention values have not been established to normalize technical variation. To address this limitation, this study combined the use of hydrolyzed dextran as an internal standard and Skyline software for post-acquisition normalization to reduce retention time and peak area technical variation in PGC-based glycan analyses. This approach allowed assignment of system-independent retention values that are applicable to typical PGC-based glycan separations and supported the construction of a library containing >300 PGC-separated glycan structures with normalized glucose unit (GU) retention values. To enable the automation of this normalization method, a spectral MS/MS library was developed of the dextran ladder, achieving confident discrimination against isomeric glycans. The utility of this approach is demonstrated in two ways. First, to inform the search space for bioinformatically predicted but unobserved glycan structures, predictive models for two structural modifications, core-fucosylation and bisecting GlcNAc, were developed based on the GU library. Second, the applicability of this method for the analysis of complex biological samples is evidenced by the ability to discriminate between cell culture and tissue sample types by the normalized intensity of N-glycan structures alone. Overall, the methods and data described here are expected to support the future development of more automated approaches to glycan identification and quantitation.
    DOI:  https://doi.org/10.1039/c9an00486f
  51. Expert Rev Mol Diagn. 2019 May 04.
    Kontostathi G, Makridakis M, Zoidakis J, Vlahou A.
      INTRODUCTION: Multiple (or selected) reaction monitoring-mass spectrometry (MRM/SRM) is a targeted proteomic method that can be used for relative and absolute quantification. Multiple reports exist supporting the potential of the approach in proteomic biomarker validation. Areas covered: To get an overview of the applications of MRM in protein quantification in plasma, a search in MedLine/PubMed was performed using the key words: "MRM/SRM plasma proteomic/proteomics/proteome". The retrieved studies were further filtered to focus on disease biomarkers and main results are summarized. Expert opinion: MRM is increasingly employed for the quantification of both well-established but also newly discovered putative biomarkers and occasionally their post-translationally modified forms in plasma. Fractionation is regularly required for the detection of low abundance proteins. Standardized procedures to facilitate assay establishment and marker quantification have been proposed and, in few cases, implemented. Nevertheless, in most cases, absolute quantification is not performed. To advance, multiple technical issues including the regular use of standard labelled peptides and appropriate quality controls to monitor assay performance should be considered. Additionally, clinical aspects involving careful study design to address biomarker clinical use should also be considered.
    Keywords:  MRM; SRM; biomarkers; drugs; plasma; quantification
    DOI:  https://doi.org/10.1080/14737159.2019.1615448
  52. J Proteome Res. 2019 May 10.
    Cao Z, Kamlage B, Wagner-Golbs A, Maisha M, Sun J, Schnackenberg LK, Pence L, Schmitt TC, Daniels JR, Rogstad S, Beger RD, Yu LR.
      Discrepancies in blood sample collection and processing could have a significant impact on levels of metabolites, peptides and protein biomarkers of inflammation in the blood, thus sample quality control is critical for successful biomarker identification and validation. In this study, we analyzed the effects of several pre-analytical processing conditions, including different storage times and temperatures for blood or plasma samples and different centrifugation forces, on the levels of metabolites, peptides and inflammation biomarkers in human plasma samples using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Temperature was found to be the major factor for metabolite variation, and both time and temperature were identified as major factors for peptide variation. For inflammation biomarkers, temperature played different roles depending on the sample type (blood or plasma). Low-temperature affected inflammation biomarkers in blood, while room temperature impacted inflammation biomarkers in plasma.
    DOI:  https://doi.org/10.1021/acs.jproteome.8b00903
  53. Clin Chim Acta. 2019 May 03. pii: S0009-8981(19)31844-3. [Epub ahead of print]
    Bai F, Tu T, Qin F, Ma Y, Liu N, Liu Y, Liao X, Zhou S, Liu Q.
      BACKGROUND: Previous studies have suggested that proteomic modifications are closely associated with cardiovascular diseases. The aim of this study was to identify potential mechanisms by profiling the changes in succinylated protein expression in left appendage tissues from patients with valvular heart disease and atrial fibrillation (AF).METHODS: Using dimethyl labeling for relative and absolute quantification-coupled high-performance liquid chromatography-tandem mass spectrometry, we analyzed the proteomics profiles and succinylation events in 18 left atrial appendage tissue samples from patients who underwent cardiac valvular surgery, including nine patients with permanent AF and nine patients with sinus rhythm (SR).
    RESULTS: In total, after setting the quantification ratio > 1.3 and < 1:1.3 representing the up- and downregulated cutoff values, respectively, 132 proteins were classified as targets of upregulation and 117 proteins as targets of downregulation. Within these proteins, 246 sites exhibited upregulated succinylation and 45 sites exhibited downregulated succinylation. Protein-protein interaction networks showed that the proteins exhibiting lysine succinylation and AF status were highly enriched in energy metabolism, extracellular matrix-related, and cellular structure-related proteins. These results were confirmed by western blot.
    CONCLUSIONS: The differences in succinylation level of energy metabolism-related proteins indicates the possible involvement of these proteins in AF of valvular heart disease patients, and provide insight for further analysis of their biological functions.
    Keywords:  Atrial fibrillation; Isobaric tag for relative and absolute quantification; Proteomics; Succinylation
    DOI:  https://doi.org/10.1016/j.cca.2019.05.002
  54. J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Apr 27. pii: S1570-0232(19)30232-6. [Epub ahead of print]1120 16-23
    Liu TT, Cheong LZ, Man QQ, Zheng X, Zhang J, Song S.
      Simultaneous profiling of serum vitamin D (VD) metabolites with similar structures is a big challenge. Thus, we developed and validated a SFC-MS/MS method, which is capable of eluting hydrophobic molecules, for quantification of VD2/VD3, 25-OH VD2/VD3, 3-epi-25-OH VD2/VD3, 1,25-(OH)2 VD2/VD3 and 24,25-(OH)2 VD2/VD3. VD metabolites were extracted from human serum using acetonitrile solvent. Column stationary phase, elution gradients, flow rate, column temperature, ion-source type and buffer system in post-column make-up solvent were optimized. Baseline separation of 10 VD metabolites can be achieved using PFP column within 10 min; and detection performed under positive electrospray ionization mode allowed quantification of VD metabolites in serum matrix with a limit of quantification (LOQ) varrying from 0.071 to 0.704 ng/mL. The accuracy was controlled with relative bias lower than 5.5% for QC and NIST samples. The developed method showed excellent intra-assay (0.52-7.93% RSD) and inter-assay (1.35-9.04% RSD) precision. The methodology shows enhanced efficiency and sensitivity as compared to LC-MS/MS method using the same column and mass spectrometer, along with significant correlation and low mean difference bias on measurements. For analysis of trace 1,25-(OH)2 VD2 and 1,25-(OH)2 VD3 in normal human serum or plasma, further improvement of LOQ (like derivatization) should be considered. In conclusion, the use of supercritical fluid not only enhanced safety with reduced solvent cost, but also improved retention and sensitivity as compared to LC-MS/MS method. The developed SFC-MS/MS method is appropriate for high throughput analysis of multiple VD metabolites in human serum with reduced solvent and economic cost.
    Keywords:  Metabolites; Serum; Supercritical fluid chromatography; Vitamin D
    DOI:  https://doi.org/10.1016/j.jchromb.2019.04.050
  55. Methods Mol Biol. 2019 ;1985 303-319
    Lipka E.
      Nowadays polysaccharide chiral stationary phases are very popular and this originates for many reasons: (1) their wide applications window, (2) numerous different chemistry availability either in coated or in immobilized versions, and (3) large loading capability useful for preparative scale. Indeed chiral separations remain a hot topic (particularly in the pharmaceutical market) and in this field supercritical fluid chromatography is emerging rapidly. However, its use is more complex than high-performance liquid chromatography. The presented example illustrates analytical scale chiral separation method development and the effect of each operating parameter, i.e., flow rate, outlet pressure, and temperature variations on a chiral separation.
    Keywords:  Chiral stationary phase; Enantioseparation; Polysaccharide-based stationary phase; Supercritical fluid chromatography
    DOI:  https://doi.org/10.1007/978-1-4939-9438-0_16
  56. Signal Transduct Target Ther. 2019 ;4 13
    Wu C, Zheng L.
      
    Keywords:  Gastrointestinal cancer; Molecular medicine; Systems biology
    DOI:  https://doi.org/10.1038/s41392-019-0046-9
  57. Analyst. 2019 May 08.
    Frick AA, Weyermann C.
      More detailed fundamental information is required about latent fingermark composition in order to better understand fingermark properties and their impact on detection efficiency, and the physical and chemical changes that occur with time following deposition. The composition of the glyceride fraction of latent fingermark lipids in particular is relatively under-investigated due in part to their high structural variability and the limitations of the analytical methods most frequently utilised to investigate fingermark composition. Here, we present an ultra performance liquid chromatography-ion mobility spectroscopy-quadrupole time-of-flight mass spectrometry (UPLC-IMS-QToF-MSE) method to characterise glycerides in charged latent fingermarks using data-independent acquisition. Di- and triglycerides were identified in fingermark samples from a population of 10 donors, through a combination of in silico fragmentation and monitoring for fatty acid neutral losses. 23 diglycerides and 85 families of triglycerides were identified, with significant diversity in chain length and unsaturation. 21 of the most abundant triglyceride families were found to be common to most or all donors, presenting potential targets for further studies to monitor chemical and physical changes in latent fingermarks over time. Differences in relative peak intensities may be indicative of inter- and intra-donor variability. While this study represents a promising step to obtaining more in-depth information about fingermark composition, it also highlights the complex nature of these traces.
    DOI:  https://doi.org/10.1039/c9an00521h
  58. Biochem Biophys Res Commun. 2019 May 02. pii: S0006-291X(19)30829-0. [Epub ahead of print]
    Li W, Zhang J, Lv Y, Sheibani N.
      Liver sinusoidal endothelial cells are the border patrol in the liver. Their open transcellular fenestrations allow the transfer of small and dissolved substances from the blood into the liver parenchymal cells. Fenestrations are dynamic structures, and many drugs and diseases alter their size and number, thus making them an important target for modulation. There is an urgent need to understand how various diseases, toxic substances, and physiological conditions influence liver endothelial cell fenestrations, and how these changes affects liver function. This work represents a straightforward quantitative proteomics study of the in vivo arsenic-stressed liver sinusoidal endothelial cells using a reverse super-SILAC based method. The aim of this study was to identify proteins, which are up- or down-regulated in response to arsenic. This knowledge will aid in identification of potential targets and mechanisms of arsenic toxicity and novel ways to reverse these changes.
    Keywords:  Arsenic; Mass spectrometry; Mouse liver sinusoidal endothelial cell; Proteomics; Super-SILAC
    DOI:  https://doi.org/10.1016/j.bbrc.2019.04.172
  59. Methods Mol Biol. 2019 ;1975 321-330
    Sun Z, Zhao J, Yu H, Zhang C, Li H, Zeng Z, Zhang J.
      Stem cell research has been greatly facilitated by comprehensive and integrative multi-omics studies. As a unique approach of functional analysis, metabolomics measures many metabolites and activities of metabolic pathways which can directly indicate cellular energetic status, cell proliferation and fitness, and stem cell fate choices such as self-renewal versus differentiation. Here we describe the methods of applying metabolomics, 13C-labeled glucose and glutamine tracing with mouse embryonic stem cells (ES cells), metabolite analysis using mass spectrometry tools, and the following statistical and computational modeling analysis. Integration of these methods into the more common gene expression and epigenetics analysis toolbox will help to generate a more complete picture and in-depth understanding of one's stem cells of interest.
    Keywords:  13C-tracing analysis; Mass spectroscopy; Metabolic pathway; Metabolite; Metabolomics; Statistical analysis
    DOI:  https://doi.org/10.1007/978-1-4939-9224-9_15