bims-mascan 2021-09-19 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2021–09–19
25 papers selected by
Giovanny Rodríguez Blanco, University of Edinburgh

Stable isotope tracing to assess tumor metabolism in vivo.
Quantitative global lipidomics analysis of patients with ovarian cancer versus benign adnexal mass.
Accurate quantification of endogenous N-acylethanolamides by chemical isotope labeling coupled with liquid chromatography-tandem mass spectrometry.
Optimization of a liquid chromatography-ion mobility-high resolution mass spectrometry platform for untargeted lipidomics and application to HepaRG cell extracts.
Pharmacologic approaches to amino acid depletion for cancer therapy.
Glutamine anaplerosis is required for amino acid biosynthesis in human meningiomas.
PCK2 opposes mitochondrial respiration and maintains the redox balance in starved lung cancer cells.
Time-resolved in vivo ubiquitinome profiling by DIA-MS reveals USP7 targets on a proteome-wide scale.
Ferroptosis and Liver Fibrosis.
Optimization and validation of capillary electrophoresis- and gas chromatography-tandem mass spectrometry methods for the analysis of intermediate metabolites in glycolysis and pentose phosphate pathways within biological samples.
Chemical Proportionality within Molecular Networks.
Monitoring the Cellular Metabolism of a Membrane-Permeant Photo-Caged Phosphatidylinositol 3,4,5-Trisphosphate Derivative.
Pharmacokinetic and metabolic analysis of an Alzheimer's disease therapeutic in rat serum via microfluidic CZE-MS.
NADPH debt drives redox bankruptcy: SLC7A11/xCT-mediated cystine uptake as a double-edged sword in cellular redox regulation.
Diagnostic approach to thyroid cancer based on amino acid metabolomics in saliva by ultra-performance liquid chromatography with high resolution mass spectrometry.
Classification and biomarker identification of prostate tissue from TRAMP mice with hyperpolarized 13C-SIRA.
Discovery of Potential Lipid Biomarkers for Human Colorectal Cancer by In-Capillary Extraction Nanoelectrospray Ionization Mass Spectrometry.
Targeted fatty acid metabolomics to discover Parkinson's disease associated metabolic alteration.
Bifunctionalized isotopologues as calibrants for standard-free quantitation of drug metabolites in plasma by liquid chromatography coupled with radiometry and mass spectrometry.
Exposure to ambient fine particulate matter impedes the function of spleen in the mouse metabolism of high-fat diet.
Regulation of Extracellular Matrix Production in Activated Fibroblasts: Roles of Amino Acid Metabolism in Collagen Synthesis.
Combined Metabolic and Chemical (CoMetChem) Labeling Using Stable Isotopes-a Strategy to Reveal Site-Specific Histone Acetylation and Deacetylation Rates by LC-MS.
In silico prediction, LC-HRMS/MS analysis, and targeted/untargeted data-mining workflow for the profiling of phenylfentanyl in vitro metabolites.
Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.
Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue.

Nat Protoc. 2021 Sep 17.

Stable isotope tracing to assess tumor metabolism in vivo.

Brandon Faubert, Alpaslan Tasdogan, Sean J Morrison, Thomas P Mathews, Ralph J DeBerardinis.

Cancer cells undergo diverse metabolic adaptations to meet the energetic demands imposed by dysregulated growth and proliferation. Assessing metabolism in intact tumors allows the investigator to observe the combined metabolic effects of numerous cancer cell-intrinsic and -extrinsic factors that cannot be fully captured in culture models. We have developed methods to use stable isotope-labeled nutrients (e.g., [13C]glucose) to probe metabolic activity within intact tumors in vivo, in mice and humans. In these methods, the labeled nutrient is introduced to the circulation through an intravenous catheter prior to surgical resection of the tumor and adjacent nonmalignant tissue. Metabolism within these tissues during the infusion transfers the isotope label into metabolic intermediates from pathways supplied by the infused nutrient. Extracting metabolites from surgical specimens and analyzing their isotope labeling patterns provides information about metabolism in the tissue. We provide detailed information about this technique, from introduction of the labeled tracer through data analysis and interpretation, including streamlined approaches to quantify isotope labeling in informative metabolites extracted from tissue samples. We focus on infusions with [13C]glucose and the application of mass spectrometry to assess isotope labeling in intermediates from central metabolic pathways, including glycolysis, the tricarboxylic acid cycle and nonessential amino acid synthesis. We outline practical considerations to apply these methods to human subjects undergoing surgical resections of solid tumors. We also discuss the method's versatility and consider the relative advantages and limitations of alternative approaches to introduce the tracer, harvest the tissue and analyze the data.

DOI: https://doi.org/10.1038/s41596-021-00605-2
Sci Rep. 2021 Sep 13. 11(1): 18156

Quantitative global lipidomics analysis of patients with ovarian cancer versus benign adnexal mass.

Matthew F Buas, Charles W Drescher, Nicole Urban, Christopher I Li, Lisa Bettcher, Nitai C Hait, Kirsten B Moysich, Kunle Odunsi, Daniel Raftery, Li Yan.

Altered lipid metabolism has emerged as an important feature of ovarian cancer (OC), yet the translational potential of lipid metabolites to aid in diagnosis and triage remains unproven. We conducted a multi-level interrogation of lipid metabolic phenotypes in patients with adnexal masses, integrating quantitative lipidomics profiling of plasma and ascites with publicly-available tumor transcriptome data. Using Sciex Lipidyzer, we assessed concentrations of > 500 plasma lipids in two patient cohorts-(i) a pilot set of 100 women with OC (50) or benign tumor (50), and (ii) an independent set of 118 women with malignant (60) or benign (58) adnexal mass. 249 lipid species and several lipid classes were significantly reduced in cases versus controls in both cohorts (FDR < 0.05). 23 metabolites-triacylglycerols, phosphatidylcholines, cholesterol esters-were validated at Bonferroni significance (P < 9.16 × 10-5). Certain lipids exhibited greater alterations in early- (diacylglycerols) or late-stage (lysophospholipids) cases, and multiple lipids in plasma and ascites were positively correlated. Lipoprotein receptor gene expression differed markedly in OC versus benign tumors. Importantly, several plasma lipid species, such as DAG(16:1/18:1), improved the accuracy of CA125 in differentiating early-stage OC cases from benign controls, and conferred a 15-20% increase in specificity at 90% sensitivity in multivariate models adjusted for age and BMI. This study provides novel insight into systemic and local lipid metabolic differences between OC and benign disease, further implicating altered lipid uptake in OC biology, and advancing plasma lipid metabolites as a complementary class of circulating biomarkers for OC diagnosis and triage.

DOI: https://doi.org/10.1038/s41598-021-97433-x
Anal Chim Acta. 2021 Sep 22. pii: S0003-2670(21)00665-6. [Epub ahead of print]1179 338839

Accurate quantification of endogenous N-acylethanolamides by chemical isotope labeling coupled with liquid chromatography-tandem mass spectrometry.

Yan-Qing Chen, Hong Shen, Ru-Jie Yang, Jian-Bo Wan.

  N-acylethanolamides (NAEs) are a class of naturally occurring lipid molecules with pleiotropic activities ranging from energy homeostasis to analgesic functioning. However, the comprehensive quantitation of endogenous NAEs is challenged by the sub-trace level (nM) in complex biological samples and the limited availability of stable isotope labeled internal standards (SIL-IS). Herein, a sensitive method was developed to accurately determine 20 NAEs in biological samples by chemical isotope labeling strategy coupled with liquid chromatography - tandem mass spectrometry (LC-MS/MS). A pair of efficient derivatization reagents, acetyl chloride-d0 (ACC-d0) and acetyl chloride-d3 (ACC-d3), were used to label NAEs in biological samples and NAE standard mixture, respectively. The heavily labeled NAE derivatives of the standard substances were used as one-to-one internal standards to minimize the matrix effects and potential ion suppression in MS analysis. Although no chemical moiety with high ionization capability was introduced, the detection sensitivity of the derivatized NAEs were substantially enhanced, as evidenced by 6- to 170-fold increase in LOQs, compared to non-derivatized NAEs. The derivatized NAEs provided the stable and abundant specific product ions in MS/MS spectrum, which were used as the quantitation ions for multiple reaction monitoring (MRM) analysis. The validated LC-MS/MS method was also successfully applied to determine NAEs in serum samples and liver tissues from control and alcohol-fed mice, which shown its practicability in the analysis of endogenous NAE in biological samples. Collectively, the proposed method offers a sensitive and accurate quantification of endogenous NAEs, which may facilitate the understanding of NAE metabolisms and their functions in the physiological and pathological processes.

Keywords:  Acetyl chloride; Chemical isotope labeling; LC-MS/MS; N-acylethanolamides

DOI:  https://doi.org/10.1016/j.aca.2021.338839
Talanta. 2021 Dec 01. pii: S0039-9140(21)00729-3. [Epub ahead of print]235 122808

Optimization of a liquid chromatography-ion mobility-high resolution mass spectrometry platform for untargeted lipidomics and application to HepaRG cell extracts.

Katyeny Manuela da Silva, Elias Iturrospe, Joris Heyrman, Jeremy P Koelmel, Matthias Cuykx, Tamara Vanhaecke, Adrian Covaci, Alexander L N van Nuijs.

  Analytical methods to evaluate the lipidome of biological samples need to provide high data quality to ensure comprehensive profiling and reliable structural elucidation. In this perspective, liquid chromatography-high resolution mass spectrometry (LC-HRMS) is the state-of-the-art technique for lipidomic analysis of biological samples. There are thousands of lipids in most biological samples, and therefore separation methods before introduction to the mass spectrometer is key for relative quantitation and identification. Chromatographic methods differ across laboratories, without any consensus on the best methodologies. Therefore, we designed an experiment to determine the optimal LC methodology, and assessed the value of ion mobility for an additional dimension of separation. To apply an untargeted method for hypothesis generation focused on lipidomics, LC-HRMS parameters were optimized based on the measurement of 50 panel lipids covering key human metabolic pathways. Reversed-phase liquid chromatography columns were compared based on a quality scoring system considering the signal-to-noise ratio, peak shape, and retention factor. Furthermore, drift tube ion mobility spectrometry (DTIMS) was implemented to increase peak capacity and confidence during annotation by providing collision cross section (CCS) values for the analytes under investigation. However, hyphenating DTIMS to LC-HRMS may result in a reduced sensitivity due to impaired duty cycles. To increase the signal intensity, a Box-Behnken design (BBD) was used to optimize four key factors, i.e. drift entrance voltage, drift exit voltage, rear funnel entrance, and rear funnel exit voltages. Application of a maximized desirability function provided voltages for the above-mentioned parameters resulting in higher signal intensity compared to each combination of parameters used during the BBD. In addition, the influence of single pulse and Hadamard 4-bit multiplexed modes on signal intensity was explored and different trap filling and release times of ions were evaluated. The optimized LC-DTIM-HRMS platform was applied to extracts from HepaRG cells and resulted in 3912 high-quality features (<30% median relative standard deviation; n = 6, t = 24 h). From these features, 436 lipid species could be annotated (i.e., matching based on accurate mass <5 ppm, isotopic pattern, in-silico MS/MS fragmentation, and in-silico CCS database matching <3%). The application of LC-DTIM-HRMS for untargeted analysis workflows is growing and the platform optimization, as described here, can be used to guide the method development and CCS database comparison for high confidence lipid annotation.

Keywords:  Collision cross section; Hadamard multiplexing ion mobility; High-resolution mass spectrometry; Human HepaRG cells; Liver intracellular extracts; Untargeted lipidomics

DOI:  https://doi.org/10.1016/j.talanta.2021.122808
Mol Carcinog. 2021 Sep 17.

Pharmacologic approaches to amino acid depletion for cancer therapy.

Carly S Wilder, Zhao Chen, John DiGiovanni.

  Cancer cells undergo metabolic reprogramming to support increased demands in bioenergetics and biosynthesis and to maintain reactive oxygen species at optimum levels. As metabolic alterations are broadly observed across many cancer types, metabolic reprogramming is considered a hallmark of cancer. A metabolic alteration commonly seen in cancer cells is an increased demand for certain amino acids. Amino acids are involved in a wide range of cellular functions, including proliferation, redox balance, bioenergetic and biosynthesis support, and homeostatic functions. Thus, targeting amino acid dependency in cancer is an attractive strategy for a number of cancers. In particular, pharmacologically mediated amino acid depletion has been evaluated as a cancer treatment option for several cancers. Amino acids that have been investigated for the feasibility of drug-induced depletion in preclinical and clinical studies for cancer treatment include arginine, asparagine, cysteine, glutamine, lysine, and methionine. In this review, we will summarize the status of current research on pharmacologically mediated amino acid depletion as a strategy for cancer treatment and potential chemotherapeutic combinations that synergize with amino acid depletion to further inhibit tumor growth and progression.

Keywords:  amino acid depletion; amino acid metabolism; cancer; cancer therapy; metabolic reprogramming

DOI:  https://doi.org/10.1002/mc.23349
Neuro Oncol. 2021 Sep 13. pii: noab219. [Epub ahead of print]

Glutamine anaplerosis is required for amino acid biosynthesis in human meningiomas.

Omkar B Ijare, Shashank Hambarde, Fabio Henrique Brasil da Costa, Sophie Lopez, Martyn A Sharpe, Santosh A Helekar, Gilbert Hangel, Wolfgang Bogner, Georg Widhalm, Robert M Bachoo, David S Baskin, Kumar Pichumani.

   BACKGROUND: We postulate that meningiomas undergo distinct metabolic reprogramming in tumorigenesis and unravelling their metabolic phenotypes provide new therapeutic insights. Glutamine catabolism is key to the growth and proliferation of tumors. Here, we investigated the metabolomics of freshly resected meningiomas and glutamine metabolism in patient-derived meningioma cells.
METHODS: 1H NMR spectroscopy of tumor tissues from 33 meningioma patients was used to differentiate the metabolite profiles of grade-I and grade-II meningiomas. Glutamine metabolism was examined using 13C/ 15N glutamine tracer, in five patient-derived meningioma cells.
RESULTS: Alanine, lactate, glutamate, glutamine, and glycine were predominantly elevated only in grade-II meningiomas by 74%, 76%, 35%, 75% and 33% respectively, with alanine, and glutamine being statistically significant (p ≤ 0.02). 13C/ 15N glutamine tracer experiments revealed that both grade-I and -II meningiomas actively metabolize glutamine to generate various key carbon intermediates including alanine and proline that are necessary for the tumor growth. Also, it is shown that glutaminase (GLS1) inhibitor, CB-839 is highly effective in downregulating glutamine metabolism and decreasing proliferation in meningioma cells.
CONCLUSION: Alanine and glutamine/glutamate are mainly elevated in grade-II meningiomas. Grade-I meningiomas possess relatively higher glutamine metabolism providing carbon/nitrogen for the biosynthesis of key nonessential amino acids. GLS1 inhibitor (CB-839) would be very effective in downregulating glutamine metabolic pathways in grade-I meningiomas leading to decreased cellular proliferation.

Keywords:  alanine; glutamine; meningioma; metabolic flux analysis; metabolomics

DOI:  https://doi.org/10.1093/neuonc/noab219
Free Radic Biol Med. 2021 Sep 11. pii: S0891-5849(21)00720-6. [Epub ahead of print]

PCK2 opposes mitochondrial respiration and maintains the redox balance in starved lung cancer cells.

Gabriele Bluemel, Mélanie Planque, Corina T Madreiter-Sokolowski, Theresa Haitzmann, Andelko Hrzenjak, Wolfgang F Graier, Sarah-Maria Fendt, Horst Olschewski, Katharina Leithner.

  Cancer cells frequently lack nutrients like glucose, due to insufficient vascular networks. Mitochondrial phosphoenolpyruvate carboxykinase, PCK2, has recently been found to mediate partial gluconeogenesis and hence anabolic metabolism in glucose starved cancer cells. Here we show that PCK2 acts as a regulator of mitochondrial respiration and maintains the redox balance in nutrient-deprived human lung cancer cells. PCK2 silencing increased the abundance and interconversion of tricarboxylic acid (TCA) cycle intermediates, augmented mitochondrial respiration and enhanced glutathione oxidation under glucose and serum starvation, in a PCK2 re-expression reversible manner. Moreover, enhancing the TCA cycle by PCK2 inhibition severely reduced colony formation of lung cancer cells under starvation. As a conclusion, PCK2 contributes to maintaining a reduced glutathione pool in starved cancer cells besides mediating the biosynthesis of gluconeogenic/glycolytic intermediates. The study sheds light on adaptive responses in cancer cells to nutrient deprivation and shows that PCK2 confers protection against respiration-induced oxidative stress.

Keywords:  Adaptation; Cancer metabolism; Gluconeogenesis; Metabolic flexibility; Mitochondria; Redox balance; Respiration

DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.09.007
Nat Commun. 2021 Sep 13. 12(1): 5399

Time-resolved in vivo ubiquitinome profiling by DIA-MS reveals USP7 targets on a proteome-wide scale.

Martin Steger, Vadim Demichev, Mattias Backman, Uli Ohmayer, Phillip Ihmor, Stefan Müller, Markus Ralser, Henrik Daub.

Mass spectrometry (MS)-based ubiquitinomics provides system-level understanding of ubiquitin signaling. Here we present a scalable workflow for deep and precise in vivo ubiquitinome profiling, coupling an improved sample preparation protocol with data-independent acquisition (DIA)-MS and neural network-based data processing specifically optimized for ubiquitinomics. Compared to data-dependent acquisition (DDA), our method more than triples identification numbers to 70,000 ubiquitinated peptides in single MS runs, while significantly improving robustness and quantification precision. Upon inhibition of the oncology target USP7, we simultaneously record ubiquitination and consequent changes in abundance of more than 8,000 proteins at high temporal resolution. While ubiquitination of hundreds of proteins increases within minutes of USP7 inhibition, we find that only a small fraction of those are ever degraded, thereby dissecting the scope of USP7 action. Our method enables rapid mode-of-action profiling of candidate drugs targeting DUBs or ubiquitin ligases at high precision and throughput.

DOI: https://doi.org/10.1038/s41467-021-25454-1
Int J Med Sci. 2021 ;18(15): 3361-3366

Ferroptosis and Liver Fibrosis.

Qi Pan, Yi Luo, Qiang Xia, Kang He.

  Ferroptosis is an iron-dependent form of regulated cell death, which is characterized by a large amount of lipid peroxide accumulation and the imbalance of redox state in cells. Ferroptosis is usually accompanied with the dysfunction of lipid repair enzyme (glutathione peroxidase 4, GPX4), large masses of iron accumulation and lipid peroxidation of polyunsaturated fatty acids (PUFAs). Ferroptosis is related to several signaling pathways, including amino acid and iron metabolism, ferritinophagy, cell adhesion and p53 and Keap1/Nrf2 signaling pathways. A number of studies have indicated that ferroptosis is closely associated with acute renal failure, tumor, ischemia and reperfusion injury, neurodegenerative diseases and liver fibrosis. Liver fibrosis, which has long been a global health problem, still lacks effective treatment till now, and the discovery of ferroptosis provides a new insight into addressing this issue.

Keywords:  ferroptosis; hepatic stellate cells; liver fibrosis

DOI:  https://doi.org/10.7150/ijms.62903
J Chromatogr A. 2021 Sep 04. pii: S0021-9673(21)00655-5. [Epub ahead of print]1656 462531

Optimization and validation of capillary electrophoresis- and gas chromatography-tandem mass spectrometry methods for the analysis of intermediate metabolites in glycolysis and pentose phosphate pathways within biological samples.

Miso Nam, Min-Sun Kim, Geum-Sook Hwang.

  Highly selective methods for the analysis of intermediate metabolites involved in glycolysis and phosphate pentose pathways are essential for metabolism and metabolic flux studies. However, the successful separation of phosphorylated compounds is difficult due to their high polarity, as well as their structural isomers. In this study, phosphorylated compounds in spiked serum samples were analyzed using capillary electrophoresis tandem mass spectrometry (CE-MS/MS) and gas chromatography (GC)-MS/MS. Following liquid-liquid extraction, ultrafiltration and derivatization steps were needed to perform CE-MS/MS and GC-MS/MS, respectively. The CE-MS/MS method allowed for the identification and quantification of all 15 biologically important phosphorylated compounds, whereas only 13 compounds were identified and quantified by GC-MS/MS. Both methods demonstrated wide linear ranges, good interday (<9.6%: CE-MS/MS; <14.7%: GC-MS/MS) and intraday (<13.0%: CE-MS/MS; <14.9%: GC-MS/MS) variability, and limits of detection (LODs) in the ranges of 0.25-2 and 0.05-0.5 μmol/L for CE-MS/MS and GC-MS/MS, respectively. In the phosphorylated compound stability test, the instability of glyceraldehyde 3-phosphate (GA3P) and dihydroxyacetone phosphate (DHAP) was observed during freeze-thaw and long-term storage due to reversible isomerization. The results of CE-MS/MS and GC-MS/MS analysis showed that the concentrations of phosphorylated compounds determined using the two methods matched closely, while that of glycerol 3-phosphate (G3P) showed some variability in cell extracts. Therefore, while both CE-MS/MS and GC-MS/MS are suitable for analyzing metabolites resulting from the glycolysis and pentose phosphate pathways, additional validation is needed for some compounds, depending on the background matrix.

Keywords:  CE–MS/MS; Cell extract; GC–MS/MS; Human serum; Intermediates of glycolysis and pentose phosphate pathways; Method validation

DOI:  https://doi.org/10.1016/j.chroma.2021.462531
Anal Chem. 2021 Sep 17.

Chemical Proportionality within Molecular Networks.

Daniel Petras, Andrés Mauricio Caraballo-Rodríguez, Alan K Jarmusch, Carlos Molina-Santiago, Julia M Gauglitz, Emily C Gentry, Pedro Belda-Ferre, Diego Romero, Shirley M Tsunoda, Pieter C Dorrestein, Mingxun Wang.

Molecular networking of non-targeted tandem mass spectrometry data connects structurally related molecules based on similar fragmentation spectra. Here, we report the Chemical Proportionality (ChemProp) contextualization of molecular networks. ChemProp scores the changes of abundance between two connected nodes over sequential data series (e.g., temporal or spatial relationships), which can be displayed as a direction within the network to prioritize potential biological and chemical transformations or proportional changes of (biosynthetically) related compounds. We tested the ChemProp workflow on a ground truth data set of a defined mixture and highlighted the utility of the tool to prioritize specific molecules within biological samples, including bacterial transformations of bile acids, human drug metabolism, and bacterial natural products biosynthesis. The ChemProp workflow is freely available through the Global Natural Products Social Molecular Networking (GNPS) environment.

DOI: https://doi.org/10.1021/acs.analchem.1c01520
Chem Phys Lipids. 2021 Sep 09. pii: S0009-3084(21)00077-3. [Epub ahead of print] 105124

Monitoring the Cellular Metabolism of a Membrane-Permeant Photo-Caged Phosphatidylinositol 3,4,5-Trisphosphate Derivative.

Mevlut Citir, Rainer Müller, Sebastian Hauke, Alexis Traynor-Kaplan, Carsten Schultz.

  To deliver charged lipid derivatives to the cell interior, bioactivatable and photo-activatable protecting groups are frequently used. The intracellular metabolism of the protecting groups, as well as the lipid itself, are key factors that determine biological activity. Here, we followed the cellular metabolism of cell-permeant photo-activatable ("caged") and non-caged membrane-permeant analogs of dioctanoyl phosphatidylinositol 3,4,5-trisphosphate (diC8PIP3), carrying biodegradable protecting groups, by mass spectrometry. After successful cell entry, the photo-activatable group can be removed on demand by a light pulse. Hence, UV irradiation acts as a switch to expose the cellular metabolism to a bolus of active compound. To investigate lipid metabolites and to capture a more complete metabolome, we adapted standard extraction methods and employed multi-reaction monitoring mass spectrometry (MRM-MS). This required a previously developed permethylation method that stabilized metabolites and enhanced volatility of the phosphoinositide metabolites. The mass spectrometric analysis allowed for the monitoring of the intracellular removal of photo-activatable caging as well as biodegradable protecting groups from the membrane-permeant phosphoinositides along with cellular turnover, namely by dephosphorylation. We found that phosphate masking groups, namely acetoxymethyl esters, were rapidly removed by endogenous enzymes while butyrates masking hydroxy groups showed a longer lifetime, giving rise to trapped intermediates. We further identified key intermediate metabolites and demonstrated the beneficial effect of caging groups and their removal on the formation of favorable metabolites. Surprisingly, caging and protecting groups were found to influence each other's stability.

Keywords:  Phosphoinositides; mass spectrometry; multi-reaction monitoring; photoactivatable groups; protecting groups

DOI:  https://doi.org/10.1016/j.chemphyslip.2021.105124
Biomed Chromatogr. 2021 Sep 13. e5243

Pharmacokinetic and metabolic analysis of an Alzheimer's disease therapeutic in rat serum via microfluidic CZE-MS.

Z D Kelley, M A Lovell, B C Lynn.

  Sensitive, high-throughput methods for pharmacokinetic (PK) profiling are essential for potential therapeutics during critical stages of clinical trials. The application of a microfluidic capillary zone electrophoresis mass spectrometry (CZE-MS) method for PK profiling allows for rapid, sensitive, and in-depth analysis of multiple samples within a short timeframe. Here, a CZE-MS approach for PK analysis was compared to a traditional UHPLC-MS approach when analyzing serum extracts from rats treated with a potential Alzheimer's disease (AD) therapeutic, BNC-1. Resulting PK data generated from both methods displayed statistical similarity. Additionally, the separation efficiency attributed to the use of the CZE-MS method provided substantial metabolic regulation data that was not apparent in the UHPLC-MS method. Additionally, the coupling of the CZE-MS method to the data processing software, MZmine2, was used to monitor changes in metabolism and observe putative BNC-1-derived metabolites. The ability for fast analyses without sacrificing sensitivity or metabolic information, suggests that this CZE-MS method is an ideal method for metabolomics-inclusive, high-throughput PK profiling.

Keywords:  Microfluidics; capillary zone electrophoresis; metabolomics; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.5243
Genes Dis. 2021 Nov;8(6): 731-745

NADPH debt drives redox bankruptcy: SLC7A11/xCT-mediated cystine uptake as a double-edged sword in cellular redox regulation.

Xiaoguang Liu, Yilei Zhang, Li Zhuang, Kellen Olszewski, Boyi Gan.

  Cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11; also known as xCT) plays a key role in antioxidant defense by mediating cystine uptake, promoting glutathione synthesis, and maintaining cell survival under oxidative stress conditions. Recent studies showed that, to prevent toxic buildup of highly insoluble cystine inside cells, cancer cells with high expression of SLC7A11 (SLC7A11high) are forced to quickly reduce cystine to more soluble cysteine, which requires substantial NADPH supply from the glucose-pentose phosphate pathway (PPP) route, thereby inducing glucose- and PPP-dependency in SLC7A11high cancer cells. Limiting glucose supply to SLC7A11high cancer cells results in significant NADPH "debt", redox "bankruptcy", and subsequent cell death. This review summarizes our current understanding of NADPH-generating and -consuming pathways, discusses the opposing role of SLC7A11 in protecting cells from oxidative stress-induced cell death such as ferroptosis but promoting glucose starvation-induced cell death, and proposes the concept that SLC7A11-mediated cystine uptake acts as a double-edged sword in cellular redox regulation. A detailed understanding of SLC7A11 in redox biology may identify metabolic vulnerabilities in SLC7A11high cancer for therapeutic targeting.

Keywords:  Cysteine; Cystine; NADPH; Pentose phosphate pathway; SLC7A11; xCT

DOI:  https://doi.org/10.1016/j.gendis.2020.11.010
Talanta. 2021 Dec 01. pii: S0039-9140(21)00650-0. [Epub ahead of print]235 122729

Diagnostic approach to thyroid cancer based on amino acid metabolomics in saliva by ultra-performance liquid chromatography with high resolution mass spectrometry.

Jing Zhang, Xinxin Wen, Yuting Li, Jing Zhang, Xian Li, Cheng Qian, Yonghui Tian, Rui Ling, Yixiang Duan.

  Thyroid cancer is a malignant disease with dramatically low advanced-stage 10-year survival. Meanwhile, the metabolites in saliva are becoming a wealthy source of disease biomarkers. However, there is a lack of non-invasive analytical methods for the identification of biomarkers in saliva for the preoperative diagnosis of thyroid cancer. Therefore, we developed an ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) method to simultaneously determine the metabolic levels of 10 amino acids in saliva, aiming to study the amino acid metabolism profile to promote early diagnosis of thyroid cancer. We tested unstimulated whole saliva from patients with papillary thyroid carcinoma (PTC; n = 61) and healthy controls (HC; n = 61), and used receiver operating characteristic (ROC) curves to establish the diagnostic value of potential markers. The method validation results showed good precision, linearity (R2 > 0.99), recovery (92.2 %-110.3 %), intra- and inter-day precision (RSD < 7 % and RSD < 9 %, respectively). The concentration of 10 amino acids was significantly different between PTC and HC in human salivary analysis (P < 0.05), the area under the curve (AUC) values of a single marker for the diagnosis of PTC were ranging from 0.678 to 0.833. A panel of alanine, valine, proline, phenylalanine was selected in combination yielded the AUC of 0.936, which will improve the accuracy of early diagnosis of thyroid cancer (sensitivity: 91.2 %; specificity: 85.2 %). This study proved the possibility of salivary amino acid biomarkers for PTC early diagnosis, providing a simple auxiliary way for the non-invasive diagnosis of thyroid cancer.

Keywords:  Amino acid; Diagnostic; Metabolomics; Saliva; Thyroid cancer

DOI:  https://doi.org/10.1016/j.talanta.2021.122729
Talanta. 2021 Dec 01. pii: S0039-9140(21)00733-5. [Epub ahead of print]235 122812

Classification and biomarker identification of prostate tissue from TRAMP mice with hyperpolarized 13C-SIRA.

Anne B Frahm, Deborah Hill, Sotirios Katsikis, Trygve Andreassen, Jan Henrik Ardenkjær-Larsen, Tone Frost Bathen, Siver Andreas Moestue, Pernille Rose Jensen, Mathilde Hauge Lerche.

  Hyperpolarized 13C isotope resolved spectroscopy boosts NMR signal intensity, which improves signal detection and allows metabolic fluxes to be analyzed. Such hyperpolarized flux data may offer new approaches to tissue classification and biomarker identification that could be translated in vivo. Here we used hyperpolarized stable isotope resolved analysis (SIRA) to measure metabolite specific 13C isotopic enrichments in the central carbon metabolism of mouse prostate. Prostate and tumor tissue samples were acquired from transgenic adenocarcinomas of the mouse prostate (TRAMP) mice. Before euthanasia, mice were injected with [U-13C]glucose intraperitoneally (i.p.). Polar metabolite extracts were prepared, and hyperpolarized 1D-13C NMR spectra were obtained from normal prostate (n = 19) and cancer tissue (n = 19) samples. Binary classification and feature analysis was performed to make a separation model and to investigate differences between samples originating from normal and cancerous prostate tissue, respectively. Hyperpolarized experiments were carried out according to a standardized protocol, which showed a high repeatability (CV = 15%) and an average linewidth in the 1D-13C NMR spectra of 2 ± 0.5 Hz. The resolution of the hyperpolarized 1D-13C spectra was high with little signal overlap in the carbonyl region and metabolite identification was easily accomplished. A discrimination with 95% success rate could be made between samples originating from TRAMP mice prostate and tumor tissue based on isotopomers from uniquely identified metabolites. Hyperpolarized 13C-SIRA allowed detailed metabolic information to be obtained from tissue specimens. The positional information of 13C isotopic enrichments lead to easily interpreted features responsible for high predictive classification of tissue types. This analytical approach has matured, and the robust experimental protocols currently available allow systematic tracking of metabolite flux ex vivo.

Keywords:  Dissolution dynamic nuclear polarization; Isotopic fingerprinting; Prostate cancer; Random forest; Stable isotope resolved analysis; Support vector machines

DOI:  https://doi.org/10.1016/j.talanta.2021.122812
Anal Chem. 2021 Sep 15.

Discovery of Potential Lipid Biomarkers for Human Colorectal Cancer by In-Capillary Extraction Nanoelectrospray Ionization Mass Spectrometry.

Jiewei Deng, Yunyun Yang, Zhaolei Zeng, Xue Xiao, Jiajie Li, Tiangang Luan.

Discovering cancer biomarkers is of significance for clinical medicine and disease diagnosis. In this article, we develop an in-capillary extraction nanoelectrospray ionization mass spectrometry (ICE-nanoESI-MS) method to rapidly and in situ investigate human colorectal cancer for discovering lipid biomarkers. The ICE-nanoESI-MS method is performed using a tungsten microdissecting probe for in situ microsampling of surgical human colorectal cancer tumors and their paired distal noncancerous tissues during/after surgery. After sampling, the tungsten probe and the adhered tissues are inserted into a nanospray tip prefilled with some solvent for simultaneous in-capillary extraction and nanoESI-MS detection under ambient and open-air conditions. Online coupling of the Paternò-Büchi reaction and radical-direct fragmentation with ICE-nanoESI-MS is easily realized, which provides the opportunity to precisely determine carbon-carbon double bond (C═C) locations and stereospecific numbering (sn) positions of lipid biomarkers. Subsequently, a total of 12 pairs of colorectal cancer tumors and distal noncancerous tissues from different patients are investigated by our proposed ICE-nanoESI-MS method. A significant increase in lysophospholipids and fatty acids as well as a significant decrease in ceramides are discovered, and lysophospholipids are found as the potential biomarkers related to the formation and pathogenesis of human colorectal cancer.

DOI: https://doi.org/10.1021/acs.analchem.1c03249
J Mass Spectrom. 2021 Oct;56(10): e4781

Targeted fatty acid metabolomics to discover Parkinson's disease associated metabolic alteration.

Junjie Zhang, Lulu Liu, Lijiang Zhang, Simei Chen, Yusen Chen, Chun Cai.

  The pathogenesis of Parkinson's disease (PD) remains to be elucidated, and the metabolomics analysis has the potential to identify metabolic profiles that are involved in PD pathogenesis. Here we applied a target metabolomics approach to measure the plasma levels of 158 fatty acid metabolites in a discovery cohort including 42 PD patients and 54 health volunteers, and found two upregulated (arachidonic acid and 13-hydroxy-octadecatrienoic acid) and eleven down-regulated (docosahexaenoic acid, lyso-platelet-activating factor, 12-hydroxy-eicosatetraenoic acid, dihydroxy-eicosatrienoic acids, dihidroxy-octadecenoic acids, 17,18-dihydroxy-eicosatetraenoic acid, and hydroperoxy-octadecadienoic acids) metabolites as primary candidate marker of PD. A support vector machine algorithm with primary candidate marker was used in an independent validation cohort to identify PD. Arachidonic acid and 13-hydroxy-octadecatrienoic acid were evaluated as an effective tool in that area under the receiver operating characteristic curve reached 0.995 and 0.912 in the validation set for diagnosing PD from healthy volunteers. Besides, the sensitivity and specificity of arachidonic acid as diagnostic factor of PD in validation set were 100% and 94.10%. Similarly, the sensitivity and specificity of 13-hydroxy-octadecatrienoic acid were 100% and 82.40% for identifying PD. This target fatty acid metabolomics demonstrated a series of plasma fatty acid metabolite as PD candidate marker with high efficiency and provided insights into the understanding of PD metabolic regulation.

Keywords:  13-hydroxy-octadecatrienoic acid; Parkinson's disease; arachidonic acid; machine learning; metabolome

DOI:  https://doi.org/10.1002/jms.4781
Biomed Chromatogr. 2021 Sep 13. e5242

Bifunctionalized isotopologues as calibrants for standard-free quantitation of drug metabolites in plasma by liquid chromatography coupled with radiometry and mass spectrometry.

Yong Gong, Jie Chen, Heng-Keang Lim, Naidong Weng, Rhys Salter.

  The following method describes a novel workflow that eliminates the need of authentic reference standards for the quantitation of drug metabolites in biological samples using a single multi-isotopically labeled compound bearing both radio and stable isotopes. The resulting radio and stable bifunctionalized isotopologue (RADSTIL) of parent drug is employed as a substrate for in vitro biotransformation to targeted RADSTILs of metabolites as calibrants. Inclusion of a radio label enables both radiometric and mass spectrometric detection. Addition of stable labels ensures subsequent isotopic interference-free quantitation of unlabeled metabolites in preclinical and clinical samples. This affords a more accurate quantitation workflow compared to current semi-quantitation method, which utilizes isotopic interfering radio isotopologues of metabolites alone as calibrants. The proof-of-concept is illustrated with (14 C,13 C2 )-acetaminophen where in vitro biotransformation produced (14 C,13 C2 )-sulfate and (14 C,13 C2 )-glucuronide calibrants. Absolute quantitation of the acetaminophen metabolites was then achieved by liquid chromatography coupled with radiometry and mass spectrometry (LC-RAD/MS). Quantitative data obtained by this method fell within 82-86% of the values from conventional LC-MS/MS method.

Keywords:  LC-MS/MS; biotransformation; calibrant; isotopologue; metabolite quantitation

DOI:  https://doi.org/10.1002/bmc.5242
J Hazard Mater. 2021 Sep 09. pii: S0304-3894(21)02097-5. [Epub ahead of print]423(Pt B): 127129

Exposure to ambient fine particulate matter impedes the function of spleen in the mouse metabolism of high-fat diet.

Zhao He, Hongna Zhang, Yuanyuan Song, Zhu Yang, Zongwei Cai.

  Epidemiological and experimental evidence has been associating the exposure with ambient fine particulate matter (PM2.5) with metabolic malfunctions such as obesity and cardiovascular disease. As the blood-filter and the important lymphatic organ, spleen participates in the regulation of metabolic balance. In this work, liquid chromatography-mass spectrometry (LC-MS)-based lipidomics, metabolomics and proteomics were performed to study the effects of PM2.5 exposure and high-fat diet (HFD) induced obesity on mice spleen. By comparing the differences in lipids, metabolites, and proteins in the spleens from PM2.5 and HFD treated mice, we discovered the individual and combined effects of the two risk factors. The results showed the PM2.5 exposure altered energy metabolism of the mice, as evidenced by the upregulation of TCA cycle. In addition, the metabolism of branched-chain amino acids was also significantly changed, which might be related to the preventive function of spleen in lipid metabolism. The PM2.5-induced metabolic changes in spleen could further aggravate the adverse impacts of HFD on mice, resulting in impeded splenic metabolism of lipids. This study revealed the effects of PM2.5 and obesity mice spleen, which might be of great significance to public health.

Keywords:  HFD; LC-MS; Lipidomics; Metabolomics; Mouse spleen; PM(2.5); Proteomics

DOI:  https://doi.org/10.1016/j.jhazmat.2021.127129
Front Oncol. 2021 ;11 719922

Regulation of Extracellular Matrix Production in Activated Fibroblasts: Roles of Amino Acid Metabolism in Collagen Synthesis.

Emily J Kay, Grigorios Koulouras, Sara Zanivan.

  Cancer associated fibroblasts (CAFs) are a major component of the tumour microenvironment in most tumours, and are key mediators of the response to tissue damage caused by tumour growth and invasion, contributing to the observation that tumours behave as 'wounds that do not heal'. CAFs have been shown to play a supporting role in all stages of tumour progression, and this is dependent on the highly secretory phenotype CAFs develop upon activation, of which extracellular matrix (ECM) production is a key element. A collagen rich, stromal ECM has been shown to influence tumour growth and metastasis, exclude immune cells and impede drug delivery, and is associated with poor prognosis in many cancers. CAFs also extensively remodel their metabolism to support cancer cells, however, it is becoming clear that metabolic rewiring also supports intrinsic functions of activated fibroblasts, such as increased ECM production. In this review, we summarise how fibroblasts metabolically regulate ECM production, focussing on collagen production, at the transcriptional, translational and post-translational level, and discuss how this can provide possible strategies for effectively targeting CAF activation and formation of a tumour-promoting stroma.

Keywords:  CAF; amino acids; extracellular matrix; fibroblasts; metabolism; tumour microenvironment

DOI:  https://doi.org/10.3389/fonc.2021.719922
Anal Chem. 2021 Sep 14.

Combined Metabolic and Chemical (CoMetChem) Labeling Using Stable Isotopes-a Strategy to Reveal Site-Specific Histone Acetylation and Deacetylation Rates by LC-MS.

Alienke van Pijkeren, Jörn Dietze, Alejandro Sánchez Brotons, Anna-Sophia Egger, Tim Lijster, Andrei Barcaru, Madlen Hotze, Philipp Kobler, Frank J Dekker, Peter Horvatovich, Barbro N Melgert, Mathias Ziegler, Kathrin Thedieck, Ines Heiland, Rainer Bischoff, Marcel Kwiatkowski.

Histone acetylation is an important, reversible post-translational protein modification and a hallmark of epigenetic regulation. However, little is known about the dynamics of this process, due to the lack of analytical methods that can capture site-specific acetylation and deacetylation reactions. We present a new approach that combines metabolic and chemical labeling (CoMetChem) using uniformly 13C-labeled glucose and stable isotope-labeled acetic anhydride. Thereby, chemically equivalent, fully acetylated histone species are generated, enabling accurate relative quantification of site-specific lysine acetylation dynamics in tryptic peptides using high-resolution mass spectrometry. We show that CoMetChem enables site-specific quantification of the incorporation or loss of lysine acetylation over time, allowing the determination of reaction rates for acetylation and deacetylation. Thus, the CoMetChem methodology provides a comprehensive description of site-specific acetylation dynamics.

DOI: https://doi.org/10.1021/acs.analchem.1c01359
Talanta. 2021 Dec 01. pii: S0039-9140(21)00661-5. [Epub ahead of print]235 122740

In silico prediction, LC-HRMS/MS analysis, and targeted/untargeted data-mining workflow for the profiling of phenylfentanyl in vitro metabolites.

Annagiulia Di Trana, Pietro Brunetti, Raffaele Giorgetti, Enrico Marinelli, Simona Zaami, Francesco Paolo Busardò, Jeremy Carlier.

  Illicit fentanyl and analogues have been involved in many fatalities and cases of intoxication across the United States over the last decade, and are becoming a health concern in Europe. New potent analogues emerge onto the drug market every year to circumvent analytical detection and legislation, and little pharmacological/toxicological data are available when the substances first appear. However, pharmacokinetic data are crucial to determine specific biomarkers of consumption in clinical and forensic settings, considering the low active doses and the rapid metabolism of fentanyl analogues. Phenylfentanyl is a novel analogue that was first detected in seized material in 2017, and little is currently known about this substance and its metabolism. We studied phenylfentanyl metabolic fate using in silico predictions with GLORYx freeware, human hepatocyte incubations, and liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS). We applied a specific targeted/untargeted workflow using data-mining software to allow the rapid and partially automated screening of LC-HRMS/MS raw data. Approximately 90,000 substances were initially individuated after 3-h incubation with hepatocytes, and 115 substances were automatically selected for a manual check by the operators. Finally, 13 metabolites, mostly produced by N-dealkylation, amide hydrolysis, oxidation, and combinations thereof, were identified. We suggest phenylnorfentanyl as the main biological marker of phenylfentanyl use, and we proposed the inclusion of its fragmentation pattern in mzCloud and HighResNPS online libraries. Other major metabolites include N-Phenyl-1-(2-phenylethyl)-4-piperidinamine (4-ANPP), 1-(2-phenylethyl)-4-piperidinol, and other non-specific metabolites. Phase II transformations were infrequent, and the hydrolysis of the biological samples is not required to increase the detection capability of non-conjugated metabolites. The overall workflow is easily adaptable for the metabolite profiling of other novel psychoactive substances.

Keywords:  Data-dependent analysis; Hepatocyte metabolism; In silico prediction; Liquid chromatography-High-resolution tandem mass spectrometry; Phenylfentanyl; Software-assisted data mining

DOI:  https://doi.org/10.1016/j.talanta.2021.122740
Redox Biol. 2021 Sep 08. pii: S2213-2317(21)00286-X. [Epub ahead of print]46 102127

Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

Anita Ayer, Daniel J Fazakerley, Cacang Suarna, Ghassan J Maghzal, Diba Sheipouri, Kevin J Lee, Michelle C Bradley, Lucía Fernández-Del-Rio, Sergey Tumanov, Stephanie My Kong, Jelske N van der Veen, Andrian Yang, Joshua W K Ho, Steven G Clarke, David E James, Ian W Dawes, Dennis E Vance, Catherine F Clarke, René L Jacobs, Roland Stocker.

  Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.

Keywords:  Coenzyme Q; Insulin resistance; Mitochondria; PEMT; Reactive oxygen species; S-adenosylhomocysteine; S-adenosylmethionine

DOI:  https://doi.org/10.1016/j.redox.2021.102127
J Vis Exp. 2021 Aug 28.

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue.

Saicharan Ghantasala, Medha Gayathri J Pai, Sanjeeva Srivastava.

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

DOI: https://doi.org/10.3791/61833