bims-mascan 2022-08-07 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2022‒08‒07
twenty-six papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh

Segment Scan Mass Spectral Acquisition for Increasing the Metabolite Detectability in Chemical Isotope Labeling Liquid Chromatography-Mass Spectrometry Metabolome Analysis.
Serine metabolism remodeling after platinum-based chemotherapy identifies vulnerabilities in a subgroup of resistant ovarian cancers.
Optimization of Ultrafast Proteomics Using an LC-Quadrupole-Orbitrap Mass Spectrometer with Data-Independent Acquisition.
Alterations of Plasma Lipids in Adult Women With Major Depressive Disorder and Bipolar Depression.
Emerging metabolomic tools to study cancer metastasis.
Spectral binning as an approach to post-acquisition processing of high resolution FIE-MS metabolome fingerprinting data.
High-throughput proteomic sample preparation using pressure cycling technology.
13C metabolic flux analysis clarifies distinct metabolic phenotypes of cancer cell spheroid mimicking tumor hypoxia.
Single-Cell Metabolomics in Hematopoiesis and Hematological Malignancies.
Enhancing the Signal-to-Noise of Diagnostic Fragment Ions of Unsaturated Glycerophospholipids via Precursor Exclusion Ultraviolet Photodissociation Mass Spectrometry (PEx-UVPD-MS).
Sample Preparation for Rapid Lipid Analysis in Drosophila Brain using Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Imaging.
Targeting of the Lipid Metabolism Impairs Resistance to BRAF Kinase Inhibitor in Melanoma.
Systematic Investigation of LC Miniaturization to Increase Sensitivity in Wide-Target LC-MS-Based Trace Bioanalysis of Small Molecules.
Intracellular Delivery of Glutathione Peroxidase Degrader Induces Ferroptosis In Vivo.
Lipid Mass Tags via Aziridination for Probing Unsaturated Lipid Isomers and Accurate Relative Quantification.
Quaterization Derivatization with Bis(Pyridine) Iodine Tetrafluoroboride: High-Sensitivity Mass Spectrometric Analysis of Unsaturated Fatty Acids in Human Thyroid Tissues.
Nucleotide imbalance decouples cell growth from cell proliferation.
In-Depth Mass Spectrometry-Based Proteomics of Formalin-Fixed, Paraffin-Embedded Tissues with a Spatial Resolution of 50-200 μm.
Argininosuccinate synthase 1, arginine deprivation therapy and cancer management.
A non-canonical vitamin K cycle is a potent ferroptosis suppressor.
Targeting acetyl-CoA metabolism attenuates the formation of fear memories through reduced activity-dependent histone acetylation.
Aberrant iron distribution via hepatocyte-stellate cell axis drives liver lipogenesis and fibrosis.
Association of amino acid metabolites with osteoporosis, a metabolomic approach: Bushehr elderly health program.
High-throughput proteomics: a methodological mini-review.
Improved Ion Mobility Separation and Structural Characterization of Steroids using Derivatization Methods.
mTORC2 facilitates liver regeneration through the sphingolipid-induced PPAR-α-fatty acid oxidation.

Anal Chem. 2022 Aug 04.

Segment Scan Mass Spectral Acquisition for Increasing the Metabolite Detectability in Chemical Isotope Labeling Liquid Chromatography-Mass Spectrometry Metabolome Analysis.

Chu-Fan Wang, Liang Li.

We report a segmented spectrum scan method using Orbitrap MS in chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS) for improving the metabolite detection efficiency. In this method, the full m/z range is divided into multiple segments with the scanning of each segment to produce multiple narrow-range spectra during the LC data acquisition. These segmented spectra are separately processed to extract the peak pair information with each peak pair arising from a differentially labeled metabolite in the analysis of a mixture of 13C and 12C reagent-labeled samples. The sublists of peak pairs are merged to form the final peak pair list from the LC-MS run. Various experimental conditions, including automatic gain control (AGC) values, mass resolutions, segment m/z widths, number of segments, and total data acquisition time in the LC run, were examined to arrive at an optimal setting in the segment scan for increasing the number of detectable metabolites while maintaining the same analysis time as in the full scan. The optimal method used a segment width of 120 m/z with 60k resolution for a 16 min CIL LC-MS run. Using dansyl-labeled human urine samples as an example, we demonstrated that this method could detect 5867 peak pairs or metabolites (not features), compared to 3765 peak pairs detectable in a full scan, representing a 56% gain. Out of 5867 peak pairs, 5575 (95.0%) could be identified or mass-matched. The relative quantification accuracy was slightly reduced (81% peak pairs were within ±25% of the expected peak ratio of 1.0 in full, compared to 87% in the full scan) due to the inclusion of more low-abundance peak pairs in the segment scan. The peak ratio measurement precision was not significantly affected by the segment scan. We also showed the increase of the peak pair number detectable from 3843 in the full scan to 7273 (89% gain) using the Orbitrap operated at 120k resolution with a 60 m/z segment width when multiple repeat sample injections were used. Thus, segment scan Orbitrap MS is an enabling method for detecting coeluting metabolites in CIL LC-MS for increasing the metabolomic coverage.

DOI: https://doi.org/10.1021/acs.analchem.2c02220
Nat Commun. 2022 Aug 05. 13(1): 4578

Serine metabolism remodeling after platinum-based chemotherapy identifies vulnerabilities in a subgroup of resistant ovarian cancers.

Tom Van Nyen, Mélanie Planque, Lilian van Wagensveld, Joao A G Duarte, Esther A Zaal, Ali Talebi, Matteo Rossi, Pierre-René Körner, Lara Rizzotto, Stijn Moens, Wout De Wispelaere, Regina E M Baiden-Amissah, Gabe S Sonke, Hugo M Horlings, Guy Eelen, Emanuele Berardi, Johannes V Swinnen, Celia R Berkers, Peter Carmeliet, Diether Lambrechts, Ben Davidson, Reuven Agami, Sarah-Maria Fendt, Daniela Annibali, Frédéric Amant.

Resistance to platinum-based chemotherapy represents a major clinical challenge for many tumors, including epithelial ovarian cancer. Patients often experience several response-relapse events, until tumors become resistant and life expectancy drops to 12-15 months. Despite improved knowledge of the molecular determinants of platinum resistance, the lack of clinical applicability limits exploitation of many potential targets, leaving patients with limited options. Serine biosynthesis has been linked to cancer growth and poor prognosis in various cancer types, however its role in platinum-resistant ovarian cancer is not known. Here, we show that a subgroup of resistant tumors decreases phosphoglycerate dehydrogenase (PHGDH) expression at relapse after platinum-based chemotherapy. Mechanistically, we observe that this phenomenon is accompanied by a specific oxidized nicotinamide adenine dinucleotide (NAD+) regenerating phenotype, which helps tumor cells in sustaining Poly (ADP-ribose) polymerase (PARP) activity under platinum treatment. Our findings reveal metabolic vulnerabilities with clinical implications for a subset of platinum resistant ovarian cancers.

DOI: https://doi.org/10.1038/s41467-022-32272-6
J Proteome Res. 2022 Aug 01.

Optimization of Ultrafast Proteomics Using an LC-Quadrupole-Orbitrap Mass Spectrometer with Data-Independent Acquisition.

Masaki Ishikawa, Ryo Konno, Daisuke Nakajima, Mari Gotoh, Keiko Fukasawa, Hironori Sato, Ren Nakamura, Osamu Ohara, Yusuke Kawashima.

  Proteomics has become an increasingly important tool in medical and medicinal applications. It is necessary to improve the analytical throughput for these applications, particularly in large-scale drug screening to enable measurement of a large number of samples. In this study, we aimed to establish an ultrafast proteomic method based on 5-min gradient LC and quadrupole-Orbitrap mass spectrometer (Q-Orbitrap MS). We precisely optimized data-independent acquisition (DIA) parameters for 5-min gradient LC and reached a depth of >5000 and 4200 proteins from 1000 and 31.25 ng of HEK293T cell digest in a single-shot run, respectively. The throughput of our method enabled the measurement of approximately 80 samples/day, including sample loading, column equilibration, and wash running time. We demonstrated that our method is applicable for the screening of chemical responsivity via a cell stimulation assay. These data show that our method enables the capture of biological alterations in proteomic profiles with high sensitivity, suggesting the possibility of large-scale screening of chemical responsivity.

Keywords:  HT proteomics; Q-Orbitrap MS; Short LC gradients; data-independent acquisition

DOI:  https://doi.org/10.1021/acs.jproteome.2c00121
Front Psychiatry. 2022 ;13 927817

Alterations of Plasma Lipids in Adult Women With Major Depressive Disorder and Bipolar Depression.

Ting Zhang, Lin Guo, Rui Li, Fei Wang, Wen-Mao Yang, Jia-Bin Yang, Zhi-Quan Cui, Cui-Hong Zhou, Yi-Huan Chen, Huan Yu, Zheng-Wu Peng, Qing-Rong Tan.

  Lipidomics has been established as a potential tool for the investigation of mental diseases. However, the composition analysis and the comparison of the peripheral lipids regarding adult women with major depressive depression (MDD) or bipolar depression (BPD) has been poorly addressed. In the present study, age-matched female individuals with MDD (n = 28), BPD (n = 22) and healthy controls (HC, n = 25) were enrolled. Clinical symptoms were assessed and the plasma samples were analyzed by comprehensive lipid profiling based on liquid chromatography-mass spectrometry (LC/MS). We found that the composition of lipids was remarkably changed in the patients with MDD and BPD when compared to HC or compared to each other. Moreover, we identified diagnostic potential biomarkers comprising 20 lipids that can distinguish MDD from HC (area under the curve, AUC = 0.897) and 8 lipids that can distinguish BPD from HC (AUC = 0.784), as well as 13 lipids were identified to distinguish MDD from BPD with moderate reliability (AUC = 0.860). This study provides further understanding of abnormal lipid metabolism in adult women with MDD and BPD and may develop lipid classifiers able to effectively discriminate MDD from BPD and HC.

Keywords:  bipolar depression; depression; lipidomics; plasma lipid; women

DOI:  https://doi.org/10.3389/fpsyt.2022.927817
Trends Cancer. 2022 Jul 28. pii: S2405-8033(22)00156-X. [Epub ahead of print]

Emerging metabolomic tools to study cancer metastasis.

Luiza Martins Nascentes Melo, Nicholas P Lesner, Marie Sabatier, Jessalyn M Ubellacker, Alpaslan Tasdogan.

  Metastasis is responsible for 90% of deaths in patients with cancer. Understanding the role of metabolism during metastasis has been limited by the development of robust and sensitive technologies that capture metabolic processes in metastasizing cancer cells. We discuss the current technologies available to study (i) metabolism in primary and metastatic cancer cells and (ii) metabolic interactions between cancer cells and the tumor microenvironment (TME) at different stages of the metastatic cascade. We identify advantages and disadvantages of each method and discuss how these tools and technologies will further improve our understanding of metastasis. Studies investigating the complex metabolic rewiring of different cells using state-of-the-art metabolomic technologies have the potential to reveal novel biological processes and therapeutic interventions for human cancers.

Keywords:  analytical techniques; cancer metabolism; cancer metastasis; metabolomics

DOI:  https://doi.org/10.1016/j.trecan.2022.07.003
Metabolomics. 2022 Aug 02. 18(8): 64

Spectral binning as an approach to post-acquisition processing of high resolution FIE-MS metabolome fingerprinting data.

Jasen P Finch, Thomas Wilson, Laura Lyons, Helen Phillips, Manfred Beckmann, John Draper.

  INTRODUCTION: Flow infusion electrospray high resolution mass spectrometry (FIE-HRMS) fingerprinting produces complex, high dimensional data sets which require specialist in-silico software tools to process the data prior to analysis.OBJECTIVES: Present spectral binning as a pragmatic approach to post-acquisition procession of FIE-HRMS metabolome fingerprinting data.
METHODS: A spectral binning approach was developed that included the elimination of single scan m/z events, the binning of spectra and the averaging of spectra across the infusion profile. The modal accurate m/z was then extracted for each bin. This approach was assessed using four different biological matrices and a mix of 31 known chemical standards analysed by FIE-HRMS using an Exactive Orbitrap. Bin purity and centrality metrics were developed to objectively assess the distribution and position of accurate m/z within an individual bin respectively.
RESULTS: The optimal spectral binning width was found to be 0.01 amu. 80.8% of the extracted accurate m/z matched to predicted ionisation products of the chemical standards mix were found to have an error of below 3 ppm. The open-source R package binneR was developed as a user friendly implementation of the approach. This was able to process 100 data files using 4 Central Processing Units (CPU) workers in only 55 seconds with a maximum memory usage of 1.36 GB.
CONCLUSION: Spectral binning is a fast and robust method for the post-acquisition processing of FIE-HRMS data. The open-source R package binneR allows users to efficiently process data from FIE-HRMS experiments with the resources available on a standard desktop computer.

Keywords:  Mass spectrometry; Metabolomic fingerprinting; Post-acquisition processing; Software

DOI:  https://doi.org/10.1007/s11306-022-01923-6
Nat Protoc. 2022 Aug 05.

High-throughput proteomic sample preparation using pressure cycling technology.

Xue Cai, Zhangzhi Xue, Chunlong Wu, Rui Sun, Liujia Qian, Liang Yue, Weigang Ge, Xiao Yi, Wei Liu, Chen Chen, Huanhuan Gao, Jing Yu, Luang Xu, Yi Zhu, Tiannan Guo.

High-throughput lysis and proteolytic digestion of biopsy-level tissue specimens is a major bottleneck for clinical proteomics. Here we describe a detailed protocol of pressure cycling technology (PCT)-assisted sample preparation for proteomic analysis of biopsy tissues. A piece of fresh frozen or formalin-fixed paraffin-embedded tissue weighing ~0.1-2 mg is placed in a 150 μL pressure-resistant tube called a PCT-MicroTube with proper lysis buffer. After closing with a PCT-MicroPestle, a batch of 16 PCT-MicroTubes are placed in a Barocycler, which imposes oscillating pressure to the samples from one atmosphere to up to ~3,000 times atmospheric pressure. The pressure cycling schemes are optimized for tissue lysis and protein digestion, and can be programmed in the Barocycler to allow reproducible, robust and efficient protein extraction and proteolysis digestion for mass spectrometry-based proteomics. This method allows effective preparation of not only fresh frozen and formalin-fixed paraffin-embedded tissue, but also cells, feces and tear strips. It takes ~3 h to process 16 samples in one batch. The resulting peptides can be analyzed by various mass spectrometry-based proteomics methods. We demonstrate the applications of this protocol with mouse kidney tissue and eight types of human tumors.

DOI: https://doi.org/10.1038/s41596-022-00727-1
Metab Eng. 2022 Jul 31. pii: S1096-7176(22)00095-7. [Epub ahead of print]

13C metabolic flux analysis clarifies distinct metabolic phenotypes of cancer cell spheroid mimicking tumor hypoxia.

Shingo Noguchi, Kenichi Wakita, Fumio Matsuda, Hiroshi Shimizu.

  Cancer cells adapt their intracellular energy metabolism to the oxygen-deprived tumor microenvironment (TME) to ensure tumor progression. This adaptive mechanism has focused attention on the metabolic phenotypes of tumor cells under hypoxic TME for developing novel cancer therapies. Although widely used monolayer (2D) culture does not fully reflect in vivo hypoxic TME, spheroid (3D) culture can produce a milieu similar to the TME in vivo. However, how different metabolic phenotypes are expressed in 3D cultures mimicking tumor hypoxia compared with 2D cultures under hypoxia remains unclear. To address this issue, we investigated the metabolic phenotypes of 2D- and 3D-cultured cancer cells by 13C-metabolic flux analysis (13C-MFA). Principal component analysis of 13C mass isotopomer distributions clearly demonstrated distinct metabolic phenotypes of 3D-cultured cells. 13C-MFA clarified that 3D culture significantly upregulated pyruvate carboxylase flux in line with the pyruvate carboxylase protein expression level. On the other hand, 3D culture downregulated glutaminolytic flux. Consistent with our findings, 3D-cultured cells are more resistant to a glutaminase inhibitor than 2D-cultured cells. This study suggests the importance of considering the metabolic characteristics of the particular in vitro model used for research on cancer metabolism.

Keywords:  (13)C-metabolic flux analysis; 3D culture; Cancer metabolism; Hypoxic tumor microenvironment; Spheroid

DOI:  https://doi.org/10.1016/j.ymben.2022.07.008
Front Oncol. 2022 ;12 931393

Single-Cell Metabolomics in Hematopoiesis and Hematological Malignancies.

Fengli Zuo, Jing Yu, Xiujing He.

  Aberrant metabolism contributes to tumor initiation, progression, metastasis, and drug resistance. Metabolic dysregulation has emerged as a hallmark of several hematologic malignancies. Decoding the molecular mechanism underlying metabolic rewiring in hematological malignancies would provide promising avenues for novel therapeutic interventions. Single-cell metabolic analysis can directly offer a meaningful readout of the cellular phenotype, allowing us to comprehensively dissect cellular states and access biological information unobtainable from bulk analysis. In this review, we first highlight the unique metabolic properties of hematologic malignancies and underscore potential metabolic vulnerabilities. We then emphasize the emerging single-cell metabolomics techniques, aiming to provide a guide to interrogating metabolism at single-cell resolution. Furthermore, we summarize recent studies demonstrating the power of single-cell metabolomics to uncover the roles of metabolic rewiring in tumor biology, cellular heterogeneity, immunometabolism, and therapeutic resistance. Meanwhile, we describe a practical view of the potential applications of single-cell metabolomics in hematopoiesis and hematological malignancies. Finally, we present the challenges and perspectives of single-cell metabolomics development.

Keywords:  amino acids metabolism; glucose metabolism; hematological malignancies; hematopoiesis; lipid-related metabolism; metabolic reprogramming; single-cell metabolomics

DOI:  https://doi.org/10.3389/fonc.2022.931393
Anal Chem. 2022 Aug 02.

Enhancing the Signal-to-Noise of Diagnostic Fragment Ions of Unsaturated Glycerophospholipids via Precursor Exclusion Ultraviolet Photodissociation Mass Spectrometry (PEx-UVPD-MS).

Samuel W J Shields, James D Sanders, Jennifer S Brodbelt.

Understanding and elucidating the diverse structures and functions of lipids has motivated the development of many innovative tandem mass spectrometry (MS/MS) strategies. Higher-energy activation methods, such as ultraviolet photodissociation (UVPD), generate unique fragment ions from glycerophospholipids that can be used to perform in-depth structural analysis and facilitate the deconvolution of isomeric lipid structures in complex samples. Although detailed characterization is central to the correlation of lipid structure to biological function, it is often impeded by the lack of sufficient instrument sensitivity for highly bioactive but low-abundance phospholipids. Here, we present precursor exclusion (PEx) UVPD, a simple yet powerful technique to enhance the signal-to-noise (S/N) of informative low-abundance fragment ions produced from UVPD of glycerophospholipids. Through the exclusion of the large population of undissociated precursor ions with an MS3 strategy, the S/N of diagnostic fragment ions from PC 18:0/18:2(9Z, 12Z) increased up to an average of 13x for PEx-UVPD compared to UVPD alone. These enhancements were extended to complex mixtures of lipids from bovine liver extract to confidently identify 35 unique structures using liquid chromatography PEx-UVPD. This methodology has the potential to advance lipidomics research by offering deeper structure elucidation and confident identification of biologically active lipids.

DOI: https://doi.org/10.1021/acs.analchem.2c02128
J Vis Exp. 2022 Jul 14.

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain using Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Yuki X Chen, Kelly Veerasammy, Jun Yin, Tenzin Choetso, Tiffany Zhong, Muniyat A Choudhury, Cory Weng, Ethan Xu, Mayan A Hein, Rinat Abzalimov, Ye He.

Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired from lipidomics is valuable in studying the pathways involved in development, disease, and cellular metabolism. Many tools and instrumentations have aided lipidomics projects, most notably various combinations of mass spectrometry and liquid chromatography techniques. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has recently emerged as a powerful imaging technique that complements conventional approaches. This novel technique provides unique information on the spatial distribution of lipids within tissue compartments, which was previously unattainable without the use of excessive modifications. The sample preparation of the MALDI MSI approach is critical and, therefore, is the focus of this paper. This paper presents a rapid lipid analysis of a large number of Drosophila brains embedded in optimal cutting temperature compound (OCT) to provide a detailed protocol for the preparation of small tissues for lipid analysis or metabolite and small molecule analysis through MALDI MSI.

DOI: https://doi.org/10.3791/63930
Front Cell Dev Biol. 2022 ;10 927118

Targeting of the Lipid Metabolism Impairs Resistance to BRAF Kinase Inhibitor in Melanoma.

Elisabetta Vergani, Giovanni L Beretta, Mariachiara Aloisi, Matteo Costantino, Cristina Corno, Simona Frigerio, Stella Tinelli, Matteo Dugo, Felice Maria Accattatis, Agnese Granata, Lorenzo Arnaboldi, Monica Rodolfo, Paola Perego, Laura Gatti.

  Drug resistance limits the achievement of persistent cures for the treatment of melanoma, in spite of the efficacy of the available drugs. The aim of the present study was to explore the involvement of lipid metabolism in melanoma resistance and assess the effects of its targeting in cellular models of melanoma with acquired resistance to the BRAF-inhibitor PLX4032/Vemurafenib. Since transcriptional profiles pointed to decreased cholesterol and fatty acids synthesis in resistant cells as compared to their parental counterparts, we examined lipid composition profiles of resistant cells, studied cell growth dependence on extracellular lipids, assessed the modulation of enzymes controlling the main nodes in lipid biosynthesis, and evaluated the effects of targeting Acetyl-CoA Acetyltransferase 2 (ACAT2), the first enzyme in the cholesterol synthesis pathway, and Acyl-CoA Cholesterol Acyl Transferase (ACAT/SOAT), which catalyzes the intracellular esterification of cholesterol and the formation of cholesteryl esters. We found a different lipid composition in the resistant cells, which displayed reduced saturated fatty acids (SFA), increased monounsaturated (MUFA) and polyunsaturated (PUFA), and reduced cholesteryl esters (CE) and triglycerides (TG), along with modulated expression of enzymes regulating biosynthetic nodes of the lipid metabolism. The effect of tackling lipid metabolism pathways in resistant cells was evidenced by lipid starvation, which reduced cell growth, increased sensitivity to the BRAF-inhibitor PLX4032, and induced the expression of enzymes involved in fatty acid and cholesterol metabolism. Molecular targeting of ACAT2 or pharmacological inhibition of SOAT by avasimibe showed antiproliferative effects in melanoma cell lines and a synergistic drug interaction with PLX4032, an effect associated to increased ferroptosis. Overall, our findings reveal that lipid metabolism affects melanoma sensitivity to BRAF inhibitors and that extracellular lipid availability may influence tumor cell response to treatment, a relevant finding in the frame of personalized therapy. In addition, our results indicate new candidate targets for drug combination treatments.

Keywords:  BRAF inhibitors; avasimibe; drug resistance; lipid metabolism; melanoma

DOI:  https://doi.org/10.3389/fcell.2022.927118
Front Mol Biosci. 2022 ;9 857505

Systematic Investigation of LC Miniaturization to Increase Sensitivity in Wide-Target LC-MS-Based Trace Bioanalysis of Small Molecules.

Veronika Fitz, Yasin El Abiead, Daniel Berger, Gunda Koellensperger.

  Covering a wide spectrum of molecules is essential for global metabolome assessment. While metabolomics assays are most frequently carried out in microbore LC-MS analysis, reducing the size of the analytical platform has proven its ability to boost sensitivity for specific -omics applications. In this study, we elaborate the impact of LC miniaturization on exploratory small-molecule LC-MS analysis, focusing on chromatographic properties with critical impact on peak picking and statistical analysis. We have assessed a panel of small molecules comprising endogenous metabolites and environmental contaminants covering three flow regimes-analytical, micro-, and nano-flow. Miniaturization to the micro-flow regime yields moderately increased sensitivity as compared to the nano setup, where median sensitivity gains around 80-fold are observed in protein-precipitated blood plasma extract. This gain resulting in higher coverage at low µg/L concentrations is compound dependent. At the same time, the nano-LC-high-resolution mass spectrometry (HRMS) approach reduces the investigated chemical space as a consequence of the trap-and-elute nano-LC platform. Finally, while all three setups show excellent retention time stabilities, rapid gradients jeopardize the peak area repeatability of the nano-LC setup. Micro-LC offers the best compromise between improving signal intensity and metabolome coverage, despite the fact that only incremental gains can be achieved. Hence, we recommend using micro-LC for wide-target small-molecule trace bioanalysis and global metabolomics of abundant samples.

Keywords:  LC-MS; chromatography; coverage; exposomics; metabolomics; miniaturization; sensitivity

DOI:  https://doi.org/10.3389/fmolb.2022.857505
Angew Chem Int Ed Engl. 2022 Aug 04.

Intracellular Delivery of Glutathione Peroxidase Degrader Induces Ferroptosis In Vivo.

Tianli Luo, Qizhen Zheng, Leihou Shao, Tianyu Ma, Lanqun Mao, Ming Wang.

  F erroptosis is a new form of regulated, non-apoptotic cell death driven by iron-dependent phospholipid peroxidation. Its therapeutic potential is however, greatly limited by the low efficiency of regulating cell ferroptosis in vivo. Herein, we report a PROTAC-based protein degrader that depletes endogenous glutathione peroxidase 4 (GPX4) and induces cancer cell ferroptosis. We demonstrate that a rationally designed GPX 4 degrader, dGPX4, can deplete tumor cell GPX4 via proteasomal protein degradation, showing a five-fold enhancement of ferroptosis induction efficiency compared to that of GPX4 inhibition using ML162. Moreover, we show that the intracellular delivery of dGPX4 using biodegradable lipid nanoparticles (dGPX4@401-TK-12) induces cell-selective ferroptosis by targeting cancer cell microenvironment. The in vivo administration of dGPX4@401-TK-12 effectively suppresses tumor growth without appreciable side effects. We anticipate the protein degradation strategy described herein could be easily expanded to other essential regulatory proteins of ferroptosis for developing targeted cancer therapeutics.

Keywords:  Ferroptosis; Lipid nanoparticles; PROTAC; drug delivery

DOI:  https://doi.org/10.1002/anie.202206277
Angew Chem Int Ed Engl. 2022 Aug 06.

Lipid Mass Tags via Aziridination for Probing Unsaturated Lipid Isomers and Accurate Relative Quantification.

Tingyuan Yang, Shuli Tang, Syuan-Ting Kuo, Dallas Freitas, Madison Edwards, Hongying Wang, Yuxiang Sun, Xin Yan.

  Knowing concentrations of lipids is essential for understanding their physiological functions and discovering new disease biomarkers. However, it is highly challenging to accurately quantify lipids due to structural diversity and multiple isomeric forms of lipids. To address these critical gaps, we develop a novel aziridine-based isobaric tag labeling strategy that allows (i) determination of lipid double-bond positional isomers, (ii) accurate relative quantification of unsaturated lipids, and (iii) improvement of ionization efficiencies of nonpolar lipids. The power of this method is demonstrated in characterization and quantification of various categories of lipids such as fatty acids, phosphoglycerol lipids, cholesteryl esters (CE), and glycerides. 17 CE lipid isomers were identified and quantified simultaneously from Alzheimer's disease (AD) mouse serum without using lipid standards. Among them, 6 CE isomers showed significant changes in concentrations in AD serum.

Keywords:  aziridination; double bond positional isomer; isobaric tag; lipid isomer; lipid quantification

DOI:  https://doi.org/10.1002/anie.202207098
Anal Chem. 2022 Aug 02.

Quaterization Derivatization with Bis(Pyridine) Iodine Tetrafluoroboride: High-Sensitivity Mass Spectrometric Analysis of Unsaturated Fatty Acids in Human Thyroid Tissues.

Jie Cheng, Yuling Li, Yunjun Wang, Jing Zhang, Tuanqi Sun, Li Zhang, Yinlong Guo.

Accurate quantification of disease-related unsaturated fatty acids (UFAs) in biomedical samples plays an important role in clinical diagnosis. Here, we reported a quaterization derivatization-stable isotope labeling strategy for accurate quantitative analysis of UFAs by high-performance liquid chromatography-mass spectrometry. [d0]/[d10]-Bis(pyridine) iodine tetrafluoroboride ([d0]/[d10]-IPy2BF4) was employed as the carbon-carbon double bond derivatization reagent with high efficiency and high specificity, to introduce a charge tag on UFAs and avoid the interference of saturated fatty acids. After labeling, the detection sensitivity was significantly enhanced by up to three orders of magnitude compared to intact UFAs. The standard curves showed good linearity (R2 > 0.999) over a wide concentration range. This strategy was successfully applied to determine the content of 12 UFAs in human thyroid carcinoma and para-carcinoma tissues. A significant difference was found in the content of several UFAs between these two kinds of tissues (p < 0.05). These results indicated that the proposed strategy may be valuable for the discovery of abnormal UFA content in early clinical diagnosis.

DOI: https://doi.org/10.1021/acs.analchem.2c01519
Nat Cell Biol. 2022 Aug 04.

Nucleotide imbalance decouples cell growth from cell proliferation.

Frances F Diehl, Teemu P Miettinen, Ryan Elbashir, Christopher S Nabel, Alicia M Darnell, Brian T Do, Scott R Manalis, Caroline A Lewis, Matthew G Vander Heiden.

Nucleotide metabolism supports RNA synthesis and DNA replication to enable cell growth and division. Nucleotide depletion can inhibit cell growth and proliferation, but how cells sense and respond to changes in the relative levels of individual nucleotides is unclear. Moreover, the nucleotide requirement for biomass production changes over the course of the cell cycle, and how cells coordinate differential nucleotide demands with cell cycle progression is not well understood. Here we find that excess levels of individual nucleotides can inhibit proliferation by disrupting the relative levels of nucleotide bases needed for DNA replication and impeding DNA replication. The resulting purine and pyrimidine imbalances are not sensed by canonical growth regulatory pathways like mTORC1, Akt and AMPK signalling cascades, causing excessive cell growth despite inhibited proliferation. Instead, cells rely on replication stress signalling to survive during, and recover from, nucleotide imbalance during S phase. We find that ATR-dependent replication stress signalling is activated during unperturbed S phases and promotes nucleotide availability to support DNA replication. Together, these data reveal that imbalanced nucleotide levels are not detected until S phase, rendering cells reliant on replication stress signalling to cope with this metabolic problem and disrupting the coordination of cell growth and division.

DOI: https://doi.org/10.1038/s41556-022-00965-1
J Proteome Res. 2022 Aug 02.

In-Depth Mass Spectrometry-Based Proteomics of Formalin-Fixed, Paraffin-Embedded Tissues with a Spatial Resolution of 50-200 μm.

Andikan J Nwosu, Santosh A Misal, Thy Truong, Richard H Carson, Kei G I Webber, Nathaniel B Axtell, Yiran Liang, S Madisyn Johnston, Kenneth L Virgin, Ethan G Smith, George V Thomas, Terry Morgan, John C Price, Ryan T Kelly.

  Formalin-fixed, paraffin-embedded (FFPE) tissues are banked in large repositories to cost-effectively preserve valuable specimens for later study. With the rapid growth of spatial proteomics, FFPE tissues can serve as a more accessible alternative to more commonly used frozen tissues. However, extracting proteins from FFPE tissues is challenging due to cross-links formed between proteins and formaldehyde. Here, we have adapted the nanoPOTS sample processing workflow, which was previously applied to single cells and fresh-frozen tissues, to profile protein expression from FFPE tissues. Following the optimization of extraction solvents, times, and temperatures, we identified an average of 1312 and 3184 high-confidence master proteins from 10 μm thick FFPE-preserved mouse liver tissue squares having lateral dimensions of 50 and 200 μm, respectively. The observed proteome coverage for FFPE tissues was on average 88% of that achieved for similar fresh-frozen tissues. We also characterized the performance of our fully automated sample preparation and analysis workflow, termed autoPOTS, for FFPE spatial proteomics. This modified nanodroplet processing in one pot for trace samples (nanoPOTS) and fully automated processing in one pot for trace sample (autoPOTS) workflows provides the greatest coverage reported to date for high-resolution spatial proteomics applied to FFPE tissues. Data are available via ProteomeXchange with identifier PXD029729.

Keywords:  FFPE; LCM; autoPOTS; mass spectrometry; nanodroplet; spatial proteomics

DOI:  https://doi.org/10.1021/acs.jproteome.2c00409
Front Pharmacol. 2022 ;13 935553

Argininosuccinate synthase 1, arginine deprivation therapy and cancer management.

Naihui Sun, Xing Zhao.

  Metabolic reprogramming is an emerging hallmark of tumor cells. In order to survive in the nutrient-deprived environment, tumor cells rewire their metabolic phenotype to provide sufficient energy and build biomass to sustain their transformed state and promote malignant behaviors. Amino acids are the main compositions of protein, which provide key intermediate substrates for the activation of signaling pathways. Considering that cells can synthesize arginine via argininosuccinate synthase 1 (ASS1), arginine is regarded as a non-essential amino acid, making arginine depletion as a promising therapeutic strategy for ASS1-silencing tumors. In this review, we summarize the current knowledge of expression pattern of ASS1 and related signaling pathways in cancer and its potential role as a novel therapeutic target in cancer. Besides, we outline how ASS1 affects metabolic regulation and tumor progression and further discuss the role of ASS1 in arginine deprivation therapy. Finally, we review approaches to target ASS1 for cancer therapies.

Keywords:  amino acid; arginine; metabolic reprogramming; prognosis; resistance

DOI:  https://doi.org/10.3389/fphar.2022.935553
Nature. 2022 Aug 03.

A non-canonical vitamin K cycle is a potent ferroptosis suppressor.

Eikan Mishima, Junya Ito, Zijun Wu, Toshitaka Nakamura, Adam Wahida, Sebastian Doll, Wulf Tonnus, Palina Nepachalovich, Elke Eggenhofer, Maceler Aldrovandi, Bernhard Henkelmann, Ken-Ichi Yamada, Jonas Wanninger, Omkar Zilka, Emiko Sato, Regina Feederle, Daniela Hass, Adriano Maida, André Santos Dias Mourão, Andreas Linkermann, Edward K Geissler, Kiyotaka Nakagawa, Takaaki Abe, Maria Fedorova, Bettina Proneth, Derek A Pratt, Marcus Conrad.

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.

DOI: https://doi.org/10.1038/s41586-022-05022-3
Proc Natl Acad Sci U S A. 2022 Aug 09. 119(32): e2114758119

Targeting acetyl-CoA metabolism attenuates the formation of fear memories through reduced activity-dependent histone acetylation.

Desi C Alexander, Tanya Corman, Mariel Mendoza, Andrew Glass, Tal Belity, Ranran Wu, Rianne R Campbell, Joseph Han, Ashley A Keiser, Jeffrey Winkler, Marcelo A Wood, Thomas Kim, Benjamin A Garcia, Hagit Cohen, Philipp Mews, Gabor Egervari, Shelley L Berger.

  Histone acetylation is a key component in the consolidation of long-term fear memories. Histone acetylation is fueled by acetyl-coenzyme A (acetyl-CoA), and recently, nuclear-localized metabolic enzymes that produce this metabolite have emerged as direct and local regulators of chromatin. In particular, acetyl-CoA synthetase 2 (ACSS2) mediates histone acetylation in the mouse hippocampus. However, whether ACSS2 regulates long-term fear memory remains to be determined. Here, we show that Acss2 knockout is well tolerated in mice, yet the Acss2-null mouse exhibits reduced acquisition of long-term fear memory. Loss of Acss2 leads to reductions in both histone acetylation and expression of critical learning and memory-related genes in the dorsal hippocampus, specifically following fear conditioning. Furthermore, systemic administration of blood-brain barrier-permeable Acss2 inhibitors during the consolidation window reduces fear-memory formation in mice and rats and reduces anxiety in a predator-scent stress paradigm. Our findings suggest that nuclear acetyl-CoA metabolism via ACSS2 plays a critical, previously unappreciated, role in the formation of fear memories.

Keywords:  epigenetics; fear conditioning; histone acetylation; learning and memory; mass spectrometry

DOI:  https://doi.org/10.1073/pnas.2114758119
Cell Metab. 2022 Aug 02. pii: S1550-4131(22)00305-9. [Epub ahead of print]34(8): 1201-1213.e5

Aberrant iron distribution via hepatocyte-stellate cell axis drives liver lipogenesis and fibrosis.

Hong Gao, Zhongmou Jin, Gautam Bandyopadhyay, Gaowei Wang, Dinghong Zhang, Karina Cunha E Rocha, Xiao Liu, Huayi Zhao, Tatiana Kisseleva, David A Brenner, Michael Karin, Wei Ying.

  Hepatocytes have important roles in liver iron homeostasis, abnormalities in which are tightly associated with liver steatosis and fibrosis. Here, we show that non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are characterized by iron-deficient hepatocytes and iron overload in hepatic stellate cells (HSCs). Iron deficiency enhances hepatocyte lipogenesis and insulin resistance through HIF2α-ATF4 signaling. Elevated secretion of iron-containing hepatocyte extracellular vesicles (EVs), which are normally cleared by Kupffer cells, accounts for hepatocyte iron deficiency and HSC iron overload in NAFLD/NASH livers. Iron accumulation results in overproduction of reactive oxygen species that promote HSC fibrogenic activation. Conversely, blocking hepatocyte EV secretion or depleting EV iron cargo restores liver iron homeostasis, concomitant with mitigation of NAFLD/NASH-associated liver steatosis and fibrosis. Taken together, these studies show that iron distribution disorders contribute to the development of liver metabolic diseases.

Keywords:  NAFLD; NASH; extracellular vesicle; hepatic stellate cell; hepatocyte; iron; liver fibrosis; liver steatosis

DOI:  https://doi.org/10.1016/j.cmet.2022.07.006
Metabolomics. 2022 Aug 01. 18(8): 63

Association of amino acid metabolites with osteoporosis, a metabolomic approach: Bushehr elderly health program.

Nekoo Panahi, Noushin Fahimfar, Shahin Roshani, Babak Arjmand, Safoora Gharibzadeh, Gita Shafiee, Eugenia Migliavacca, Denis Breuille, Jerome N Feige, Yohan Grzywinski, John Corthesy, Farideh Razi, Ramin Heshmat, Iraj Nabipour, Farshad Farzadfar, Akbar Soltani, Bagher Larijani, Afshin Ostovar.

  INTRODUCTION AND OBJECTIVES: Amino acids are the most frequently reported metabolites associated with low bone mineral density (BMD) in metabolomics studies. We aimed to evaluate the association between amino acid metabolic profile and bone indices in the elderly population.METHODS: 400 individuals were randomly selected from 2384 elderly men and women over 60 years participating in the second stage of the Bushehr elderly health (BEH) program, a population-based prospective cohort study that is being conducted in Bushehr, a southern province of Iran. Frozen plasma samples were used to measure 29 amino acid and derivatives metabolites using the UPLC-MS/MS-based targeted metabolomics platform. We conducted Elastic net regression analysis to detect the metabolites associated with BMD of different sites and lumbar spine trabecular bone score, and also to examine the ability of the measured metabolites to differentiate osteoporosis.
RESULTS: We adjusted the analysis for possible confounders (age, BMI, diabetes, smoking, physical activity, vitamin D level, and sex). Valine, leucine, isoleucine, and alanine in women and tryptophan in men were the most important amino acids inversely associated with osteoporosis (OR range from 0.77 to 0.89). Sarcosine, followed by tyrosine, asparagine, alpha aminobutyric acid, and ADMA in women and glutamine in men and when both women and men were considered together were the most discriminating amino acids detected in individuals with osteoporosis (OR range from 1.15 to 1.31).
CONCLUSION: We found several amino acid metabolites associated with possible bone status in elderly individuals. Further studies are required to evaluate the utility of these metabolites as clinical biomarkers for osteoporosis prediction and their effect on bone health as dietary supplements.

Keywords:  Amino acid; Bone mineral density; Elderly; Metabolomics; Osteoporosis; Trabecular bone score

DOI:  https://doi.org/10.1007/s11306-022-01919-2
Lab Invest. 2022 Aug 03.

High-throughput proteomics: a methodological mini-review.

Miao Cui, Chao Cheng, Lanjing Zhang.

Proteomics plays a vital role in biomedical research in the post-genomic era. With the technological revolution and emerging computational and statistic models, proteomic methodology has evolved rapidly in the past decade and shed light on solving complicated biomedical problems. Here, we summarize scientific research and clinical practice of existing and emerging high-throughput proteomics approaches, including mass spectrometry, protein pathway array, next-generation tissue microarrays, single-cell proteomics, single-molecule proteomics, Luminex, Simoa and Olink Proteomics. We also discuss important computational methods and statistical algorithms that can maximize the mining of proteomic data with clinical and/or other 'omics data. Various principles and precautions are provided for better utilization of these tools. In summary, the advances in high-throughput proteomics will not only help better understand the molecular mechanisms of pathogenesis, but also to identify the signature signaling networks of specific diseases. Thus, modern proteomics have a range of potential applications in basic research, prognostic oncology, precision medicine, and drug discovery.

DOI: https://doi.org/10.1038/s41374-022-00830-7
J Am Soc Mass Spectrom. 2022 Aug 01.

Improved Ion Mobility Separation and Structural Characterization of Steroids using Derivatization Methods.

Diana C Velosa, Andrew J Dunham, Marcus E Rivera, Shon P Neal, Christopher D Chouinard.

  Steroids are an important class of biomolecules studied for their role in metabolism, development, nutrition, and disease. Although highly sensitive GC- and LC-MS/MS-based methods have been developed for targeted quantitation of known steroid metabolites, emerging techniques including ion mobility (IM) have shown promise in improved analysis and capacity to better identify unknowns in complex biological samples. Herein, we couple LC-IM-MS/MS with structurally selective reactions targeting hydroxyl and carbonyl functional groups to improve IM resolution and structural elucidation. We demonstrate that 1,1-carbonyldiimidazole derivatization of hydroxyl stereoisomer pairs such as testosterone/epitestosterone and androsterone/epiandrosterone results in increased IM resolution with ΔCCS > 15%. Additionally, performing this in parallel with derivatization of the carbonyl group by Girard's Reagent P resulted in unique products based on relative differences in number of each functional group and C17 alkylation. These changes could be easily deciphered using the combination of retention time, collision cross section, accurate mass, and MS/MS fragmentation pattern. Derivatization by Girard's Reagent P, which contains a fixed charge quaternary amine, also increased the ionization efficiency and could be explored for its potential benefit to sensitivity. Overall, the combination of these simple and easy derivatization reactions with LC-IM-MS/MS analysis provides a method for improved analysis of known target analytes while also yielding critical structural information that can be used for identification of potential unknowns.

Keywords:  Derivatization; Ion Mobility-Mass Spectrometry; Shift Reagents; Steroids

DOI:  https://doi.org/10.1021/jasms.2c00164
Cell Mol Gastroenterol Hepatol. 2022 Aug 02. pii: S2352-345X(22)00169-2. [Epub ahead of print]

mTORC2 facilitates liver regeneration through the sphingolipid-induced PPAR-α-fatty acid oxidation.

Lingling Zhang, Yanqiu Li, Ying Wang, Yugang Qiu, Hanchuan Mou, Yuanyao Deng, Jiyuan Yao, Zhiqing Xia, Wenzhe Zhang, Di Zhu, Zeyu Qiu, Zhongjie Lu, Jirong Wang, Zhouxin Yang, GenXiang Mao, Dan Chen, Leimin Sun, Leiming Liu, Zhenyu Ju.

  BACKGROUND & AIMS: During liver regeneration following partial hepatectomy, the function and metabolic pathways governing transient lipid droplet accumulation in hepatocytes remain obscure. Mammalian target of rapamycin 2 (mTORC2) facilitates de novo synthesis of hepatic lipids. Under normal conditions and in tumorigenesis, decreased levels of triglyceride (TG) and fatty acids (FAs) are observed in the mTORC2-deficient liver. However, during liver regeneration, their levels increase in the absence of mTORC2.METHODS: Rictor liver-specific knockout (R-LKO) and control mice underwent partial hepatectomy, followed by measurement of TG and FA contents during liver regeneration. FA metabolism was evaluated by analyzing the expression of FA metabolism-related genes and proteins. Intraperitoneal injection of the peroxisome proliferator-activated receptor α (PPAR-α) agonist, p53 inhibitor, and AKT activator was performed to verify the regulatory pathways involved. Lipid mass spectrometry was performed to identify the potential PPAR-α activators.
RESULTS: The expression of FA metabolism-related genes and proteins suggested that FAs are mainly transported into hepatocytes during liver regeneration. The PPAR-α pathway is significantly downregulated in the mTORC2-deficient liver, resulting in the accumulation of TGs. The PPAR-α agonist WY-14643 rescued deficient liver regeneration and survival in mTORC2-deficient mice. Furthermore, lipidomic analysis suggested that mTORC2 deficiency substantially reduced glucosylceramide (GluCer) content. GluCer activated PPAR-α. GluCer treatment in vivo restored the regenerative ability and survival rates in the mTORC2-deficient group.
CONCLUSIONS: Our data suggest that FAs are mainly transported into hepatocytes during liver regeneration, and their metabolism is facilitated by mTORC2 through the GluCer-PPAR-α pathway, thereby establishing a novel role for mTORC2 in lipid metabolism.

Keywords:  fatty acids; hepatectomy; metabolism; proliferation; triglyceride

DOI:  https://doi.org/10.1016/j.jcmgh.2022.07.011