bims-mascan 2023-08-20 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2023–08–20
23 papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz

LC-MS-Based Redox Phosphoipidomics Analysis in Ferroptosis.
Stratifying Ferroptosis Sensitivity in Cells and Tissues with PALP.
Lipidomics Analysis in Ferroptosis.
Analysis of polar primary metabolites in biological samples using targeted metabolomics and LC-MS.
Linking MS1 and MS2 signals in positive and negative modes of LC-HRMS in untargeted metabolomics using the ROIMCR approach.
Improving the Data Quality of Untargeted Metabolomics through a Targeted Data-Dependent Acquisition Based on an Inclusion List of Differential and Preidentified Ions.
Probing Lipid Peroxidation in Ferroptosis: Emphasizing the Utilization of C11-BODIPY-Based Protocols.
IQMMA: Efficient MS1 Intensity Extraction Pipeline Using Multiple Feature Detection Algorithms for DDA Proteomics.
Lipid droplets control mitogenic lipid mediator production in human cancer cells.
An automated spray-capillary platform for the microsampling and CE-MS analysis of picoliter- and nanoliter-volume samples.
Extracellular vesicles: Emerging mediators of cell communication in gastrointestinal cancers exhibiting metabolic abnormalities.
Evaluation of Data-Dependent MS/MS Acquisition Parameters for Non-Targeted Metabolomics and Molecular Networking of Environmental Samples: Focus on the Q Exactive Platform.
A Taxonomically-informed Mass Spectrometry Search Tool for Microbial Metabolomics Data.
Improving Quantitative Accuracy in Nontargeted Lipidomics by Evaluating Adduct Formation.
FASN deficiency induces a cytosol-to-mitochondria citrate flux to mitigate detachment-induced oxidative stress.
FAM120A couples SREBP-dependent transcription and splicing of lipogenesis enzymes downstream of mTORC1.
A pathogen-specific isotope tracing approach reveals metabolic activities and fluxes of intracellular Salmonella.
In-depth mass spectrometry analysis of rhGH administration altered energy metabolism and steroidogenesis.
Pyruvate anaplerosis is a targetable vulnerability in persistent leukaemic stem cells.
Application of a porous graphitic carbon column to carbon and nitrogen isotope analysis of underivatized individual amino acids using high-performance liquid chromatography coupled with elemental analyzer/isotope ratio mass spectrometry.
A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples.
Protocol to track the biosynthesis of cholesterol in cultured HCC cells using 13C compound-specific stable isotopic tracers.
A robust method for measuring aminoacylation through tRNA-Seq.

Methods Mol Biol. 2023 ;2712 81-90

LC-MS-Based Redox Phosphoipidomics Analysis in Ferroptosis.

Wan-Yang Sun, Rong Wang, Rong-Rong He.

  Ferroptosis is a regulated form of cell death characterized by the accumulation of oxidized phospholipids, particularly oxidized phosphatidylethanolamines (PE), which serve as important biomarkers in the progression of various diseases. To facilitate the comprehensive investigation of ferroptosis in biological systems, we present a robust and versatile untargeted redox phospholipidomics method employing normal-phase liquid chromatography-mass spectrometry (LC-MS). This high-throughput technique enables the identification and quantification of dozens of oxidized phospholipid species in a single run, providing valuable insights into the molecular mechanisms underlying ferroptosis. It has been successfully applied to diverse biological samples, including human patients, animals, and cell cultures, and offers a powerful tool for investigating the roles of oxidized phospholipids in the development and progression of various diseases.

Keywords:  Ferroptosis; Lipid peroxidation; Liquid chromatography-mass spectrometry; Oxidized phospholipid; Redox phospholipidomics

DOI:  https://doi.org/10.1007/978-1-0716-3433-2_8
Methods Mol Biol. 2023 ;2712 9-17

Stratifying Ferroptosis Sensitivity in Cells and Tissues with PALP.

Fengxiang Wang, Nathchar Naowarojna, Yilong Zou.

  Ferroptosis is emerging as a promising strategy for suppressing multiple types of human cancers. Rapid and accurate assessment of the relative sensitivity to ferroptosis in biological samples will accelerate the development of ferroptosis-targeted therapies. We previously demonstrated that photochemical activation of membrane lipid peroxidation (PALP) that uses high-power lasers to induce localized polyunsaturated fatty acyl (PUFA)-lipid peroxidation can efficiently report ferroptosis sensitivity in live cells and tissues in situ. Here, we describe the experimental details for PALP analysis, including preparation of tissue sections, preparation of fluorescent lipid peroxidation reporter, sample staining, lipid peroxidation induced by laser source, and data processing. We envision predicting the relative sensitivity to ferroptosis of cellular and tissue samples is potentially useful for basic research and clinical investigations.

Keywords:  Ferroptosis sensitivity; Lipid peroxidation; Live-cell imaging; Polyunsaturated-lipids; Tissue in situ imaging; Tumor

DOI:  https://doi.org/10.1007/978-1-0716-3433-2_2
Methods Mol Biol. 2023 ;2712 149-156

Lipidomics Analysis in Ferroptosis.

Zhi Lin, Minghua Yang.

  Ferroptosis is a form of regulated cell death that occurs due to abnormal lipid metabolism. Lipids, which have been identified in over 45,000 different molecular species, play essential roles in modulating basic life processes. The process of ferroptosis is highly reliant on various lipid species, with polyunsaturated fatty acids (PUFAs) playing a central role in driving this process. Recent advances in mass spectrometry-based lipidomics have led to a surge in studies on ferroptosis. To explore the mechanism of lipid homeostasis in ferroptosis, the development of lipidomics techniques is critical. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) are the most widely used analytical techniques in lipidomics. These techniques offer deeper insights into the complex lipid mechanisms that underlie ferroptosis.

Keywords:  Ferroptosis; Lipidomics; Mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-3433-2_13
STAR Protoc. 2023 Aug 16. pii: S2666-1667(23)00367-2. [Epub ahead of print]4(3): 102400

Analysis of polar primary metabolites in biological samples using targeted metabolomics and LC-MS.

Evgenia Turtoi, Jeremy Jeudy, Sylvain Henry, Ikrame Dadi, Gilles Valette, Christine Enjalbal, Andrei Turtoi.

  Primary metabolites are molecules of essential biochemical reactions that define the biological phenotype. All primary metabolites cannot be measured in a single analysis. In this protocol, we outline the multiplexed and quantitative measurement of 106 metabolites that cover the central part of primary metabolism. The protocol includes several sample preparation techniques and one liquid chromatography-mass spectrometry method. Then, we describe the steps of the bioinformatic data analysis to better understand the metabolic perturbations that may occur in a biological system. For complete details on the use and execution of this protocol, please refer to: Costanza et al.,1 Blomme et al.,2 Blomme et al.,3 Guillon et al.,4 Stuani et al.5.

Keywords:  Mass Spectrometry; Metabolomics

DOI:  https://doi.org/10.1016/j.xpro.2023.102400
Anal Bioanal Chem. 2023 Aug 17.

Linking MS1 and MS2 signals in positive and negative modes of LC-HRMS in untargeted metabolomics using the ROIMCR approach.

Flávia Yoshie Yamamoto, Carlos Pérez-López, Ana Lopez-Antia, Silvia Lacorte, Denis Moledo de Souza Abessa, Romà Tauler.

  Data-independent acquisition (DIA) mode in liquid chromatography (LC) high-resolution mass spectrometry (HRMS) has emerged as a powerful strategy in untargeted metabolomics for detecting a broad range of metabolites. However, the use of this approach also represents a challenge in the analysis of the large datasets generated. The regions of interest (ROI) multivariate curve resolution (MCR) approach can help in the identification and characterization of unknown metabolites in their mixtures by linking their MS1 and MS2 DIA spectral signals. In this study, it is proposed for the first time the analysis of MS1 and MS2 DIA signals in positive and negative electrospray ionization modes simultaneously to increase the coverage of possible metabolites present in biological systems. In this work, this approach has been tested for the detection and identification of the amino acids present in a standard mixture solution and in fish embryo samples. The ROIMCR analysis allowed for the identification of all amino acids present in the analyzed mixtures in both positive and negative modes. The methodology allowed for the direct linking and correspondence between the MS signals in their different acquisition modes. Overall, this approach confirmed the advantages and possibilities of performing the proposed ROIMCR simultaneous analysis of mass spectrometry signals in their differing acquisition modes in untargeted metabolomics studies.

Keywords:  All-ion fragmentation (AIF); Metabolite coverage improvement; Positive and negative acquisition modes; ROIMCR, Regions of interest multivariate curve resolution; Untargeted metabolomics

DOI:  https://doi.org/10.1007/s00216-023-04893-3
Anal Chem. 2023 Aug 18.

Improving the Data Quality of Untargeted Metabolomics through a Targeted Data-Dependent Acquisition Based on an Inclusion List of Differential and Preidentified Ions.

Yuhao Zhang, Jingyu Liao, Wanqi Le, Gaosong Wu, Weidong Zhang.

Metabolomics based on high-resolution mass spectrometry has become a powerful technique in biomedical research. The development of various analytical tools and online libraries has promoted the identification of biomarkers. However, how to make mass spectrometry collect more data information is an important but underestimated research topic. Herein, we combined full-scan and data-dependent acquisition (DDA) modes to develop a new targeted DDA based on the inclusion list of differential and preidentified ions (dpDDA). In this workflow, the MS1 datasets for statistical analysis and metabolite preidentification were first obtained using full-scan, and then, the MS/MS datasets for metabolite identification were obtained using targeted DDA of quality control samples based on the inclusion list. Compared with the current methods (DDA, data-independent acquisition, targeted DDA with time-staggered precursor ion list, and iterative exclusion DDA), dpDDA showed better stability, higher characteristic ion coverage, higher differential metabolites' MS/MS coverage, and higher quality MS/MS spectra. Moreover, the same trend was verified in the analysis of large-scale clinical samples. More surprisingly, dpDDA can distinguish patients with different severities of coronary heart disease (CHD) based on the Canadian Cardiovascular Society angina classification, which we cannot distinguish through conventional metabolomics data collection. Finally, dpDDA was employed to differentiate CHD from healthy control, and targeted metabolomics confirmed that dpDDA could identify a more complete metabolic pathway network. At the same time, four unreported potential CHD biomarkers were identified, and the area under the receiver operating characteristic curve was greater than 0.85. These results showed that dpDDA would expand the discovery of biomarkers based on metabolomics, more comprehensively explore the key metabolites and their association with diseases, and promote the development of precision medicine.

DOI: https://doi.org/10.1021/acs.analchem.3c02888
Methods Mol Biol. 2023 ;2712 61-72

Probing Lipid Peroxidation in Ferroptosis: Emphasizing the Utilization of C11-BODIPY-Based Protocols.

Zhangshuai Dai, Wanting Zhang, Liqun Zhou, Junqi Huang.

  Ferroptosis is a form of regulated cell death that relies on iron and is characterized by the accumulation of lipid peroxides, resulting in oncotic cell swelling and eventual disruption of cellular membranes. Lipid peroxidation, a hallmark of ferroptosis, refers to the oxidative deterioration of lipids that contain carbon-carbon double bonds, particularly polyunsaturated fatty acids (PUFAs). Understanding the molecular mechanisms underlying the interplay between ferroptosis and lipid peroxidation and identifying reliable techniques for assessing lipid peroxidation levels are crucial for further advancements in this field of research. Various methods have been developed to detect lipid peroxidation levels, including C11-BODIPY (BODIPY™ 581/591 C11), liperfluo, 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), Click-iT LAA (linoleamide alkyne), and liquid chromatography-mass spectrometry (LC-MS)-based epilipidomics (redox lipidomics). Currently, one of the most commonly used and effective methods is the C11-BODIPY assay, which utilizes a fluorescent probe that selectively sensitizes lipid peroxidation in cell membranes. Incorporating advanced techniques such as flow cytometry and fluorescence microscopy with C11-BODIPY dye is essential for accurate assessment of lipid peroxidation levels in ferroptosis. This chapter aims to provide comprehensive experimental protocols for detecting lipid peroxidation levels indicative of ferroptosis using C11-BODIPY staining and subsequent detection via flow cytometry and fluorescence microscopy.

Keywords:  C11-BODIPY; Ferroptosis; Flow cytometry; Fluorescence microscopy; Lipid peroxidation

DOI:  https://doi.org/10.1007/978-1-0716-3433-2_6
J Proteome Res. 2023 Aug 14.

IQMMA: Efficient MS1 Intensity Extraction Pipeline Using Multiple Feature Detection Algorithms for DDA Proteomics.

Valeriy I Postoenko, Leyla A Garibova, Lev I Levitsky, Julia A Bubis, Mikhail V Gorshkov, Mark V Ivanov.

  One of the key steps in data dependent acquisition (DDA) proteomics is detection of peptide isotopic clusters, also called "features", in MS1 spectra and matching them to MS/MS-based peptide identifications. A number of peptide feature detection tools became available in recent years, each relying on its own matching algorithm. Here, we provide an integrated solution, the intensity-based Quantitative Mix and Match Approach (IQMMA), which integrates a number of untargeted peptide feature detection algorithms and returns the most probable intensity values for the MS/MS-based identifications. IQMMA was tested using available proteomic data acquired for both well-characterized (ground truth) and real-world biological samples, including a mix of Yeast and E. coli digests spiked at different concentrations into the Human K562 digest used as a background, and a set of glioblastoma cell lines. Three open-source feature detection algorithms were integrated: Dinosaur, biosaur2, and OpenMS FeatureFinder. None of them was found optimal when applied individually to all the data sets employed in this work; however, their combined use in IQMMA improved efficiency of subsequent protein quantitation. The software implementing IQMMA is freely available at https://github.com/PostoenkoVI/IQMMA under Apache 2.0 license.

Keywords:  bioinformatics; feature detection; mass spectrometry; protein quantitation

DOI:  https://doi.org/10.1021/acs.jproteome.3c00075
Mol Metab. 2023 Aug 14. pii: S2212-8778(23)00125-4. [Epub ahead of print] 101791

Lipid droplets control mitogenic lipid mediator production in human cancer cells.

Eva Jarc Jovičić, Anja Pucer Janež, Thomas O Eichmann, Špela Koren, Vesna Brglez, Paul M Jordan, Jana Gerstmeier, Duško Lainšček, Anja Golob-Urbanc, Roman Jerala, Gérard Lambeau, Oliver Werz, Robert Zimmermann, Toni Petan.

   OBJECTIVES: Polyunsaturated fatty acids (PUFAs) are structural components of membrane phospholipids and precursors of oxygenated lipid mediators with diverse functions, including the control of cell growth, inflammation and tumourigenesis. However, the molecular pathways that control the availability of PUFAs for lipid mediator production are not well understood. Here, we investigated the crosstalk of three pathways in the provision of PUFAs for lipid mediator production: (i) secreted group X phospholipase A2 (GX sPLA2) and (ii) cytosolic group IVA PLA2 (cPLA2α), both mobilizing PUFAs from membrane phospholipids, and (iii) adipose triglyceride lipase (ATGL), which mediates the degradation of triacylglycerols (TAGs) stored in cytosolic lipid droplets (LDs).
METHODS: We combined lipidomic and functional analyses in cancer cell line models to dissect the trafficking of PUFAs between membrane phospholipids and LDs and determine the role of these pathways in lipid mediator production, cancer cell proliferation and tumour growth in vivo.
RESULTS: We demonstrate that lipid mediator production strongly depends on TAG turnover. GX sPLA2 directs ω-3 and ω-6 PUFAs from membrane phospholipids into TAG stores, whereas ATGL is required for their entry into lipid mediator biosynthetic pathways. ATGL controls the release of PUFAs from LD stores and their conversion into cyclooxygenase- and lipoxygenase-derived lipid mediators under conditions of nutrient sufficiency and during serum starvation. In starving cells, ATGL also promotes the incorporation of LD-derived PUFAs into phospholipids, representing substrates for cPLA2α. Furthermore, we demonstrate that the built-up of TAG stores by acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is required for the production of mitogenic lipid signals that promote cancer cell proliferation and tumour growth.
CONCLUSION: This study shifts the paradigm of PLA2-driven lipid mediator signalling and identifies LDs as central lipid mediator production hubs. Targeting DGAT1-mediated LD biogenesis is a promising strategy to restrict lipid mediator production and tumour growth.

Keywords:  adipose triglyceride lipase; cancer; diacylglycerol acyltransferase; lipid droplets; lipid mediators; phospholipase A(2)

DOI:  https://doi.org/10.1016/j.molmet.2023.101791
Anal Bioanal Chem. 2023 Aug 15.

An automated spray-capillary platform for the microsampling and CE-MS analysis of picoliter- and nanoliter-volume samples.

Jiaxue Li, Lushuang Huang, Yanting Guo, Kellye A Cupp-Sutton, Si Wu.

  Capillary electrophoresis mass spectrometry (CE-MS) is an emerging analytical tool for microscale biological sample analysis that offers high separation resolution, low detection limit, and low sample consumption. We recently developed a novel microsampling device, "spray-capillary," for quantitative low-volume sample extraction (as low as 15 pL/s) and online CE-MS analysis. This platform can efficiently analyze picoliter samples (e.g., single cells) with minimal sample loss and no additional offline sample-handling steps. However, our original spray-capillary-based experiments required manual manipulation of the sample inlet for sample collection and separation, which is time consuming and requires proficiency in device handling. To optimize the performance of spray-capillary CE-MS analysis, we developed an automated platform for robust, high-throughput analysis of picoliter samples using a commercially available CE autosampler. Our results demonstrated high reproducibility among 50 continuous runs using the standard peptide angiotensin II (Ang II), with an RSD of 14.70% and 0.62% with respect to intensity and elution time, respectively. We also analyzed Ang II using varying injection times to evaluate the capability of the spray-capillary to perform quantitative sampling and found high linearity for peptide intensity with respect to injection time (R2 > 0.99). These results demonstrate the capability of the spray-capillary sampling platform for high-throughput quantitative analysis of low-volume, low-complexity samples using pressure elution (e.g., direct injection). To further evaluate and optimize the automated spray-capillary platform to analyze complex biological samples, we performed online CE-MS analysis on Escherichia coli lysate digest spiked with Ang II using varying injection times. We maintained high linearity of intensity with respect to injection time for Ang II and E. coli peptides (R2 > 0.97 in all cases). Furthermore, we observed good CE separation and high reproducibility between automated runs. Overall, we demonstrated that the automated spray-capillary CE-MS platform can efficiently and reproducibly sample picoliter and nanoliter biological samples for high-throughput proteomics analysis.

Keywords:  CE-MS; Low-volume sampling; Mass-limited proteomics; Proteomics

DOI:  https://doi.org/10.1007/s00216-023-04870-w
Cytokine Growth Factor Rev. 2023 Aug 06. pii: S1359-6101(23)00039-4. [Epub ahead of print]

Extracellular vesicles: Emerging mediators of cell communication in gastrointestinal cancers exhibiting metabolic abnormalities.

Ghazaleh Pourali, Nima Zafari, Hamid Fiuji, Jyotsna Batra, Elham Nazari, Majid Khazaei, Seyed Mahdi Hassanian, Mahrou Vahabi, MohammadAli Kiani, Majid Ghayour-Mobarhan, Godefridus J Peters, Gordon A Ferns, Alfred King-Yin Lam, Elisa Giovannetti, Amir Avan.

  There is a complex interaction between pro-tumoural and anti-tumoural networks in the tumour microenvironment (TME). Throughout tumourigenesis, communication between malignant cells and various cells of the TME contributes to metabolic reprogramming. Tumour Dysregulation of metabolic pathways offer an evolutional advantage in the TME and enhance the tumour progression, invasiveness, and metastasis. Therefore, understanding these interactions within the TME is crucial for the development of innovative cancer treatments. Extracellular vesicles (EVs) serve as carriers of various materials that include microRNAs, proteins, and lipids that play a vital role in the communication between tumour cells and non-tumour cells. EVs are actively involved in the metabolic reprogramming process. This review summarized recent findings regarding the involvement of EVs in the metabolic reprogramming of various cells in the TME of gastrointestinal cancers. Additionally, we highlight identified microRNAs involved in the reprogramming process in this group of cancers and explained the abnormal tumour metabolism targeted by exosomal cargos as well as the novel potential therapeutic approaches.

Keywords:  Exosomal cargos; Extracellular vesicles; Gastrointestinal cancers; Hepatopancreatobiliary cancer; Metabolic reprogramming; Tumour microenvironment

DOI:  https://doi.org/10.1016/j.cytogfr.2023.08.001
Anal Chem. 2023 Aug 14.

Evaluation of Data-Dependent MS/MS Acquisition Parameters for Non-Targeted Metabolomics and Molecular Networking of Environmental Samples: Focus on the Q Exactive Platform.

Paolo Stincone, Abzer K Pakkir Shah, Robin Schmid, Lana G Graves, Stilianos P Lambidis, Ralph R Torres, Shu-Ning Xia, Vidit Minda, Allegra T Aron, Mingxun Wang, Chambers C Hughes, Daniel Petras.

Non-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely used tool for metabolomics analysis, enabling the detection and annotation of small molecules in complex environmental samples. Data-dependent acquisition (DDA) of product ion spectra is thereby currently one of the most frequently applied data acquisition strategies. The optimization of DDA parameters is central to ensuring high spectral quality, coverage, and number of compound annotations. Here, we evaluated the influence of 10 central DDA settings of the Q Exactive mass spectrometer on natural organic matter samples from ocean, river, and soil environments. After data analysis with classical and feature-based molecular networking using MZmine and GNPS, we compared the total number of network nodes, multivariate clustering, and spectrum quality-related metrics such as annotation and singleton rates, MS/MS placement, and coverage. Our results show that automatic gain control, microscans, mass resolving power, and dynamic exclusion are the most critical parameters, whereas collision energy, TopN, and isolation width had moderate and apex trigger, monoisotopic selection, and isotopic exclusion minor effects. The insights into the data acquisition ergonomics of the Q Exactive platform presented here can guide new users and provide them with initial method parameters, some of which may also be transferable to other sample types and MS platforms.

DOI: https://doi.org/10.1021/acs.analchem.3c01202
Res Sq. 2023 Aug 03. pii: rs.3.rs-3189768. [Epub ahead of print]

A Taxonomically-informed Mass Spectrometry Search Tool for Microbial Metabolomics Data.

Pieter Dorrestein, Simone Zuffa, Robin Schmid, Anelize Bauermeister, Paulo Wender Portal Gomes, Andres Caraballo-Rodriguez, Yasin El Abiead, Allegra Aron, Emily Gentry, Jasmine Zemlin, Michael Meehan, Nicole Avalon, Robert Cichewicz, Ekaterina Buzun, Marvic Carrillo-Terrazas, Chia-Yun Hsu, Renee Oles, Adriana Vasquez Ayala, Jiaqi Zhao, Hiutung Chu, Mirte Kuijpers, Sara Jackrel, Fidele Tugizimana, Lerato Nephali, Ian Dubery, Ntakadzeni Madala, Eduarda Moreira, Leticia Costa-Lotufo, Norberto Lopes, Paula Rezende-Teixeira, Paula Jimenez, Bipin Rimal, Andrew Patterson, Matthew Traxler, Rita de Cassia Pessotti, Daniel Alvarado-Villalobos, Giselle Tamayo-Castillo, Priscila Chaverri, Efrain Escudero-Leyva, Luis-Manuel Quiros-Guerrero, Alexandre Bory, Juliette Joubert, Adriano Rutz, Jean-Luc Wolfender, Pierre-Marie Allard, Andreas Sichert, Sammy Pontrelli, Benjamin Pullman, Nuno Bandeira, William Gerwick, Katia Gindro, Josep Massana-Codina, Berenike Wagner, Karl Forchhammer, Daniel Petras, Nicole Aiosa, Neha Garg, Manuel Liebeke, Patric Bourceau, Kyo Bin Kang, Henna Gadhavi, Luiz Pedro de Carvalho, Mariana Silva Dos Santos, Alicia Pérez-Lorente, Carlos Molina-Santiago, Diego Romero, Raimo Franke, Mark Broenstrup, Arturo Vera Ponce De Leon, Phillip Pope, Sabina La Rosa, Giorgia La Barbera, Henrik Roager, Martin Laursen, Fabian Hammerle, Bianka Siewert, Ursula Peintner, Cuauhtemoc Licona-Cassani, Lorena Rodriguez-Orduña, Evelyn Rampler, Felina Hildebrand, Gunda Koellensperger, Harald Schoeny, Katharina Hohenwallner, Lisa Panzenboeck, Rachel Gregor, Ellis O'Neill, Eve Roxborough, Jane Odoi, Nicole Bale, Su Ding, Jaap S Sinninghe Damsté, Xueli Guan, Jerry Cui, Kou-San Ju, Denise Silva, Fernanda Silva, Gilvan da Silva, Hector Koolen, Carlismari Grundmann, Jason Clement, Hosein Mohimani, Kirk Broders, Kerry McPhail, Sidnee Ober-Singleton, Christopher Rath, Daniel McDonald, Rob Knight, Mingxun Wang.

MicrobeMASST, a taxonomically-informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbial-derived metabolites and relative producers, without a priori knowledge, will vastly enhance the understanding of microorganisms' role in ecology and human health.

DOI: https://doi.org/10.21203/rs.3.rs-3189768/v1
Anal Chem. 2023 Aug 15.

Improving Quantitative Accuracy in Nontargeted Lipidomics by Evaluating Adduct Formation.

Lauren M Bishop, Tong Shen, Oliver Fiehn.

For large-scale lipidomic analyses, accurate and reproducible quantification of endogenous lipids is crucial for comparing results within and across studies. Many lipids present in liquid chromatography-electrospray ionization-mass spectrometry form various adducts with buffer components. The mechanisms and conditions that dictate adduct formation are still poorly understood. In a positive mode, neutral lipids like mono-, di-, and triacylglycerides and cholesteryl esters typically generate [M + NH4]+ adduct ions, although [M + Na]+, [M + K]+, and other (more complex) species can also be significantly abundant in MS1 precursor ion spectra. Variations in the ratios of these adducts (within and between matrices) can lead to dramatic inaccuracies during quantification. Here, we examine 48 unique diacylglycerol (DAG) species across 2366 mouse samples for eight matrix-specific data sets of plasma, liver, kidney, brain, heart muscle, gastrocnemius muscle, gonadal, and inguinal fat. Typically, no single adduct ion species accounted for more than 60% of the total observed abundance across each data set. Even within a single matrix, DAGs showed a high variability of adduct ratios. The ratio of [M + NH4]+ adduct ions was increased for longer-chain DAGs and for polyunsaturated DAGs, at the expense of reduced ratios of [M + Na]+ adducts. When using three deuterated internal DAG standards, we found that absolute concentrations were estimated with up to 70% error when only one adduct ion was used instead of all adducts combined. Importantly, when combining [M + NH4]+ and [M + Na]+ adduct ions, quantification results were within 5% accuracy compared to all adduct ions combined. Additional variance can be caused by other factors, such as instrument conditions or matrix effects.

DOI: https://doi.org/10.1021/acs.analchem.3c01221
Cell Rep. 2023 Aug 12. pii: S2211-1247(23)00982-8. [Epub ahead of print]42(8): 112971

FASN deficiency induces a cytosol-to-mitochondria citrate flux to mitigate detachment-induced oxidative stress.

Wenting Dai, Zhichao Wang, Guan Wang, Qiong A Wang, Ralph DeBerardinis, Lei Jiang.

  Fatty acid synthase (FASN) maintains de novo lipogenesis (DNL) to support rapid growth in most proliferating cancer cells. Lipogenic acetyl-coenzyme A (CoA) is primarily produced from carbohydrates but can arise from glutamine-dependent reductive carboxylation. Here, we show that reductive carboxylation also occurs in the absence of DNL. In FASN-deficient cells, reductive carboxylation is mainly catalyzed by isocitrate dehydrogenase-1 (IDH1), but IDH1-generated cytosolic citrate is not utilized for supplying DNL. Metabolic flux analysis (MFA) shows that FASN deficiency induces a net cytosol-to-mitochondria citrate flux through mitochondrial citrate transport protein (CTP). Previously, a similar pathway has been shown to mitigate detachment-induced oxidative stress in anchorage-independent tumor spheroids. We further report that tumor spheroids show reduced FASN activity and that FASN-deficient cells acquire resistance to oxidative stress in a CTP- and IDH1-dependent manner. Collectively, these data indicate that by inducing a cytosol-to-mitochondria citrate flux, anchorage-independent malignant cells can gain redox capacity by trading off FASN-supported rapid growth.

Keywords:  CP: Metabolism; CP: Molecular biology; DNL; FASN inhibitor; IDH1 inhibitor; MFA; SLC25A1; anchorage-independent growth; cytosol-to-mitochondria citrate flux; de novo lipogenesis; metabolic flux analysis; redox; reductive carboxylation

DOI:  https://doi.org/10.1016/j.celrep.2023.112971
Mol Cell. 2023 Aug 17. pii: S1097-2765(23)00560-9. [Epub ahead of print]83(16): 3010-3026.e8

FAM120A couples SREBP-dependent transcription and splicing of lipogenesis enzymes downstream of mTORC1.

Sungyun Cho, Yujin Chun, Long He, Cuauhtemoc B Ramirez, Kripa S Ganesh, Kyungjo Jeong, Junho Song, Jin Gyu Cheong, Zhongchi Li, Jungmin Choi, Joohwan Kim, Nikos Koundouros, Fangyuan Ding, Noah Dephoure, Cholsoon Jang, John Blenis, Gina Lee.

  The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth that stimulates macromolecule synthesis through transcription, RNA processing, and post-translational modification of metabolic enzymes. However, the mechanisms of how mTORC1 orchestrates multiple steps of gene expression programs remain unclear. Here, we identify family with sequence similarity 120A (FAM120A) as a transcription co-activator that couples transcription and splicing of de novo lipid synthesis enzymes downstream of mTORC1-serine/arginine-rich protein kinase 2 (SRPK2) signaling. The mTORC1-activated SRPK2 phosphorylates splicing factor serine/arginine-rich splicing factor 1 (SRSF1), enhancing its binding to FAM120A. FAM120A directly interacts with a lipogenic transcription factor SREBP1 at active promoters, thereby bridging the newly transcribed lipogenic genes from RNA polymerase II to the SRSF1 and U1-70K-containing RNA-splicing machinery. This mTORC1-regulated, multi-protein complex promotes efficient splicing and stability of lipogenic transcripts, resulting in fatty acid synthesis and cancer cell proliferation. These results elucidate FAM120A as a critical transcription co-factor that connects mTORC1-dependent gene regulation programs for anabolic cell growth.

Keywords:  FAM120A; RNA splicing; RNA stability; SREBP; SRPK2; SRSF1; lipid metabolism; mTOR signaling

DOI:  https://doi.org/10.1016/j.molcel.2023.07.017
PLoS Biol. 2023 Aug 18. 21(8): e3002198

A pathogen-specific isotope tracing approach reveals metabolic activities and fluxes of intracellular Salmonella.

Karin Mitosch, Martin Beyß, Prasad Phapale, Bernhard Drotleff, Katharina Nöh, Theodore Alexandrov, Kiran R Patil, Athanasios Typas.

Pathogenic bacteria proliferating inside mammalian host cells need to rapidly adapt to the intracellular environment. How they achieve this and scavenge essential nutrients from the host has been an open question due to the difficulties in distinguishing between bacterial and host metabolites in situ. Here, we capitalized on the inability of mammalian cells to metabolize mannitol to develop a stable isotopic labeling approach to track Salmonella enterica metabolites during intracellular proliferation in host macrophage and epithelial cells. By measuring label incorporation into Salmonella metabolites with liquid chromatography-mass spectrometry (LC-MS), and combining it with metabolic modeling, we identify relevant carbon sources used by Salmonella, uncover routes of their metabolization, and quantify relative reaction rates in central carbon metabolism. Our results underline the importance of the Entner-Doudoroff pathway (EDP) and the phosphoenolpyruvate carboxylase for intracellularly proliferating Salmonella. More broadly, our metabolic labeling strategy opens novel avenues for understanding the metabolism of pathogens inside host cells.

DOI: https://doi.org/10.1371/journal.pbio.3002198
Talanta. 2023 Aug 09. pii: S0039-9140(23)00820-2. [Epub ahead of print]266(Pt 2): 125069

In-depth mass spectrometry analysis of rhGH administration altered energy metabolism and steroidogenesis.

Dan Zhao, Wenqian Zhuang, Yang Wang, Xin Xu, Liang Qiao.

  Growth hormone, as a proteohormone, is primarily known of its dramatic effect on longitudinal growth. Recombinant DNA technology has provided a safe, abundant and comparatively cheap supply of human GH for growth hormone-deficient individuals. However, many healthy subjects, especially athletics, administrate GH for enhanced athletic performance or strength. A better and more comprehensive understanding of rhGH effect in healthy individuals is urgent and essential. In this study, we recruited 14 healthy young male and injected rhGH once. Untargeted LC-MS metabolomics profiling of serum and urine was performed before and after the rhGH injection. The GH-induced dysregulation of energy related pathways, such as amino acid metabolism, nucleotide metabolism, glycolysis and TCA cycle, was revealed. Moreover, individuals supplemented with micro-doses of rhGH exhibited significantly changed urinary steroidal profiles, suggesting a role of rhGH in both energy metabolism and steroidogenesis. We expect that our results will be helpful to provide new evidence on the effects of rhGH injection and provide potential biomarkers for rhGH administration.

Keywords:  Energy metabolism; Growth hormone; Liquid chromatography-tandem mass spectrometry; Metabolomics; Steroidogenesis

DOI:  https://doi.org/10.1016/j.talanta.2023.125069
Nat Commun. 2023 Aug 17. 14(1): 4634

Pyruvate anaplerosis is a targetable vulnerability in persistent leukaemic stem cells.

Kevin M Rattigan, Zuzana Brabcova, Daniele Sarnello, Martha M Zarou, Kiron Roy, Ryan Kwan, Lucie de Beauchamp, Amy Dawson, Angela Ianniciello, Ahmed Khalaf, Eric R Kalkman, Mary T Scott, Karen Dunn, David Sumpton, Alison M Michie, Mhairi Copland, Saverio Tardito, Eyal Gottlieb, G Vignir Helgason.

Deregulated oxidative metabolism is a hallmark of leukaemia. While tyrosine kinase inhibitors (TKIs) such as imatinib have increased survival of chronic myeloid leukaemia (CML) patients, they fail to eradicate disease-initiating leukemic stem cells (LSCs). Whether TKI-treated CML LSCs remain metabolically deregulated is unknown. Using clinically and physiologically relevant assays, we generate multi-omics datasets that offer unique insight into metabolic adaptation and nutrient fate in patient-derived CML LSCs. We demonstrate that LSCs have increased pyruvate anaplerosis, mediated by increased mitochondrial pyruvate carrier 1/2 (MPC1/2) levels and pyruvate carboxylase (PC) activity, in comparison to normal counterparts. While imatinib reverses BCR::ABL1-mediated LSC metabolic reprogramming, stable isotope-assisted metabolomics reveals that deregulated pyruvate anaplerosis is not affected by imatinib. Encouragingly, genetic ablation of pyruvate anaplerosis sensitises CML cells to imatinib. Finally, we demonstrate that MSDC-0160, a clinical orally-available MPC1/2 inhibitor, inhibits pyruvate anaplerosis and targets imatinib-resistant CML LSCs in robust pre-clinical CML models. Collectively these results highlight pyruvate anaplerosis as a persistent and therapeutically targetable vulnerability in imatinib-treated CML patient-derived samples.

DOI: https://doi.org/10.1038/s41467-023-40222-z
Rapid Commun Mass Spectrom. 2023 Sep 15. 37(17): e9602

Application of a porous graphitic carbon column to carbon and nitrogen isotope analysis of underivatized individual amino acids using high-performance liquid chromatography coupled with elemental analyzer/isotope ratio mass spectrometry.

Yuchen Sun, Nanako O Ogawa, Naoto F Ishikawa, Thomas M Blattmann, Yoshinori Takano, Naohiko Ohkouchi.

RATIONALE: Isolation of underivatized amino acids (AAs) using high-performance liquid chromatography (HPLC) is becoming a popular method for carbon (δ13 C) and nitrogen isotope (δ15 N) analyses of AAs because of the high analytical precision and for performing dual-isotope analysis. However, some AAs in natural samples, especially small, hydrophilic AAs, are not suitably separated using reversed-phase columns (e.g., C18) and ion-exchange columns (e.g., Primesep A).
METHODS: We developed a new method for HPLC using a porous graphitic carbon column for the separation of nine hydrophilic AAs. After purification, δ13 C and δ15 N values of AAs were determined using elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). We demonstrated the application of this method by determining δ13 C and δ15 N values of individual hydrophilic AAs in a biological sample, the muscle of blue mackerel (Scomber australasicus).
RESULTS: Chromatographically, the baseline separation of hydrophilic AAs was achieved in both the standard mixture and the biological sample. We confirmed that δ13 C and δ15 N values of AA standards remained unchanged during the whole experimental procedure. The δ13 C values of AAs in mackerel muscle are also in good agreement with the values obtained using another verified method for δ13 C analysis.
CONCLUSIONS: The good separation performance of hydrophilic AAs and the reliability of δ13 C and δ15 N analyses of individual AAs using the porous graphite column offer a significant advantage over conventional settings. We suggest that, in the future, the HPLC × EA/IRMS method can be used for reliable δ13 C and δ15 N analyses of AAs in natural samples.

DOI: https://doi.org/10.1002/rcm.9602
MethodsX. 2023 Dec;11 102306

A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples.

Marta Zurawska, Mark Basik, Adriana Aguilar-Mahecha, Michal Dadlez, Dominik Domanski.

  We present a method and a simple system for high-pH RP-LC peptide fractionation of small sample amounts (30-60 µg), at micro-flow rates with micro-liter fraction collection using ammonium bicarbonate as an optimized buffer for system stability and robustness. The method is applicable to targeted mass spectrometry approaches and to in-depth proteomic studies where the amount of sample is limited. Using targeted proteomics with peptide standards, we present the method's analytical parameters, and potential in increasing the detection of low-abundance proteins that are difficult to quantify with direct targeted or global LC-MS analyses. This fractionation system increased peptide signals by up to 18-fold, while maintaining high quantitative precision, with high fractionation reproducibility across varied sample sets. In real applications, it increased the detection of targeted endogenous peptides by two-fold in a 25 cell-cycle-control protein panel, and in-depth MS analyses of nuclear extracts, it allowed the detection of up to 8,896 proteins with 138,417 peptides in 24-concatenated fractions compared to 3,344 proteins with 23,093 peptides without fractionation. In a relevant biological problem of CDK4/6-inhibitors and breast cancer, the method reproduced known information and revealed novel insights, highlighting that it can be successfully applied in studies involving low-abundance proteins and limited samples. •Tested nine high-pH buffer/solvent systems to obtain a robust, effective, and reproducible micro-flow fractionation method which was devoid of commonly encountered LC clogging/pressure issues after months of use.•Peptide enrichment method to improve detection and quantitation of low-abundance proteins in targeted and in-depth proteomic studies.•Can be applied to diverse protein samples where the available amount is limited.

Keywords:  CDK4/6 inhibitors; Cell-cycle; Global proteomics; High-pH reversed phase liquid chromatography; Micro-flow high-pH reversed-phase LC system for peptide fractionation and collection; Multiple reaction monitoring (MRM); Parallel reaction monitoring (PRM); Peptide fractionation; Proteomics; Targeted proteomics; Two-dimensional peptide separation

DOI:  https://doi.org/10.1016/j.mex.2023.102306
STAR Protoc. 2023 Aug 17. pii: S2666-1667(23)00473-2. [Epub ahead of print]4(3): 102506

Protocol to track the biosynthesis of cholesterol in cultured HCC cells using 13C compound-specific stable isotopic tracers.

Jonathan D Cybulski, Kit Sum Leung, Carmen Oi Ning Leung, David M Baker, Terence Kin Wah Lee.

  Cholesterol biosynthesis supports proliferation and drives resistance to tyrosine kinase inhibitor (TKI) therapy in hepatocellular carcinoma (HCC). Here, we present a protocol for using stable isotopic tracers to track the biosynthesis of cholesterol in cultured HCC cells. We describe steps for cell preparation, incubation, separation, and homogenization. We then detail lipid extraction and compound-specific isotope analysis for comparing and quantifying cholesterol synthesis between TKI-resistant HCC cells and their mock counterparts. This protocol can be expanded for use with other shorter-chained lipids.

Keywords:  Cancer; Cell Culture; Chemistry; Metabolism

DOI:  https://doi.org/10.1016/j.xpro.2023.102506
bioRxiv. 2023 Aug 01. pii: 2023.07.31.551363. [Epub ahead of print]

A robust method for measuring aminoacylation through tRNA-Seq.

Kristian Davidsen, Lucas B Sullivan.

Current methods to quantify the fraction of aminoacylated tRNAs, also known as the tRNA charge, are limited by issues with either low throughput, precision, and/or accuracy. Here, we present an optimized charge tRNA-Seq method that combines previous developments with newly described approaches to establish a protocol for precise and accurate tRNA charge measurements. We verify that this protocol provides robust quantification of tRNA aminoacylation and we provide an end-to-end method that scales to hundreds of samples including software for data processing. Additionally, we show that this method supports measurements of relative tRNA expression levels and can be used to infer tRNA modifications through reverse transcription misincorporations, thereby supporting multipurpose applications in tRNA biology.

DOI: https://doi.org/10.1101/2023.07.31.551363