bims-mascan 2025-01-05 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2025–01–05
eightteen papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz

diaTracer enables spectrum-centric analysis of diaPASEF proteomics data.
Glial Biologist's Guide to Mass Spectrometry-Based Lipidomics: A Tutorial From Sample Preparation to Data Analysis.
Lipid metabolism in ferroptosis: Unraveling key mechanisms and therapeutic potential in cancer.
Aspartate signalling drives lung metastasis via alternative translation.
Enalomics: A Mass Spectrometry-Based Approach for Profiling, Identifying, and Semiquantifying Enals in Biological Samples.
Concatemer-assisted stoichiometry analysis: targeted mass spectrometry for protein quantification.
A Multiyear Longitudinal Harmonization Study of Quality Controls in Mass Spectrometry Proteomics Core Facilities.
Untargeted metabolomics combined with lipidomics revealed the effects of myocardial infarction and exercise rehabilitation on blood circulation metabolism of patients based on liquid chromatography-mass spectrometry.
Universal, untargeted detection of bacteria in tissues using metabolomics workflows.
Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics.
Comprehensive Mass Spectral Libraries of Human Thyroid Tissues and Cells.
Development and thorough evaluation of a multi-omics sample preparation workflow for comprehensive LC-MS/MS-based metabolomics, lipidomics and proteomics datasets.
Development of a liquid chromatography-mass spectrometry based targeted metabolomics method for discovering diagnostic biomarkers in Kawasaki disease.
Exogenous fatty acids inhibit fatty acid synthesis by competing with endogenously generated substrates for phospholipid synthesis in Escherichia coli.
Muscle Biopsy Sample Preparation and Proteomics Analysis Based on UHPLC-MS/MS.
Standardized workflow for multiplexed charge detection mass spectrometry on orbitrap analyzers.
Streamlined Vitamin D Metabolite Fingerprinting Analysis Using Isotope-Coded Multiplexing MS with Cost-Effective One-Pot Double Derivatization.
Deficiency of the mitochondrial transporter SLC25A47 minimally impacts hepatic lipid metabolism in fasted and diet-induced obese mice.

Nat Commun. 2025 Jan 02. 16(1): 95

diaTracer enables spectrum-centric analysis of diaPASEF proteomics data.

Kai Li, Guo Ci Teo, Kevin L Yang, Fengchao Yu, Alexey I Nesvizhskii.

Data-independent acquisition has become a widely used strategy for peptide and protein quantification in liquid chromatography-tandem mass spectrometry-based proteomics studies. The integration of ion mobility separation into data-independent acquisition analysis, such as the diaPASEF technology available on Bruker's timsTOF platform, further improves the quantification accuracy and protein depth achievable using data-independent acquisition. We introduce diaTracer, a spectrum-centric computational tool optimized for diaPASEF data. diaTracer performs three-dimensional (mass to charge ratio, retention time, ion mobility) peak tracing and feature detection to generate precursor-resolved "pseudo-tandem mass spectra", facilitating direct ("spectral-library free") peptide identification and quantification from diaPASEF data. diaTracer is available as a stand-alone tool and is fully integrated into the widely used FragPipe computational platform. We demonstrate the performance of diaTracer and FragPipe using diaPASEF data from triple-negative breast cancer, cerebrospinal fluid, and plasma samples, data from phosphoproteomics and human leukocyte antigens immunopeptidomics experiments, and low-input data from a spatial proteomics study. We also show that diaTracer enables unrestricted identification of post-translational modifications from diaPASEF data using open/mass-offset searches.

DOI: https://doi.org/10.1038/s41467-024-55448-8
Glia. 2025 Jan 03.

Glial Biologist's Guide to Mass Spectrometry-Based Lipidomics: A Tutorial From Sample Preparation to Data Analysis.

Caitlin E Randolph, Katherine A Walker, Ruilin Yu, Connor Beveridge, Palak Manchanda, Gaurav Chopra.

  Neurological diseases are associated with disruptions in the brain lipidome that are becoming central to disease pathogenesis. Traditionally perceived as static structural support in membranes, lipids are now known to be actively involved in cellular signaling, energy metabolism, and other cellular activities involving membrane curvature, fluidity, fusion or fission. Glia are critical in the development, health, and function of the brain, and glial regulation plays a major role in disease. The major pathways of glial dysregulation related to function are associated with downstream products of metabolism including lipids. Taking advantage of significant innovations and technical advancements in instrumentation, lipidomics has emerged as a popular omics discipline, serving as the prevailing approach to comprehensively define metabolic alterations associated with organismal development, damage or disease. A key technological platform for lipidomics studies is mass spectrometry (MS), as it affords large-scale profiling of complex biological samples. However, as MS-based techniques are often refined and advanced, the relative comfort level among biologists with this instrumentation has not followed suit. In this review, we aim to highlight the importance of the study of glial lipids and to provide a concise record of best practices and steps for MS-based lipidomics. Specifically, we outline procedures for glia lipidomics workflows ranging from sample collection and extraction to mass spectrometric analysis to data interpretation. To ensure these approaches are more accessible, this tutorial aims to familiarize glia biologists with sample handling and analysis techniques for MS-based lipidomics, and to guide non-experts toward generating high quality lipidomics data.

Keywords:  glial lipidomics; lipid identification; lipidomics data analysis; lipids in neurological disease; mass spectrometry‐based lipidomics; sample preparation

DOI:  https://doi.org/10.1002/glia.24665
Biochim Biophys Acta Rev Cancer. 2024 Dec 31. pii: S0304-419X(24)00189-6. [Epub ahead of print]1880(1): 189258

Lipid metabolism in ferroptosis: Unraveling key mechanisms and therapeutic potential in cancer.

Jaewang Lee, Jong-Lyel Roh.

  Ferroptosis, a form of iron-dependent cell death driven by lipid peroxidation, has emerged as a critical area of research for cancer therapy. This review delves into the intricate relationship between lipid metabolism and ferroptosis, emphasizing the impact of lipidome remodeling on cancer cell susceptibility. We explore key mechanisms, such as the role of polyunsaturated fatty acids and phosphatidylethanolamines in ferroptosis induction, alongside the protective effects of monounsaturated fatty acids and their regulatory enzymes. We also discuss the influence of dietary fatty acids, lipid droplets, and the epithelial-to-mesenchymal transition on ferroptosis and cancer resistance. By integrating current findings on enzymatic regulation, lipid peroxidation pathways, and metabolic adaptations, this review highlights potential therapeutic strategies targeting lipid metabolism to enhance ferroptosis-based cancer treatments. Our goal is to provide a comprehensive overview that underscores the significance of lipid metabolic pathways in ferroptosis and their implications for developing novel cancer therapies.

Keywords:  Ferroptosis; Lipid metabolism; Lipid peroxidation; Lipid remodeling; cancer

DOI:  https://doi.org/10.1016/j.bbcan.2024.189258
Nature. 2025 Jan 01.

Aspartate signalling drives lung metastasis via alternative translation.

Ginevra Doglioni, Juan Fernández-García, Sebastian Igelmann, Patricia Altea-Manzano, Arnaud Blomme, Rita La Rovere, Xiao-Zheng Liu, Yawen Liu, Tine Tricot, Max Nobis, Ning An, Marine Leclercq, Sarah El Kharraz, Panagiotis Karras, Yu-Heng Hsieh, Fiorella A Solari, Luiza Martins Nascentes Melo, Gabrielle Allies, Annalisa Scopelliti, Matteo Rossi, Ines Vermeire, Dorien Broekaert, Ana Margarida Ferreira Campos, Patrick Neven, Marion Maetens, Karen Van Baelen, H Furkan Alkan, Mélanie Planque, Giuseppe Floris, Albert Sickmann, Alpaslan Tasdogan, Jean-Christophe Marine, Colinda L G J Scheele, Christine Desmedt, Geert Bultynck, Pierre Close, Sarah-Maria Fendt.

Lung metastases occur in up to 54% of patients with metastatic tumours1,2. Contributing factors to this high frequency include the physical properties of the pulmonary system and a less oxidative environment that may favour the survival of cancer cells3. Moreover, secreted factors from primary tumours alter immune cells and the extracellular matrix of the lung, creating a permissive pre-metastatic environment primed for the arriving cancer cells4,5. Nutrients are also primed during pre-metastatic niche formation6. Yet, whether and how nutrients available in organs in which tumours metastasize confer cancer cells with aggressive traits is mostly undefined. Here we found that pulmonary aspartate triggers a cellular signalling cascade in disseminated cancer cells, resulting in a translational programme that boosts aggressiveness of lung metastases. Specifically, we observe that patients and mice with breast cancer have high concentrations of aspartate in their lung interstitial fluid. This extracellular aspartate activates the ionotropic N-methyl-D-aspartate receptor in cancer cells, which promotes CREB-dependent expression of deoxyhypusine hydroxylase (DOHH). DOHH is essential for hypusination, a post-translational modification that is required for the activity of the non-classical translation initiation factor eIF5A. In turn, a translational programme with TGFβ signalling as a central hub promotes collagen synthesis in lung-disseminated breast cancer cells. We detected key proteins of this mechanism in lung metastases from patients with breast cancer. In summary, we found that aspartate, a classical biosynthesis metabolite, functions in the lung environment as an extracellular signalling molecule to promote aggressiveness of metastases.

DOI: https://doi.org/10.1038/s41586-024-08335-7
Anal Chem. 2025 Jan 02.

Enalomics: A Mass Spectrometry-Based Approach for Profiling, Identifying, and Semiquantifying Enals in Biological Samples.

Congyu Ma, Wen Zhang, Jialong Jing, Zhe Wang, Ning Sheng, Zhuoling An, Jinlan Zhang.

Human cells generate a bulk of aldehydes during lipid peroxidation (LPO), influencing critical cellular processes, such as oxidative stress, protein modification, and DNA damage. Enals, highly reactive α,β-unsaturated aldehydic metabolites, are implicated in various human pathologies, especially neurodegenerative disorders, cancer, and cardiovascular diseases. Despite their importance, endogenous enals remain poorly characterized, primarily due to their instability and low abundance. Herein, we introduced "enalomics," a mass spectrometry (MS)-based approach for profiling, identifying, and semiquantifying enals in biological samples. Derivatization with 2,4-dinitrophenylhydrazine and treatment with ascorbic acid stabilized enals in biological matrices and provided a unique MS fragment ([M-H-47]-) for reliable enal identification. Utilizing precursor ion scanning, dynamic multiple reaction monitoring, high-resolution MS, and mathematical correlations between retention times and carbon numbers of enals, we identified 157 enals (127 newly reported) with tissue-specific profiles in rats and 29 enals (24 newly reported) in human plasma. To the best of our knowledge, this represents the comprehensive analysis of enals, i.e., "enalomics," in biological samples. Enalomics demonstrated significant alterations in enal metabolism in rats with myocardial injury, highlighting the potential of medium- and short-chain plasma enals as sensitive diagnostic biomarkers. Further application of enalomics in patients with myocardial infarction (MI) identified 14 plasma diagnostic biomarkers. Receiver operating characteristic curves showed good discrimination (area under curve ≥ 0.8603, p ≤ 0.0043). This research advances the understanding of LPO products and emphasizes the roles of enals in human diseases, offering good prospects for early screening, diagnosis, and clinical interventions targeting LPO products in MI patients.

DOI: https://doi.org/10.1021/acs.analchem.4c02842
Life Sci Alliance. 2025 Mar;pii: e202403007. [Epub ahead of print]8(3):

Concatemer-assisted stoichiometry analysis: targeted mass spectrometry for protein quantification.

Jiaxi Cai, Yun Quan, Cindy Yuxuan Zhang, Ziyi Wang, Stephen M Hinshaw, Huilin Zhou, Raymond T Suhandynata.

Large multiprotein machines are central to many biological processes. However, stoichiometric determination of protein complex subunits in their native states presents a significant challenge. This study addresses the limitations of current tools in accuracy and precision by introducing concatemer-assisted stoichiometry analysis (CASA). CASA leverages stable isotope-labeled concatemers and liquid chromatography-parallel reaction monitoring-mass spectrometry (LC-PRM-MS) to achieve robust quantification of proteins with sub-femtomole sensitivity. As a proof of concept, CASA was applied to study budding yeast kinetochores. Stoichiometries were determined for ex vivo reconstituted kinetochore components, including the canonical H3 nucleosomes, centromeric (Cse4CENP-A) nucleosomes, centromere proximal factors (Cbf1 and CBF3 complex), inner kinetochore proteins (Mif2CENP-C, Ctf19CCAN complex), and outer kinetochore proteins (KMN network). Absolute quantification by CASA revealed Cse4CENP-A as a cell cycle-controlled limiting factor for kinetochore assembly. These findings demonstrate that CASA is applicable for stoichiometry analysis of multiprotein assemblies.

DOI: https://doi.org/10.26508/lsa.202403007
J Proteome Res. 2025 Jan 01.

A Multiyear Longitudinal Harmonization Study of Quality Controls in Mass Spectrometry Proteomics Core Facilities.

Cristina Chiva, Roger Olivella, An Staes, Teresa Mendes Maia, Christian Panse, Karel Stejskal, Thibaut Douché, Bérangère Lombard, Andrea Schuhmann, Damarys Loew, Karl Mechtler, Mariette Matondo, Mandy Rettel, Dominic Helm, Francis Impens, Simon Devos, Anna Shevchenko, Paolo Nanni, Eduard Sabidó.

  Quality control procedures play a pivotal role in ensuring the reliability and consistency of data generated in mass spectrometry-based proteomics laboratories. However, the lack of standardized quality control practices across laboratories poses challenges for data comparability and reproducibility. In response, we conducted a harmonization study within proteomics laboratories of the Core for Life alliance with the aim of establishing a common quality control framework, which facilitates comprehensive quality assessment and identification of potential sources of performance drift. Through collaborative efforts, we developed a consensus quality control standard for longitudinal assessment and adopted common processing software. We generated a 4-year longitudinal data set from multiple instruments and laboratories, which enabled us to assess intra- and interlaboratory variability, to identify causes of performance drift, and to establish community reference values for several quality control parameters. Our study enhances data comparability and reliability and fosters a culture of collaboration and continuous improvement within the proteomics community to ensure the integrity of proteomics data.

Keywords:  liquid chromatography; longitudinal study; mass spectrometry; proteomics; quality control

DOI:  https://doi.org/10.1021/acs.jproteome.4c00359
J Pharm Biomed Anal. 2024 Dec 26. pii: S0731-7085(24)00693-9. [Epub ahead of print]255 116651

Untargeted metabolomics combined with lipidomics revealed the effects of myocardial infarction and exercise rehabilitation on blood circulation metabolism of patients based on liquid chromatography-mass spectrometry.

Na Wang, Huimin Li, Hao Wu, Zilin Xia, Dabing Ren, Yunmei Zhang, Yan Zhao, Hong Zhang, Ke Zhuang, Lunzhao Yi.

  Myocardial infarction (MI) is a major cause of death worldwide. Exercise rehabilitation (ER) is a powerful tool to improve life quality and prognosis of MI patients. Herein, we developed an untargeted metabolomics combined with lipidomics method to qualitatively and quantitatively detect metabolites in plasma. A total of 475 metabolites were annotated according to MS, MS/MS, and quantified by internal standard method. Moreover, medical statistical methods combined with chemometrics were used for metabolomics data mining and interpretation of clinical issues (matched Cohort 1, n = 90, Cohort 2, n = 6). The results illustrated that abnormal lipid metabolism is the most significant metabolic disorder for MI patients. And, three metabolic pathways, bile secretion, HIF-1 signaling pathway, and glutathione metabolism were uncovered in MI patients. Furthermore, glutamine, Phenylacetylglutamine (PAGln) and lysophosphatidylcholine (LPCs) were revealed as the essential biomarkers for ER of MI patients. Our findings revealed the metabolic landscape of MI and metabolic alterations after ER, will underlay potential applications of plasma metabolites in the detection of MI and optimization of ER program.

Keywords:  Etabolomics; Exercise rehabilitation; LC-MS; Lipidomics; Myocardial infarction

DOI:  https://doi.org/10.1016/j.jpba.2024.116651
Nat Commun. 2025 Jan 02. 16(1): 165

Universal, untargeted detection of bacteria in tissues using metabolomics workflows.

Wei Chen, Min Qiu, Petra Paizs, Miriam Sadowski, Toma Ramonaite, Lieby Zborovsky, Raquel Mejias-Luque, Klaus-Peter Janßen, James Kinross, Robert D Goldin, Monica Rebec, Manuel Liebeke, Zoltan Takats, James S McKenzie, Nicole Strittmatter.

Fast and reliable identification of bacteria directly in clinical samples is a critical factor in clinical microbiological diagnostics. Current approaches require time-consuming bacterial isolation and enrichment procedures, delaying stratified treatment. Here, we describe a biomarker-based strategy that utilises bacterial small molecular metabolites and lipids for direct detection of bacteria in complex samples using mass spectrometry (MS). A spectral metabolic library of 233 bacterial species is mined for markers showing specificity at different phylogenetic levels. Using a univariate statistical analysis method, we determine 359 so-called taxon-specific markers (TSMs). We apply these TSMs to the in situ detection of bacteria using healthy and cancerous gastrointestinal tissues as well as faecal samples. To demonstrate the MS method-agnostic nature, samples are analysed using spatial metabolomics and traditional bulk-based metabolomics approaches. In this work, TSMs are found in >90% of samples, suggesting the general applicability of this workflow to detect bacterial presence with standard MS-based analytical methods.

DOI: https://doi.org/10.1038/s41467-024-55457-7
Nat Commun. 2025 Jan 02. 16(1): 73

Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics.

George S Biggs, Emma E Cawood, Aini Vuorinen, William J McCarthy, Harry Wilders, Ioannis G Riziotis, Antonie J van der Zouwen, Jonathan Pettinger, Luke Nightingale, Peiling Chen, Andrew J Powell, David House, Simon J Boulton, J Mark Skehel, Katrin Rittinger, Jacob T Bush.

Identifying pharmacological probes for human proteins represents a key opportunity to accelerate the discovery of new therapeutics. High-content screening approaches to expand the ligandable proteome offer the potential to expedite the discovery of novel chemical probes to study protein function. Screening libraries of reactive fragments by chemoproteomics offers a compelling approach to ligand discovery, however, optimising sample throughput, proteomic depth, and data reproducibility remains a key challenge. We report a versatile, label-free quantification proteomics platform for competitive profiling of cysteine-reactive fragments against the native proteome. This high-throughput platform combines SP4 plate-based sample preparation with rapid chromatographic gradients. Data-independent acquisition performed on a Bruker timsTOF Pro 2 consistently identified ~23,000 cysteine sites per run, with a total of ~32,000 cysteine sites profiled in HEK293T and Jurkat lysate. Crucially, this depth in cysteinome coverage is met with high data completeness, enabling robust identification of liganded proteins. In this study, 80 reactive fragments were screened in two cell lines identifying >400 ligand-protein interactions. Hits were validated through concentration-response experiments and the platform was utilised for hit expansion and live cell experiments. This label-free platform represents a significant step forward in high-throughput proteomics to evaluate ligandability of cysteines across the human proteome.

DOI: https://doi.org/10.1038/s41467-024-55057-5
Sci Data. 2024 Dec 28. 11(1): 1448

Comprehensive Mass Spectral Libraries of Human Thyroid Tissues and Cells.

Lu Li, Wenhao Jiang, Wenjun Wei, Gnana P Krishnamoorthy, Pingping Hu, Mengni Chen, Vera Tiedje, Adrián Acuña-Ruiz, He Wang, Zhihong Wang, Jianbiao Wang, Hanqing Liu, Wanyuan Chen, Haixia Guan, Chuang Chen, Hao Zhang, Yu Wang, James A Fagin, Tiannan Guo, Yi Zhu, Yingrui Wang, Jianqing Gao, Yaoting Sun.

Thyroid nodules are a common endocrine condition with an increasing incidence over the decades. Data-independent acquisition has been widely utilized in discovery proteomics to identify disease biomarkers and therapeutic targets. To analyze the thyroid disease-related proteome in a high-throughput, reproducible and reliable manner, we introduce thyroid-specific peptide spectral libraries. Here, we generated four deep-coverage libraries through four mass spectrometers comprising Q Exactive HF, Orbitrap Exploris 480, ZenoTOF 7600, and timsTOF Pro. These libraries encompass over 215,000 precursors, 172,000 peptides, and 12,000 proteins, derived from 245 tissue samples across nine histological types and 50 cell lines across six histological types. Moreover, the spectral libraries are applied to nine types of thyroid samples to facilitate the exploration of disease-specific proteins. Our spectral libraries serve as a valuable resource for analyzing thyroid protein content, facilitating deeper insights into thyroid disorders.

DOI: https://doi.org/10.1038/s41597-024-04322-9
Talanta. 2024 Dec 24. pii: S0039-9140(24)01824-1. [Epub ahead of print]286 127442

Development and thorough evaluation of a multi-omics sample preparation workflow for comprehensive LC-MS/MS-based metabolomics, lipidomics and proteomics datasets.

Lana Brockbals, Maiken Ueland, Shanlin Fu, Matthew P Padula.

  The importance of sample preparation selection if often overlooked particularly for untargeted multi-omics approaches that gained popularity in recent years. To minimize issues with sample heterogeneity and additional freeze-thaw cycles during sample splitting, multiple -omics datasets (e.g. metabolomics, lipidomics and proteomics) should ideally be generated from the same set of samples. For sample extraction, commonly biphasic organic solvent systems are used that require extensive multi-step protocols. Individual studies have recently also started to investigate monophasic (all-in-one) extraction procedures. The aim of the current study was to develop and systematically compare ten different mono- and biphasic extraction solvent mixtures for their potential to aid in the most comprehensive metabolomics, lipidomics and proteomics datasets. As the focus was on human postmortem tissue samples (muscle and liver tissue), four tissue homogenization parameters were also evaluated. Untargeted liquid chromatography mass spectrometry-based metabolomics, lipidomic and proteomics methods were utilized along with 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and bicinchoninic acid (BCA) assay results. Optimal homogenization was found to be achieved by bead-homogenizing 20 mg of muscle or liver tissue with 200 μL (1:10 ratio) Water:Methanol (1:2) using 3 × 30 s pulses. The supernatant of the homogenate was further extracted. Comprehensive ranking, taking nine different processing parameters into account, showed that the monophasic extraction solvents, overall, showed better scores compared to the biphasic solvent systems, despite their recommendation for one or all of the -omics extractions. The optimal extraction solvent was found to be Methanol:Acetone (9:1), resulting in the most comprehensive metabolomics, lipidomics and proteomics datasets, showing the potential to be automated, hence, allowing for high-throughput analysis of samples and opening the door for comprehensive multi-omics results from routine clinical cases in the future.

Keywords:  Compound class identification; LC-MS/MS; Multi-omics; Postmortem tissue extraction; monophasic (all-in-one) extraction; untargeted -omics

DOI:  https://doi.org/10.1016/j.talanta.2024.127442
J Chromatogr A. 2024 Dec 20. pii: S0021-9673(24)00992-0. [Epub ahead of print]1741 465619

Development of a liquid chromatography-mass spectrometry based targeted metabolomics method for discovering diagnostic biomarkers in Kawasaki disease.

Xialin Luo, Jiaqi Tian, Qing Li, Zhonggan Jin, Xiaoyu Fan, Hong Zhang, Haitao Lv, Yi Ju.

  Kawasaki disease (KD) has emerged as the leading cause of acquired heart disease in children, primarily due to the absence of highly sensitive and specific biomarkers for early and accurate diagnosis. To address this issue, a simple and comprehensive targeted metabolomics method employing ultra high-performance liquid chromatography coupled with Q-TRAP mass spectrometry has been developed to identify new metabolite biomarkers for KD. This method enables the simultaneous quantification of 276 metabolites, covering 60 metabolic pathways, with a particular emphasis on metabolites relevant to KD. The use of nine ISs and commercial quality control samples significantly enhances both accuracy and precision. Through validation and application to serum samples from patients with KD, seventeen differential serum metabolites were identified. The altered metabolites are primarily associated with three functional metabolic pathways: tricarboxylic acid cycle, tryptophan metabolism, and bile acid metabolism, all of which are believed to be involved in the inflammatory and immune responses in KD patients. Ultimately, eight differential metabolites (indole-3-propionic acid, thiamine, indolepyruvic acid, levodopa, l-selenomethionine, isocitric acid, trans-aconitate, and N-acetylasparagine) were identified that could potentially serve as diagnostic biomarkers with the area under the curve values exceeding 0.9. Our targeted metabolomics approach demonstrates applicability in identifying potential metabolite biomarkers for KD and holds great promise in unraveling the intricate pathophysiology of the disease.

Keywords:  Kawasaki disease; Liquid chromatography; Mass spectrometry; Metabolite biomarkers; Metabolomics

DOI:  https://doi.org/10.1016/j.chroma.2024.465619
FEBS Lett. 2024 Dec 30.

Exogenous fatty acids inhibit fatty acid synthesis by competing with endogenously generated substrates for phospholipid synthesis in Escherichia coli.

Stefan Pieter Hendrik van den Berg, Adja Zoumaro-Djayoon, Flora Yang, Gregory Bokinsky.

  Exogenous fatty acids are directly incorporated into bacterial membranes, heavily influencing cell envelope properties, antibiotic susceptibility, and bacterial ecology. Here, we quantify fatty acid biosynthesis metabolites and enzymes of the fatty acid synthesis pathway to determine how exogenous fatty acids inhibit fatty acid synthesis in Escherichia coli. We find that acyl-CoA synthesized from exogenous fatty acids rapidly increases concentrations of long-chain acyl-acyl carrier protein (acyl-ACP), which inhibits fatty acid synthesis initiation. Accumulation of long-chain acyl-ACP is caused by competition with acyl-CoA for phospholipid synthesis enzymes. Furthermore, we find that transcriptional regulation rebalances saturated and unsaturated acyl-ACP while maintaining overall expression levels of fatty acid synthesis enzymes. Rapid feedback inhibition of fatty acid synthesis by exogenous fatty acids thus allows E. coli to benefit from exogenous fatty acids while maintaining fatty acid synthesis capacity. We hypothesize that this indirect feedback mechanism is ubiquitous across bacterial species.

Keywords:  acyl carrier protein; exogenous fatty acids; fatty acid synthesis; homeoviscous adaptation; post‐translational regulation

DOI:  https://doi.org/10.1002/1873-3468.15092
Bio Protoc. 2024 Dec 20. 14(24): e5137

Muscle Biopsy Sample Preparation and Proteomics Analysis Based on UHPLC-MS/MS.

Jiawei Du, Jinghua Hou, Hezhang Yun, Yafeng Song.

  Proteomics analysis is crucial for understanding the molecular mechanisms underlying muscle adaptations to different types of exercise, such as concentric and eccentric training. Traditional methods like two-dimensional gel electrophoresis and standard mass spectrometry have been used to analyze muscle protein content and modifications. This protocol details the preparation of muscle samples for proteomics analysis using ultra-high-performance liquid chromatography (UHPLC). It includes steps for muscle biopsy collection, protein extraction, digestion, and UHPLC-based analysis. The UHPLC method offers high-resolution separation of complex protein mixtures, providing more detailed and accurate proteomic profiles compared to conventional techniques. This protocol significantly enhances sensitivity, reproducibility, and efficiency, making it ideal for comprehensive muscle proteomics studies. Key features • Developed for analyzing muscle adaptations in response to concentric and eccentric training, applicable to various physiology exercise studies. • Utilizes UHPLC-MS/MS for high-resolution separation and detailed proteomic profiling. • Requires access to advanced UHPLC-MS/MS equipment and muscle biopsy collection tools. • The protocol can be completed within one week, including sample preparation and analysis.

Keywords:  Concentric exercise; Eccentric exercise; Muscle biopsy; Muscle proteomics; UHPLC-MS/MS

DOI:  https://doi.org/10.21769/BioProtoc.5137
Nat Protoc. 2025 Jan 02.

Standardized workflow for multiplexed charge detection mass spectrometry on orbitrap analyzers.

Pei Su, John P McGee, Michael A R Hollas, Ryan T Fellers, Kenneth R Durbin, Joseph B Greer, Bryan P Early, Ping F Yip, Vlad Zabrouskov, Kristina Srzentić, Michael W Senko, Philip D Compton, Neil L Kelleher, Jared O Kafader.

Individual ion mass spectrometry (I2MS) is the Orbitrap-based extension of the niche mass spectrometry technique known as charge detection mass spectrometry (CDMS). While traditional CDMS analysis is performed on in-house-built instruments such as the electrostatic linear ion trap, I2MS extends CDMS analysis to Orbitrap analyzers, allowing charge detection analysis to be available to the scientific community at large. I2MS simultaneously measures the mass-to-charge ratios (m/z) and charges (z) of hundreds to thousands of individual ions within one acquisition event, creating a spectral output directly into the mass domain without the need for further spectral deconvolution. A mass distribution or 'profile' can be created for any desired sample regardless of composition or heterogeneity. To assist in reducing I2MS analysis to practice, we developed this workflow for data acquisition and subsequent data analysis, which includes (i) protein sample preparation, (ii) attenuation of ion signals to obtain individual ions, (iii) the creation of a charge-calibration curve from standard proteins with known charge states and finally (iv) producing a meaningful mass spectral output from a complex or unknown sample by using the STORIboard software. This protocol is suitable for users with prior experience in mass spectrometry and bioanalytical chemistry. First, the analysis of protein standards in native and denaturing mode is presented, setting the foundation for the analysis of complex mixtures that are intractable via traditional mass spectrometry techniques. Examples of complex mixtures included here demonstrate the relevant analysis of an intact human monoclonal antibody and its intricate glycosylation patterns.

DOI: https://doi.org/10.1038/s41596-024-01091-y
ACS Omega. 2024 Dec 24. 9(51): 50660-50670

Streamlined Vitamin D Metabolite Fingerprinting Analysis Using Isotope-Coded Multiplexing MS with Cost-Effective One-Pot Double Derivatization.

Pascal Schorr, Caroline S Stokes, Dietrich A Volmer.

In this study, we extended a previously developed one-pot double derivatization reaction to establish the first routine isotope-coded multiplex derivatization for vitamin D and its metabolites for application in clinical environments, using commercial reagents, without the need for specialized reagents and advanced synthesis requirements. The original derivatization process consisted of using both a Cookson-type reagent and derivatization of hydroxyl groups. Initially, the analytes are derivatized by a Diels-Alder reaction using 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), followed by acetylation using acetic anhydride, catalyzed by 4-dimethylaminopyridine at room temperature. To enable sample multiplexing, we utilized acetic anhydride as well as the d 3- isotopologue of acetic anhydride, generating d 3- and d 6-products of the investigated vitamin D3 metabolites. This approach not only allowed for the simultaneous measurement of two samples within a single LC-MS/MS run but also improved the LC separation of the important 25-hydroxyvitamin D3 epimers (3α-25(OH)D3 and 3β-25(OH)D3) on a conventional C-18 column, addressing a significant challenge in vitamin D analysis. Typically, the separation of these epimers after PTAD derivatization cannot be performed on C-18 columns, necessitating the use of pentafluorophenylpropyl (PFP) stationary phases. However, PFP columns are not as stable as C-18 in long-term use, wherein the acetylation of the C-3 hydroxyl group provided a solution by enhancing chromatographic selectivity and achieving the baseline separation of the metabolites 24,25(OH)2D3, 3α-25(OH)D3, 3β-25(OH)D3, and vitamin D3 using a C-18 column with methanol/water gradient elution. The described duplex derivatization was tested on 40 serum samples of patients with chronic liver diseases (CLD). Additionally, the method was evaluated in terms of linearity, accuracy, precision, and interferences between heavy and light tag samples using both commercial quality control samples and in-house quality control and calibration samples.

DOI: https://doi.org/10.1021/acsomega.4c08675
Mol Metab. 2024 Dec 31. pii: S2212-8778(24)00223-0. [Epub ahead of print] 102092

Deficiency of the mitochondrial transporter SLC25A47 minimally impacts hepatic lipid metabolism in fasted and diet-induced obese mice.

Brecht Attema, Montserrat A de la Rosa Rodriguez, Evert M van Schothorst, Sander Grefte, Guido Jej Hooiveld, Sander Kersten.

The peroxisome proliferator-activated receptor-alpha (PPARα) plays a central role in lipid metabolism in the liver by stimulating the expression of hundreds of genes. Accordingly, regulation by PPARα could be a screening tool to identify novel genes involved in hepatic lipid metabolism. Previously, the mitochondrial transporter SLC25A47 was suggested to play a role in energy metabolism and liver-specific uncoupling, but further research is lacking. Here, we identify SLC25A47 as a PPARα-regulated and fasting-induced gene in human and mouse hepatocytes. We explored the potential role of SLC25A47 using mice overexpressing and lacking SLC25A47. Adenoviral-mediated overexpression of SLC25A47 minimally impacted metabolic parameters during fasting and high-fat feeding. During high-fat feeding, SLC25A47 ablation also did not influence any metabolic parameters, apart from a minor improvement in glucose tolerance. In fasted mice, SLC25A47 ablation was associated with modest, reproducible, and likely indirect reductions in plasma triglycerides and glycerol. SLC25A47 ablation did not influence energy expenditure. Depending on the nutritional status, metabolomics showed modest alterations in plasma, liver, and hepatic mitochondrial levels of various metabolites related to amino acid metabolism, TCA cycle, and fatty acid metabolism. No major and consistent alterations in levels of specific metabolites were found that establish the substrate for and function of SLC25A47. Collectively, our results hint at a role of SLC25A47 in amino acid and fatty acid metabolism, yet suggest that SLC25A47 is dispensable for hepatic lipid homeostasis during fasting and high-fat feeding.

DOI: https://doi.org/10.1016/j.molmet.2024.102092