bims-mascan 2023-06-04 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2023–06–04
twenty-six papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz

Quantification and Tracing of Stable Isotope into Cysteine and Glutathione.
MetaboMSDIA: A tool for implementing data-independent acquisition in metabolomic-based mass spectrometry analysis.
Targeted Quantification of Amino Acids by Dansylation.
Integrative processing of untargeted metabolomic and lipidomic data using MultiABLER.
13C Isotope Labeling and Mass Spectrometric Isotope Enrichment Analysis in Acute Brain Slices.
Equivalent carbon number based targeted odd chain fatty acyl lipidomics reveals triacylglycerol profiling in clinical colon cancer.
mpwR: an R package for comparing performance of mass spectrometry-based proteomic workflows.
Pancreatic tumors exhibit myeloid-driven amino acid stress and upregulate arginine biosynthesis.
Evaluation of DDA Library-Free Strategies for Phosphoproteomics and Ubiquitinomics Data-Independent Acquisition Data.
Targeted Lipidomics Reveals a Novel Role for Glucosylceramides in Glucose Response.
Quantitation of Glutathione and Oxidized Glutathione Ratios from Biological Matrices Using LC-MS/MS.
An Introduction to Mass Spectrometry-Based Proteomics.
Retinyl Ester Analysis by Orbitrap Mass Spectrometry.
Metabolomics Peak Analysis Computational Tool (MPACT): An Advanced Informatics Tool for Metabolomics and Data Visualization of Molecules from Complex Biological Samples.
Lipid metabolism and tumor immunotherapy.
Improved Label-Free Quantification of Intact Proteoforms Using Field Asymmetric Ion Mobility Spectrometry.
Profiling of branched chain and straight chain saturated fatty acids by ultra-high performance liquid chromatography-mass spectrometry.
An In Silico Infrared Spectral Library of Molecular Ions for Metabolite Identification.
Orchestral role of lipid metabolic reprogramming in T-cell malignancy.
ALDH1L2 regulation of formate, formyl-methionine, and ROS controls cancer cell migration and metastasis.
Bridging the False Discovery Gap.
Quantitative Phosphoproteome Analysis of the Interaction Between Fusarium graminearum and Triticum aestivum.
MagMet: A fully automated web server for targeted nuclear magnetic resonance metabolomics of plasma and serum.
Targeting Glutamine Metabolism as an Attractive Therapeutic Strategy for Acute Myeloid Leukemia.
UHPLC-HRMS-Based Analysis of S-Hydroxymethyl-Glutathione, GSH, and GSSG in Human Cells.
Dried blood spot analysis with liquid chromatography and mass spectrometry: Trends in clinical chemistry.

Methods Mol Biol. 2023 ;2675 51-63

Quantification and Tracing of Stable Isotope into Cysteine and Glutathione.

Yun Pyo Kang, Gina M DeNicola.

  The analysis of metabolic perturbation in biological samples is crucial to understand mechanisms of metabolic diseases. Here, we describe a protocol for quantitative stable isotope-labeled metabolite tracing of cysteine metabolism in cultured cells. This protocol relies on an extraction protocol to derivatize free thiols to prevent oxidation. In addition, the quantitative tracing of serine into multiple pathways, including the glutathione synthesis pathway, allows for the interrogation of cysteine and glutathione synthesis. This protocol provides a flexible framework that can be adapted to interrogate many metabolites and pathways of interest.

Keywords:  Cell culture; Cysteine; Mass spectrometry; Quantification; Serine; Stable isotope standard; Stable isotope tracing

DOI:  https://doi.org/10.1007/978-1-0716-3247-5_5
Anal Chim Acta. 2023 Jul 25. pii: S0003-2670(23)00529-9. [Epub ahead of print]1266 341308

MetaboMSDIA: A tool for implementing data-independent acquisition in metabolomic-based mass spectrometry analysis.

Carlos Augusto Ledesma-Escobar, Feliciano Priego-Capote, Mónica Calderón-Santiago.

  Data-dependent acquisition (DDA) is the most widely used mode in untargeted metabolomic analysis despite its limited tandem mass spectrometry (MS2) detection coverage. We present MetaboMSDIA for complete processing of data-independent acquisition (DIA) files by the extraction of multiplexed MS2 spectra and further identification of metabolites in open libraries. In the analysis of polar extracts from lemon and olive fruits, DIA allows one to obtain multiplexed MS2 spectra for 100% of precursor ions compared to 64% of precursor ions from average MS2 acquisition in DDA. MetaboMSDIA is compatible with MS2 repositories and homemade libraries prepared by analysis of standards. An additional option is based on filtering molecular entities by searching for selective fragmentation patterns according to selective neutral losses or product ions to target the annotation of families of metabolites. Combining both options, the applicability of MetaboMSDIA was tested by annotating 50 and 35 metabolites in polar extracts from lemon and olive fruit, respectively. MetaboMSDIA is particularly proposed to increase the acquisition coverage in untargeted metabolomics and to improve spectral quality, which are two critical pillars for the tentative annotation of metabolites. The R script used in MetaboMSDIA workflow is available at github repository (https://github.com/MonicaCalSan/MetaboMSDIA).

Keywords:  Data-independent acquisition; Mass spectrometry; Multiplexed MS2 spectra; R package; metabolites annotation

DOI:  https://doi.org/10.1016/j.aca.2023.341308
Methods Mol Biol. 2023 ;2675 65-76

Targeted Quantification of Amino Acids by Dansylation.

Yuanyuan Liu, Haoqing Chen, Dylan Dodd.

  Quantification of amino acids in biological samples is a critical tool for studying metabolism. Although many methods for amino acid analysis exist, important considerations include ease of sample preparation, dynamic range, reproducibility, instrument availability, and throughput. Here, we present a simple, rapid, and robust method for the analysis of amino acids by chemical derivatization and liquid chromatography-mass spectrometry (LC-MS). We provide a detailed protocol for the analysis of 20 proteinogenic amino acids in biological samples which will enable straightforward implementation on modern LC-MS instruments.

Keywords:  Amino acid analysis; Dansylation; Liquid chromatography-mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-3247-5_6
iScience. 2023 Jun 16. 26(6): 106881

Integrative processing of untargeted metabolomic and lipidomic data using MultiABLER.

Ian C H Lee, Sergey Tumanov, Jason W H Wong, Roland Stocker, Joshua W K Ho.

  Mass spectrometry (MS)-based untargeted metabolomic and lipidomic approaches are being used increasingly in biomedical research. The adoption and integration of these data are critical to the overall multi-omic toolkit. Recently, a sample extraction method called Multi-ABLE has been developed, which enables concurrent generation of proteomic and untargeted metabolomic and lipidomic data from a small amount of tissue. The proteomics field has a well-established set of software for processing of acquired data; however, there is a lack of a unified, off-the-shelf, ready-to-use bioinformatics pipeline that can take advantage of and prepare concurrently generated metabolomic and lipidomic data for joint downstream analyses. Here we present an R pipeline called MultiABLER as a unified and simple upstream processing and analysis pipeline for both metabolomics and lipidomics datasets acquired using liquid chromatography-tandem mass spectrometry. The code is available via an open-source license at https://github.com/holab-hku/MultiABLER.

Keywords:  Biological sciences research methodologies; Biological sciences tools; Lipidomics; Metabolomics

DOI:  https://doi.org/10.1016/j.isci.2023.106881
Methods Mol Biol. 2023 ;2675 181-194

13C Isotope Labeling and Mass Spectrometric Isotope Enrichment Analysis in Acute Brain Slices.

Elisa Motori, Patrick Giavalisco.

  Feeding of stable 13C-labeled compounds coupled to mass spectrometric analysis has enabled the characterization of dynamic metabolite partitioning in various experimental conditions. This information is particularly relevant for the study and functional understanding of brain metabolic heterogeneity. We here describe a protocol for the analysis of metabolic enrichment analysis upon feeding of murine acute cerebellar slices with 13C-labeled substrates.

Keywords:  13C labeling; Brain slices; Cellular metabolism; Cerebellum; Isotope enrichment; Liquid chromatography; Mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-3247-5_14
J Lipid Res. 2023 May 29. pii: S0022-2275(23)00066-4. [Epub ahead of print] 100393

Equivalent carbon number based targeted odd chain fatty acyl lipidomics reveals triacylglycerol profiling in clinical colon cancer.

Jiangang Zhang, Shuai Yang, Jingchun Wang, Yanquan Xu, Huakan Zhao, Juan Lei, Yu Zhou, Yu Chen, Lei Wu, Yongsheng Li.

  Odd chain fatty acids (OCFAs) present in very low level at nearly 1% of total fatty acids in human plasma and thus their functions were usually ignored. Recent epidemiological studies have shown that OCFAs are inversely associated with a variety of disease risks. However, the contribution of OCFAs incorporated into complex lipids remains elusive. Here, we developed a targeted odd chain fatty acyl containing lipidomics method based on equivalent carbon number and retention time prediction. The method displayed good reproducibility and robustness as shown by peak width at half height within 0.7 min and coefficient of variation (CV) under 20%. A total number of 776 lipid species with odd chain fatty acyl residues could be detected in the electrospray ionization (ESI) mode of reverse phase liquid chromatography-mass spectrometry, of which 309 lipids were further validated using multiple MRM transitiions. Using this method, we quantified odd chain fatty acyl containing lipidome in tissues from 12 colon cancer patients, revealing the remodeling of triacylglycerol (TAG). The dynamics of odd chain fatty acyl lipids were further consolidated by the association with genomic and proteomic feature of altered catabolism of branched chain amino acids and TAG endogenous synthesis in colon cancer. This lipidomics approach will be applicable for screening of dysregulated odd chain fatty acyl lipids, which enriches and improves the methods for diagnosis and prognosis evaluation of cancer using lipidomics.

Keywords:  Odd chain fatty acids; colon cancer; equivalent carbon number; lipidomics; liquid chromatography-mass spectrometry

DOI:  https://doi.org/10.1016/j.jlr.2023.100393
Bioinformatics. 2023 Jun 02. pii: btad358. [Epub ahead of print]

mpwR: an R package for comparing performance of mass spectrometry-based proteomic workflows.

Oliver Kardell, Stephan Breimann, Stefanie M Hauck.

SUMMARY: mpwR is an R package for a standardized comparison of mass spectrometry (MS)-based proteomic label-free workflows recorded by data-dependent or data-independent spectral acquisition. The user-friendly design allows easy access to compare the influence of sample preparation procedures, combinations of liquid chromatography (LC)-MS setups, as well as intra- and inter-software differences on critical performance measures across an unlimited number of analyses. mpwR supports outputs of commonly used software for bottom-up proteomics, such as ProteomeDiscoverer, Spectronaut, MaxQuant and DIA-NN.
AVAILABILITY: mpwR is available as an open-source R package. Release versions can be accessed on CRAN (https://CRAN.R-project.org/package=mpwR) for all major operating systems. The development version is maintained on GitHub (https://github.com/okdll/mpwR) and full documentation with examples and workflow templates is provided via the package website (https://okdll.github.io/mpwR/).

DOI: https://doi.org/10.1093/bioinformatics/btad358
Elife. 2023 May 31. pii: e81289. [Epub ahead of print]12

Pancreatic tumors exhibit myeloid-driven amino acid stress and upregulate arginine biosynthesis.

Juan J Apiz Saab, Lindsey N Dzierozynski, Patrick B Jonker, Roya AminiTabrizi, Hardik Shah, Rosa Elena Menjivar, Andrew J Scott, Zeribe C Nwosu, Zhou Zhu, Riona N Chen, Moses Oh, Colin Sheehan, Daniel R Wahl, Marina Pasca di Magliano, Costas A Lyssiotis, Kay F Macleod, Christopher R Weber, Alexander Muir.

  Nutrient stress in the tumor microenvironment requires cancer cells to adopt adaptive metabolic programs for survival and proliferation. Therefore, knowledge of microenvironmental nutrient levels and how cancer cells cope with such nutrition is critical to understand the metabolism underpinning cancer cell biology. Previously, we performed quantitative metabolomics of the interstitial fluid (the local perfusate) of murine pancreatic ductal adenocarcinoma (PDAC) tumors to comprehensively characterize nutrient availability in the microenvironment of these tumors (M. R. Sullivan, Danai, et al., 2019). Here, we develop Tumor Interstitial Fluid Medium (TIFM), a cell culture medium that contains nutrient levels representative of the PDAC microenvironment, enabling us to study PDAC metabolism ex vivo under physiological nutrient conditions. We show that PDAC cells cultured in TIFM adopt a cellular state closer to that of PDAC cells present in tumors compared to standard culture models. Further, using the TIFM model, we found arginine biosynthesis is active in PDAC and allows PDAC cells to maintain levels of this amino acid despite microenvironmental arginine depletion. We also show that myeloid derived arginase activity is largely responsible for the low levels of arginine in PDAC tumors. Altogether, these data indicate that nutrient availability in tumors is an important determinant of cancer cell metabolism and behavior, and cell culture models that incorporate physiological nutrient availability have improved fidelity to in vivo systems and enable the discovery of novel cancer metabolic phenotypes.

Keywords:  biochemistry; cancer biology; chemical biology; human; mouse

DOI:  https://doi.org/10.7554/eLife.81289
J Proteome Res. 2023 May 31.

Evaluation of DDA Library-Free Strategies for Phosphoproteomics and Ubiquitinomics Data-Independent Acquisition Data.

Chengwen Wen, Xiurong Wu, Guanzhong Lin, Wei Yan, Guohong Gan, Xiao Xu, Xiang-Yu Chen, Xi Chen, Xianming Liu, Guo Fu, Chuan-Qi Zhong.

  Phosphoproteomics and ubiquitinomics data-independent acquisition (DIA) mass spectrometry (MS) data is typically analyzed by using a data-dependent acquisition (DDA) spectral library. The performance of various library-free strategies for analyzing phosphoproteomics and ubiquitinomics DIA MS data has not been evaluated. In this study, we systematically compare four commonly used DDA library-free approaches including Spectronaut's directDIA, DIA-Umpire, DIA-MSFragger, and in silico-predicted library for analysis of phosphoproteomics SWATH, DIA, and diaPASEF data as well as ubiquitinomics diaPASEF data. Spectronaut's directDIA shows the highest sensitivity for phosphopeptide detection not only in synthetic phosphopeptide samples but also in phosphoproteomics SWATH-MS and DIA data from real biological samples, when compared to the other three library-free strategies. For phosphoproteomics diaPASEF data, Spectronaut's directDIA and the in silico-predicted library based on DIA-NN identify almost the same number of phosphopeptides as a project-specific DDA spectral library. However, only about 30% of the total phosphopeptides are commonly identified, suggesting that the library-free strategies for phospho-diaPASEF data need further improvement in terms of sensitivity. For ubiquitinomics diaPASEF data, the in silico-predicted library performs the best among the four workflows and detects ∼50% more K-GG peptides than a project-specific DDA spectral library. Our results demonstrate that Spectronaut's directDIA is suitable for the analysis of phosphoproteomics SWATH-MS and DIA MS data, while the in silico-predicted library based on DIA-NN shows substantial advantages for ubiquitinomics diaPASEF MS data.

Keywords:  data-independent acquisition; diaPASEF; in silico-predicted library; phosphoproteomics; ubiquitinomics

DOI:  https://doi.org/10.1021/acs.jproteome.2c00735
J Lipid Res. 2023 May 26. pii: S0022-2275(23)00067-6. [Epub ahead of print] 100394

Targeted Lipidomics Reveals a Novel Role for Glucosylceramides in Glucose Response.

Mark A Xatse, Andre F C Vieira, Chloe Byrne, Carissa Perez Olsen.

  The addition of excess glucose to the diet drives a coordinated response of lipid metabolism pathways to tune the membrane composition to the altered diet. Here, we have employed targeted lipidomic approaches to quantify the specific changes in the phospholipid and sphingolipid populations that occur in elevated glucose conditions. The lipids within wildtype Caenorhabditis elegans are strikingly stable with no significant changes identified in our global mass spectrometry-based analysis. Previous work has identified ELO-5, an elongase that is critical for the synthesis of monomethyl-branched chain fatty acids (mmBCFAs), as essential for surviving elevated glucose conditions. Therefore, we performed targeted lipidomics on elo-5 RNAi-fed animals and identified several significant changes in these animals in lipid species that contain mmBCFAs as well as in species that do not contain mmBCFAs. Of particular note, we identified a specific glucosylceramide (GlcCer 17:1;O2/22:0;O) that is also significantly upregulated with glucose in wildtype animals. Furthermore, compromising the production of the glucosylceramide pool with elo-3 or cgt-3 RNAi leads to premature death in glucose-fed animals. Taken together, our lipid analysis has expanded the mechanistic understanding of metabolic rewiring with glucose feeding and has identified a new role for the GlcCer 17:1;O2/22:0;O.

Keywords:  Caenorhabditis elegans.; RNAi; elevated glucose conditions; elongase; lipid metabolism pathways; mass spectrometry; membrane composition; monomethyl-branched chain fatty acids; phospholipids; sphingolipids

DOI:  https://doi.org/10.1016/j.jlr.2023.100394
Methods Mol Biol. 2023 ;2675 133-148

Quantitation of Glutathione and Oxidized Glutathione Ratios from Biological Matrices Using LC-MS/MS.

Thomas P Mathews.

  Oxidation of glutathione (GSH) to its disulfide dimer (GSSG) is the major mechanism by which cells balance reactive oxygen species (ROS) and mitigate oxidative stress. Thus, measuring the ratio of GSH/GSSG is an ideal way to assess oxidative stress within a cell. Quantitative mass spectrometry offers an ideal method to measure the GSH/GSSG ratio and can be applied to a variety of biological matrices and disease models. The following chapter details the design, optimization, and execution of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure the GSH/GSSG ratio.

Keywords:  Glutathione; Liquid chromatography; Mass spectrometry; Oxidative stress; Oxidized glutathione; Quantitation; Reactive oxygen species

DOI:  https://doi.org/10.1007/978-1-0716-3247-5_11
J Proteome Res. 2023 Jun 01.

An Introduction to Mass Spectrometry-Based Proteomics.

Steven R Shuken.

  Mass spectrometry is unmatched in its versatility for studying practically any aspect of the proteome. Because the foundations of mass spectrometry-based proteomics are complex and span multiple scientific fields, proteomics can be perceived as having a high barrier to entry. This tutorial is intended to be an accessible illustrated guide to the technical details of a relatively simple quantitative proteomic experiment. An attempt is made to explain the relevant concepts to those with limited knowledge of mass spectrometry and a basic understanding of proteins. An experimental overview is provided, from the beginning of sample preparation to the analysis of protein group quantities, with explanations of how the data are acquired, processed, and analyzed. A selection of advanced topics is briefly surveyed and works for further reading are cited. To conclude, a brief discussion of the future of proteomics is given, considering next-generation protein sequencing technologies that may complement mass spectrometry to create a fruitful future for proteomics.

Keywords:  bottom-up; data-dependent acquisition; label-free quantification; mass spectrometry; proteomics; untargeted proteomics

DOI:  https://doi.org/10.1021/acs.jproteome.2c00838
Methods Mol Biol. 2023 ;2669 67-77

Retinyl Ester Analysis by Orbitrap Mass Spectrometry.

Jeroen W A Jansen, Maya W Haaker, Esther A Zaal, J Bernd Helms.

  Retinoids are light-sensitive molecules that are normally detected by UV absorption techniques. Here we describe the identification and quantification of retinyl ester species by high-resolution mass spectrometry. Retinyl esters are extracted by the method of Bligh and Dyer and subsequently separated by HPLC in runs of 40 min. The retinyl esters are identified and quantified by mass spectrometry analysis. This procedure enables the highly sensitive detection and characterization of retinyl esters in biological samples such as hepatic stellate cells.

Keywords:  Atmospheric pressure chemical ionization; High-performance liquid chromatography; Mass spectrometry; Orbitrap; Retinol; Retinyl esters; Vitamin A

DOI:  https://doi.org/10.1007/978-1-0716-3207-9_5
Anal Chem. 2023 Jun 01.

Metabolomics Peak Analysis Computational Tool (MPACT): An Advanced Informatics Tool for Metabolomics and Data Visualization of Molecules from Complex Biological Samples.

Robert M Samples, Sara P Puckett, Marcy J Balunas.

Untargeted metabolomics is a powerful tool for investigating chemistry of complex biological systems, but its utility is compromised by the presence of uninformative features and the limited efficiency of currently available prioritization tools. More effective filtering and prioritization tools are required to address the challenges of large untargeted metabolomics datasets. Here, we introduce Metabolomics Peak Analysis Computational Tool (MPACT), a new mass spectrometry data analysis platform employing filtering based on multiple modalities, statistical techniques incorporating multilevel replication, and interactive data visualization. We demonstrate application of MPACT to uncover hidden effects of the rare earth element cerium on tunicate-associated bacterium Streptomyces sp. PTY087I2, culminating in characterization of two thiolated compounds including a new cysteine derivative, granaticin C, and granaticin D, recently described as mycothiogranaticin A. While we demonstrate application of MPACT to microbial natural products discovery using an elicitation approach, the platform should be readily adaptable to investigation of multipartite interactions, biomarker detection, small molecules in the environment, and a wide range of other complex sample types.

DOI: https://doi.org/10.1021/acs.analchem.2c04632
Front Cell Dev Biol. 2023 ;11 1187989

Lipid metabolism and tumor immunotherapy.

Yue Wang, Zongjin Guo, Adamu Danbala Isah, Shuangwei Chen, Yongfei Ren, Huazhong Cai.

  In recent years, the relationship between lipid metabolism and tumour immunotherapy has been thoroughly investigated. An increasing number of studies have shown that abnormal gene expression and ectopic levels of metabolites related to fatty acid synthesis or fatty acid oxidation affect tumour metastasis, recurrence, and drug resistance. Tumour immunotherapy that aims to promote an antitumour immune response has greatly improved the outcomes for tumour patients. However, lipid metabolism reprogramming in tumour cells or tumour microenvironment-infiltrating immune cells can influence the antitumour response of immune cells and induce tumor cell immune evasion. The recent increase in the prevalence of obesity-related cancers has drawn attention to the fact that obesity increases fatty acid oxidation in cancer cells and suppresses the activation of immune cells, thereby weakening antitumour immunity. This article reviews the changes in lipid metabolism in cells in the tumour microenvironment and describes the relationship between lipid metabolism reprogramming in multiple cell types and tumour immunotherapy.

Keywords:  combination therapy; immune cells; immunotherapy; lipid metabolism; tumor microenvironment

DOI:  https://doi.org/10.3389/fcell.2023.1187989
Anal Chem. 2023 May 30.

Improved Label-Free Quantification of Intact Proteoforms Using Field Asymmetric Ion Mobility Spectrometry.

Jake T Kline, Michael W Belford, Jingjing Huang, Joseph B Greer, David Bergen, Ryan T Fellers, Sylvester M Greer, David M Horn, Vlad Zabrouskov, Romain Huguet, Cornelia L Boeser, Kenneth R Durbin, Luca Fornelli.

The high-throughput quantification of intact proteoforms using a label-free approach is typically performed on proteins in the 0-30 kDa mass range extracted from whole cell or tissue lysates. Unfortunately, even when high-resolution separation of proteoforms is achieved by either high-performance liquid chromatography or capillary electrophoresis, the number of proteoforms that can be identified and quantified is inevitably limited by the inherent sample complexity. Here, we benchmark label-free quantification of proteoforms of Escherichia coli by applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS). Recent advances in Orbitrap instrumentation have enabled the acquisition of high-quality intact and fragmentation mass spectra without the need for averaging time-domain transients prior to Fourier transform. The resulting speed improvements allowed for the application of multiple FAIMS compensation voltages in the same liquid chromatography-tandem mass spectrometry experiment without increasing the overall data acquisition cycle. As a result, the application of FAIMS to label-free quantification based on intact mass spectra substantially increases the number of both identified and quantified proteoforms without penalizing quantification accuracy in comparison to traditional label-free experiments that do not adopt GPF.

DOI: https://doi.org/10.1021/acs.analchem.3c01534
J Chromatogr A. 2023 May 26. pii: S0021-9673(23)00337-0. [Epub ahead of print]1703 464111

Profiling of branched chain and straight chain saturated fatty acids by ultra-high performance liquid chromatography-mass spectrometry.

Xiaoqing Fu, Nourhane Hafza, Friedrich Götz, Michael Lämmerhofer.

  Branched chain fatty acids (BCFAs) are one of the important sub categories of fatty acids (FAs) which have unique functions in nature. They are commonly analyzed by GC-MS after derivatization to methyl esters (FAMEs). On the other hand, there is a lack of isomer-selective LC-MS methods which allow the distinction of different isomers with wide coverage of carbon chain length. In this work, a systematic retention and isomer selectivity study on seven commercially available UHPLC columns (six polysaccharide columns Chiralpak IA-U, IB-U, IC-U, ID-U, IG-U and IH-U; one Acquity UPLC CSH C18 column) was performed. Various experimental factors were evaluated including column temperatures, gradient profiles and flow rates to elucidate their effects on the separation ability of homologous series of BCFAs with distinct chain lengths, different branching types and branching positions. In general, IG-U outperformed the other columns in terms of isomer selectivity especially for the short and medium-chain BCFA isomers while RP C18 showed good potential in terms of selectivity for long-chain BCFA isomers. Furthermore, after the evaluation of the chromatographic retention pattern on the various columns and method optimization, we report a methodology for untargeted isomer-selective BCFA profiling without precolumn derivatization with UHPLC-ESI-MS/MS by quadrupole-time-of-flight instrument with SWATH acquisition. The best method provides selectivity for constitutional isomers of BCFAs covering distinct chain length (C5-C20) with different branching types (methyl or ethyl) and branching positions (2Me, 3Me, 4Me, 6Me, anteiso and iso-BCFAs) with an optimized LC condition on Acquity UPLC CSH C18 column. Finally, the optimized method was applied for the BCFAs profiling in lipid extracts of Staphylococcus aureus samples. Besides, pooled human platelets and pooled human plasma were evaluated as mammalian samples for presence of BCFAs as well. The new method showed strong potential for BCFA profiling in bacterial samples including different isomers anteiso and iso-BCFAs, which could be a useful tool for related subdisciplines in metabolomics and lipidomics in particular in combination with electron-activated dissociation MS. Compared to GC, the presented isomer selective LC methods can be also of great utility for preparative purposes. Equivalent (carbon) chain length numbers were calculated for RP18 and Chiralpak IG-U and compared to those of FAMEs obtained by GC.

Keywords:  Bacterial fatty acids; Isomer; Lipidomics; Microbiome; Staphylococcus aureus

DOI:  https://doi.org/10.1016/j.chroma.2023.464111
Anal Chem. 2023 Jun 01.

An In Silico Infrared Spectral Library of Molecular Ions for Metabolite Identification.

Kas J Houthuijs, Giel Berden, Udo F H Engelke, Vasuk Gautam, David S Wishart, Ron A Wevers, Jonathan Martens, Jos Oomens.

Infrared ion spectroscopy (IRIS) continues to see increasing use as an analytical tool for small-molecule identification in conjunction with mass spectrometry (MS). The IR spectrum of an m/z selected population of ions constitutes a unique fingerprint that is specific to the molecular structure. However, direct translation of an IR spectrum to a molecular structure remains challenging, as reference libraries of IR spectra of molecular ions largely do not exist. Quantum-chemically computed spectra can reliably be used as reference, but the challenge of selecting the candidate structures remains. Here, we introduce an in silico library of vibrational spectra of common MS adducts of over 4500 compounds found in the human metabolome database. In total, the library currently contains more than 75,000 spectra computed at the DFT level that can be queried with an experimental IR spectrum. Moreover, we introduce a database of 189 experimental IRIS spectra, which is employed to validate the automated spectral matching routines. This demonstrates that 75% of the metabolites in the experimental data set are correctly identified, based solely on their exact m/z and IRIS spectrum. Additionally, we demonstrate an approach for specifically identifying substructures by performing a search without m/z constraints to find structural analogues. Such an unsupervised search paves the way toward the de novo identification of unknowns that are absent in spectral libraries. We apply the in silico spectral library to identify an unknown in a plasma sample as 3-hydroxyhexanoic acid, highlighting the potential of the method.

DOI: https://doi.org/10.1021/acs.analchem.3c01078
Front Oncol. 2023 ;13 1122789

Orchestral role of lipid metabolic reprogramming in T-cell malignancy.

Arundhati Mehta, Yashwant Kumar Ratre, Vivek Kumar Soni, Dhananjay Shukla, Subhash C Sonkar, Ajay Kumar, Naveen Kumar Vishvakarma.

  The immune function of normal T cells partially depends on the maneuvering of lipid metabolism through various stages and subsets. Interestingly, T-cell malignancies also reprogram their lipid metabolism to fulfill bioenergetic demand for rapid division. The rewiring of lipid metabolism in T-cell malignancies not only provides survival benefits but also contributes to their stemness, invasion, metastasis, and angiogenesis. Owing to distinctive lipid metabolic programming in T-cell cancer, quantitative, qualitative, and spatial enrichment of specific lipid molecules occur. The formation of lipid rafts rich in cholesterol confers physical strength and sustains survival signals. The accumulation of lipids through de novo synthesis and uptake of free lipids contribute to the bioenergetic reserve required for robust demand during migration and metastasis. Lipid storage in cells leads to the formation of specialized structures known as lipid droplets. The inimitable changes in fatty acid synthesis (FAS) and fatty acid oxidation (FAO) are in dynamic balance in T-cell malignancies. FAO fuels the molecular pumps causing chemoresistance, while FAS offers structural and signaling lipids for rapid division. Lipid metabolism in T-cell cancer provides molecules having immunosuppressive abilities. Moreover, the distinctive composition of membrane lipids has implications for immune evasion by malignant cells of T-cell origin. Lipid droplets and lipid rafts are contributors to maintaining hallmarks of cancer in malignancies of T cells. In preclinical settings, molecular targeting of lipid metabolism in T-cell cancer potentiates the antitumor immunity and chemotherapeutic response. Thus, the direct and adjunct benefit of lipid metabolic targeting is expected to improve the clinical management of T-cell malignancies.

Keywords:  T cell malignancies; fatty acids; lipid droplets; lipid metabolism; lipid rafts

DOI:  https://doi.org/10.3389/fonc.2023.1122789
Cell Rep. 2023 May 26. pii: S2211-1247(23)00573-9. [Epub ahead of print]42(6): 112562

ALDH1L2 regulation of formate, formyl-methionine, and ROS controls cancer cell migration and metastasis.

Marc Hennequart, Steven E Pilley, Christiaan F Labuschagne, Jack Coomes, Loic Mervant, Paul C Driscoll, Nathalie M Legrave, Younghwan Lee, Peter Kreuzaler, Benedict Macintyre, Yulia Panina, Julianna Blagih, David Stevenson, Douglas Strathdee, Deborah Schneider-Luftman, Eva Grönroos, Eric C Cheung, Mariia Yuneva, Charles Swanton, Karen H Vousden.

  Mitochondrial 10-formyltetrahydrofolate (10-formyl-THF) is utilized by three mitochondrial enzymes to produce formate for nucleotide synthesis, NADPH for antioxidant defense, and formyl-methionine (fMet) to initiate mitochondrial mRNA translation. One of these enzymes-aldehyde dehydrogenase 1 family member 2 (ALDH1L2)-produces NADPH by catabolizing 10-formyl-THF into CO2 and THF. Using breast cancer cell lines, we show that reduction of ALDH1L2 expression increases ROS levels and the production of both formate and fMet. Both depletion of ALDH1L2 and direct exposure to formate result in enhanced cancer cell migration that is dependent on the expression of the formyl-peptide receptor (FPR). In various tumor models, increased ALDH1L2 expression lowers formate and fMet accumulation and limits metastatic capacity, while human breast cancer samples show a consistent reduction of ALDH1L2 expression in metastases. Together, our data suggest that loss of ALDH1L2 can support metastatic progression by promoting formate and fMet production, resulting in enhanced FPR-dependent signaling.

Keywords:  ALDH1L2; CP: Cancer; CP: Metabolism; ROS; breast cancer; formate; formyl-methionine; metastasis; one-carbon metabolism; serine

DOI:  https://doi.org/10.1016/j.celrep.2023.112562
J Proteome Res. 2023 Jun 01.

Bridging the False Discovery Gap.

Arya Ebadi, Jack Freestone, William S Noble, Uri Keich.

  Controlling the false discovery rate (FDR) among discoveries from a tandem mass spectrometry proteomics experiment using target decoy competition (TDC) controls only the proportion of false discoveries in an average sense. Thus, for any particular analysis, even with a valid FDR control procedure, the proportion of false discoveries (the FDP) may be higher than the specified FDR threshold. We demonstrate this phenomenon using real data and describe two recently developed methods that help bridge the gap between controlling the expected or average rate of false discoveries and the empirical rate (FDP). The FDP Stepdown method controls the FDP at any desired confidence level, and the TDC Uniform Band provides a confidence, or upper prediction bound, on the FDP in TDC's list of discoveries.

Keywords:  false discovery proportion; false discovery rate; peptide detection; target-decoy competition

DOI:  https://doi.org/10.1021/acs.jproteome.3c00176
Methods Mol Biol. 2023 ;2659 171-182

Quantitative Phosphoproteome Analysis of the Interaction Between Fusarium graminearum and Triticum aestivum.

Boyan Liu, Danisha Johal, Reid Buchanan, Brianna Ball, Mitra Serajazari, Jennifer Geddes-McAlister.

  Quantitative proteomics is a powerful method for distinguishing protein abundance changes in a biological system across conditions. In addition to recent advances in computational power and bioinformatics methods, improvements to sensitivity and resolution of mass spectrometry (MS) instrumentation provide an innovative approach for studying host-pathogen interaction dynamics and posttranslational modifications. In this protocol, we provide a workflow for state-of-the-art MS-based proteomics to assess changes in phosphorylated protein abundance upon interaction between the worldwide cereal crop, Triticum aestivum (wheat), and the global cereal crop fungal pathogen, Fusarium graminearum, during infection. This protocol mimics a time course of infection of T. aestivum by F. graminearum in the greenhouse, and the harvested samples undergo Fe-NTA phosphoenrichment combined with label-free quantification (LFQ) for detection by liquid-chromatography (LC)-coupled with tandem MS/MS. Our approach provides an in-depth view of changes in phosphorylation from both the host and pathogen perspectives in a single experiment across infection time points and different host cultivars.

Keywords:  Bioinformatics; Fusarium graminearum; Host-pathogen interaction; Label-free quantification (LFQ); Mass spectrometry (MS); Phosphoproteome; Quantitative proteomics; Triticum aestivum

DOI:  https://doi.org/10.1007/978-1-0716-3159-1_13
Magn Reson Chem. 2023 Jun 02.

MagMet: A fully automated web server for targeted nuclear magnetic resonance metabolomics of plasma and serum.

Manoj Rout, Matthias Lipfert, Brian L Lee, Mark Berjanskii, Nazanin Assempour, Rosa Vazquez Fresno, Arnau Serra Cayuela, Ying Dong, Mathew Johnson, Honeya Shahin, Vasuk Gautam, Tanvir Sajed, Eponine Oler, Harrison Peters, Rupasri Mandal, David S Wishart.

  Nuclear magnetic resonance (NMR) spectral analysis of biofluids can be a time-consuming process, requiring the expertise of a trained operator. With NMR becoming increasingly popular in the field of metabolomics, there is a growing need to change this paradigm and to automate the process. Here we introduce MagMet, an online web server, that automates the processing and quantification of 1D 1 H NMR spectra from biofluids-specifically, human serum/plasma metabolites, including those associated with inborn errors of metabolism (IEM). MagMet uses a highly efficient data processing procedure that performs automatic Fourier Transformation, phase correction, baseline optimization, chemical shift referencing, water signal removal, and peak picking/peak alignment. MagMet then uses the peak positions, linewidth information, and J-couplings from its own specially prepared standard metabolite reference spectral NMR library of 85 serum/plasma compounds to identify and quantify compounds from experimentally acquired NMR spectra of serum/plasma. MagMet employs linewidth adjustment for more consistent quantification of metabolites from higher field instruments and incorporates a highly efficient data processing procedure for more rapid and accurate detection and quantification of metabolites. This optimized algorithm allows the MagMet webserver to quickly detect and quantify 58 serum/plasma metabolites in 2.6 min per spectrum (when processing a dataset of 50-100 spectra). MagMet's performance was also assessed using spectra collected from defined mixtures (simulating other biofluids), with >100 previously measured plasma spectra, and from spiked serum/plasma samples simulating known IEMs. In all cases, MagMet performed with precision and accuracy matching the performance of human spectral profiling experts. MagMet is available at http://magmet.ca.

Keywords:  1H NMR; automation; metabolite identification; metabolite quantification; software

DOI:  https://doi.org/10.1002/mrc.5371
Curr Treat Options Oncol. 2023 May 30.

Targeting Glutamine Metabolism as an Attractive Therapeutic Strategy for Acute Myeloid Leukemia.

Yan Xiao, Bingbing Hu, Yao Guo, Dengyang Zhang, Yuming Zhao, Yun Chen, Na Li, Liuting Yu.

   OPINION STATEMENT: Relapse after chemotherapy and hematopoietic stem cell transplantation leads to adverse prognosis for acute myeloid leukemia (AML) patients. As a "conditionally essential amino acid," glutamine contributes to the growth and proliferation of AML cells. Glutamine-target strategies as new treatment approaches have been widely explored in AML treatment to improve outcome. Glutamine-target strategies including depletion of systemic glutamine and application of glutamine uptake inhibitors, glutamine antagonists/analogues, and glutaminase inhibitors. Because glutamine metabolism involved in multiple pathways in cells and each pathway of glutamine metabolism has many regulatory factors, therefore, AML therapy targeting glutamine metabolism should focus on how to inhibit multiple metabolic pathways without affecting normal cells and host immune to achieve effective treatment for AML.

Keywords:  AML treatment; Acute myeloid leukemia; Cancer metabolism; Glutaminase inhibitor; Glutamine; Glutamine antagonist

DOI:  https://doi.org/10.1007/s11864-023-01104-0
Methods Mol Biol. 2023 ;2675 117-132

UHPLC-HRMS-Based Analysis of S-Hydroxymethyl-Glutathione, GSH, and GSSG in Human Cells.

María Eugenia Monge, Manuela R Martinefski, Mariela Bollini, Lucas B Pontel.

  Glutathione (GSH) is one of the main antioxidant molecules present in cells. It harbors a thiol group responsible for sustaining cellular redox homeostasis. This moiety can react with cellular electrophiles such as formaldehyde yielding the compound S-hydroxymethyl-GSH (HSMGSH). HSMGSH is the substrate of the enzyme alcohol dehydrogenase 5 (ADH5) and thus a key intermediate in formaldehyde metabolism. In this work, we describe a method for the chemical synthesis of HSMGSH and a pipeline to identify this compound in complex cell extracts by means of ultra-high-performance liquid chromatography coupled to high-resolution spectrometry (UHPLC-HRMS). This method also allows determining GSH and oxidized disulfide (GSSG) in the same samples, thus providing broad information about formaldehyde-GSH metabolism.

Keywords:  ADH5; Formaldehyde; Glutathione; Oxidized glutathione; S-hydroxymethyl-glutathione; UHPLC-HRMS

DOI:  https://doi.org/10.1007/978-1-0716-3247-5_10
J Sep Sci. 2023 Jun 03. e2300210

Dried blood spot analysis with liquid chromatography and mass spectrometry: Trends in clinical chemistry.

Hanne Bendiksen Skogvold, Helge Rootwelt, Léon Reubsaet, Katja Benedikte Prestø Elgstøen, Steven Ray Wilson.

  Dried blood spot samples are simple to prepare and transport, enabling safe and accessible diagnostics, both locally and globally. We review dried blood spot samples for clinical analysis, focusing on liquid chromatography-mass spectrometry as a versatile measurement tool for these samples. Dried blood spot samples can provide information for, for example, metabolomics, xenobiotic analysis, and proteomics. Targeted analyses of small molecules are the main application of dried blood spot samples and liquid chromatography-mass spectrometry, but emerging applications include untargeted metabolomics and proteomics. Applications are highly varied, including analyses related to newborn screening, diagnostics and monitoring of disease progression and treatment effects of virtually any disease, as well as studies into the physiology and effects of diet, exercise, xenobiotics, and doping. A range of dried blood spot products and methods are available, and applied liquid chromatography-mass spectrometry instrumentation is varied with regard to liquid chromatography column formats and selectivity. In addition, novel approaches such as on-paper sample preparation (e.g., selective trapping of analytes with paper-immobilized antibodies) are described. We focus on research papers published in the last 5 years.

Keywords:  dried blood spot; liquid chromatography; mass spectrometry; omics; xenobiotics

DOI:  https://doi.org/10.1002/jssc.202300210