bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2022–03–06
twelve papers selected by
Giovanny Rodríguez Blanco, University of Edinburgh



  1. Mol Cell. 2022 Mar 03. pii: S1097-2765(22)00112-5. [Epub ahead of print]82(5): 920-932.e7
      IDO1 oxidizes tryptophan (TRP) to generate kynurenine (KYN), the substrate for 1-carbon and NAD metabolism, and is implicated in pro-cancer pathophysiology and infection biology. However, the mechanistic relationships between IDO1 in amino acid depletion versus product generation have remained a longstanding mystery. We found an unrecognized link between IDO1 and cell survival mediated by KYN that serves as the source for molecules that inhibit ferroptotic cell death. We show that this effect requires KYN export from IDO1-expressing cells, which is then available for non-IDO1-expressing cells via SLC7A11, the central transporter involved in ferroptosis suppression. Whether inside the "producer" IDO1+ cell or the "receiver" cell, KYN is converted into downstream metabolites, suppressing ferroptosis by ROS scavenging and activating an NRF2-dependent, AHR-independent cell-protective pathway, including SLC7A11, propagating anti-ferroptotic signaling. IDO1, therefore, controls a multi-pronged protection pathway from ferroptotic cell death, underscoring the need to re-evaluate the use of IDO1 inhibitors in cancer treatment.
    Keywords:  GCN2; IDO1; NRF2; SLC7A11; cancer; ferroptosis; kynurenine; metabolism; tryptophan
    DOI:  https://doi.org/10.1016/j.molcel.2022.02.007
  2. Mol Syst Biol. 2022 Mar;18(3): e10798
      Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10-fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable-core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease.
    Keywords:  drug perturbation; low-flow LC-MS; proteomics at single-cell resolution; single-cell heterogeneity; systems biology
    DOI:  https://doi.org/10.15252/msb.202110798
  3. Metabolomics. 2022 Feb 28. 18(3): 16
       INTRODUCTION: Recent advances in high-throughput methodologies in the 'omics' and synthetic biology fields call for rapid and sensitive workflows in the metabolic phenotyping of complex biological samples.
    OBJECTIVE: The objective of this research was to evaluate a straightforward to implement LC-MS metabolomics method using a commercially available chromatography column that provides increased throughput. Reducing run time can potentially impact chromatography and therefore the effects of ion mobility spectrometry to expand peak capacity were also evaluated. Additional confidence provided via collision cross section measurements for detected features was also explored.
    METHODS: A rapid untargeted metabolomics workflow was developed with broad metabolome coverage, combining zwitterionic-phase hydrophilic interaction chromatography (HILIC-Z) with drift tube ion mobility-quadrupole time-of-flight (DTIM-qTOF) mass spectrometry. The analytical performance of our method was explored using extracts from complex biological samples, including a reproducibility study on chicken serum and a simple comparative study on a bacterial metabolome.
    RESULTS: The method is acronymised RHIMMS for rapid HILIC-Z ion mobility mass spectrometry. We present the RHIMMS workflow starting with data acquisition, followed by data processing and analysis. RHIMMS demonstrates improved chromatographic separation for a selection of metabolites with wide physicochemical properties while maintaining reproducibility at better than 20% over 200 injections at 3.5 min per sample for the selected metabolites, and a mean of 13.9% for the top 50 metabolites by intensity. Additionally, the combination of rapid chromatographic separation with ion mobility allows improved annotation and the ability to distinguish isobaric compounds.
    CONCLUSION: Our results demonstrate RHIMMS to be a rapid, reproducible, sensitive and high-resolution analytical platform that is highly applicable to the untargeted metabolomics analysis of complex samples.
    Keywords:  HILIC; High throughput; Ion mobility; Untargeted metabolomics
    DOI:  https://doi.org/10.1007/s11306-022-01871-1
  4. J Bone Miner Res. 2022 Feb 27.
      Enchondromas and chondrosarcomas are common cartilage neoplasms that are either benign or malignant respectively. The majority of these tumors harbor mutations in either IDH1 or IDH2. Glutamine metabolism has been implicated as a critical regulator of tumors with IDH mutations. Using genetic and pharmacological approaches, we demonstrated that glutaminase-mediated glutamine metabolism played distinct roles in enchondromas and chondrosarcomas with IDH1 or IDH2 mutations. Glutamine affected cell differentiation and viability in these tumors differently through different downstream metabolites. During murine enchondroma-like lesion development, glutamine-derived α-ketoglutarate promoted hypertrophic chondrocyte differentiation and regulated chondrocyte proliferation. Deletion of glutaminase in chondrocytes with Idh1 mutation increased the number and size of enchondroma-like lesions. In contrast, pharmacological inhibition of glutaminase in chondrosarcoma xenografts reduced overall tumor burden partially because glutamine-derived non-essential amino acids played an important role in preventing cell apoptosis. This study demonstrates that glutamine metabolism plays different roles in tumor initiation and cancer maintenance. Supplementation of α-ketoglutarate and inhibiting GLS may provide a therapeutic approach to suppress enchondroma and chondrosarcoma tumor growth respectively.
    Keywords:  CARTILAGE TUMORS; CHONDROCYTE DIFFERENTIATION; GLUTAMINE METABOLISM; GROWTH PLATE; ISOCITRATE DEHYDROGENASE
    DOI:  https://doi.org/10.1002/jbmr.4532
  5. Genes Dis. 2022 Mar;9(2): 334-346
      Ferroptosis, a new form of non-apoptotic, regulated cell death characterized by iron dependency and lipid peroxidation, is involved in many pathological conditions such as neurodegenerative diseases, heart ischemia/reperfusion injury, acute renal failure, and cancer. While metabolic dysfunctions can lead to excessive lipid peroxidation culminating in ferroptotic cell death, glutathione peroxidase 4 (GPX4) resides in the center of a network that functions to prevent lipid hydroperoxides from accumulation, thereby suppressing ferroptosis. Indeed, RSL3 and other small-molecule GPX4 inhibitors can induce ferroptosis in not only cultured cancer cells but also tumor xenografts implanted in mice. Similarly, erastin and other system Xc- inhibitors can deplete intracellular glutathione required for GPX4 function, leading to lipid peroxidation and ferroptosis. As therapy-resistant cancer cells are sensitive to GPX4-targeted therapeutic regimens, the agents capable of inducing ferroptosis hold great promises to improve current cancer therapy. This review will outline the molecular basis of ferroptosis, but focus on the strategies and the agents developed in recent years for therapeutic induction of ferroptosis. The potentials of these ferroptosis-inducing agents, which include system Xc- inhibitors, GPX4 inhibitors, and iron-based nanoparticles, in cancer therapy will be subsequently discussed.
    Keywords:  Cancer therapy; Erastin; Ferroptosis; GPX4; Lipid peroxidation; Nanomedicine; RSL3; System Xc-
    DOI:  https://doi.org/10.1016/j.gendis.2020.09.005
  6. Methods Mol Biol. 2022 ;2419 629-644
      Atherosclerosis development and progression have been linked to vascular reactive oxygen species (ROS). Plaque formation and especially instability, frequently resulting in acute coronary syndromes, have been linked to cell apoptosis and senescence, but also mainly to increased cellular oxidative stress. ROS are characterized by their high chemical reactivity and a resulting short half-life. This high reactivity usually involves reversible and/or irreversible protein modifications and specifically the covalent oxidative modification of cysteine residues. The latter can be used for the identification of protein-chemical footprints, leading to indirect monitoring of ROS. Proteomics and especially liquid chromatography tandem mass spectrometry (LC-MS/MS) approaches have emerged as a powerful tool to identify such protein modifications in biological samples (e.g., body fluids, tissues, cells). Application of a well-established quantitative thiol trapping technique termed OxICAT enables the detection and quantification of oxidative thiol modifications of thousands of proteins in a single experiment. In this chapter, a step-by-step guide for the redox proteomics analysis of atherosclerotic aortas, by utilizing the OxICAT method, as optimized by our group is provided.
    Keywords:  Cysteine oxidation; OxICAT; Oxidative modifications; Reactive oxygen species; Redox proteomics
    DOI:  https://doi.org/10.1007/978-1-0716-1924-7_39
  7. Mol Cell Proteomics. 2022 Feb 24. pii: S1535-9476(22)00027-5. [Epub ahead of print] 100219
      In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed single-cell proteomics requires high ion injection times and high-resolution spectra to quantify the single-cell signal, however the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and present a new acquisition strategy termed RETICLE (RTS Enhanced Quant of Single Cell Spectra) that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. We show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at a MS2 injection time of 750ms using a 2h gradient.
    Keywords:  SPS-MS3; TMT; isobaric tag quantification; multiplexing; real-time-search; single-cell proteomics
    DOI:  https://doi.org/10.1016/j.mcpro.2022.100219
  8. Anal Chim Acta. 2022 Mar 22. pii: S0003-2670(21)00683-8. [Epub ahead of print]1199 338857
      Phosphorylation is one of the quickest post-translational modifications that controls downstream signaling pathways regulating processes like cell proliferation, survival, and differentiation. Nowadays, mass spectrometry-based phosphoproteomics is a well-established method providing unprecedented characterization and quantification of phosphorylated proteins and peptides in complex samples. A comprehensive phosphoproteomics workflow consists of protein digestion, phosphopeptide enrichment, sample fractionation, chromatographic separation, and final detection by mass spectrometry. Each of these stages provides its own contribution to overall data variability and should be optimized thoroughly. This review aims to provide an overview of current developments in individual steps of phosphoproteomics workflow with a special focus on applied analytical methods. Recent efforts in all experimental steps are discussed. Finally, possible future development in the field of (phospho)proteomics is proposed.
    Keywords:  Mass spectrometry; Phosphoproteomics; Receptor tyrosine kinase; Signaling pathways; Workflow
    DOI:  https://doi.org/10.1016/j.aca.2021.338857
  9. Anal Chem. 2022 Mar 03.
      The structural diversity of phospholipids plays a critical role in cellular membrane dynamics, energy storage, and cellular signaling. Despite its importance, the extent of this diversity has only recently come into focus, largely owing to advances in separation science and mass spectrometry methodology and instrumentation. Characterization of glycerophospholipid (GP) isomers differing only in their acyl chain configurations and locations of carbon-carbon double bonds (C═C) remains challenging due to the need for both effective separation of isomers and advanced tandem mass spectrometry (MS/MS) technologies capable of double-bond localization. Drift tube ion mobility spectrometry (DTIMS) coupled with MS can provide both fast separation and accurate determination of collision cross section (CCS) of molecules but typically lacks the resolving power needed to separate phospholipid isomers. Ultraviolet photodissociation (UVPD) can provide unambiguous double-bond localization but is challenging to implement on the timescales of modern commercial drift tube time-of-flight mass spectrometers. Here, we present a novel method for coupling DTIMS with a UVPD-enabled Orbitrap mass spectrometer using absorption mode Fourier transform multiplexing that affords simultaneous localization of double bonds and accurate CCS measurements even when isomers cannot be fully resolved in the mobility dimension. This method is demonstrated on two- and three-component mixtures and shown to provide CCS measurements that differ from those obtained by individual analysis of each component by less than 1%.
    DOI:  https://doi.org/10.1021/acs.analchem.1c04711
  10. Bioanalysis. 2022 Mar 02.
      
    Keywords:  LC-MS; biomarker; mass spectrometry; surrogate analyte
    DOI:  https://doi.org/10.4155/bio-2022-0030
  11. Methods Mol Biol. 2022 ;2419 193-212
      Lipid particles found in circulating extracellular fluids such as blood or lymph are essential for cellular homeostasis, metabolism and survival. Such particles provide essential lipids and fats which enable cells to synthesize new membranes and regulate different biochemical pathways. Imbalance in lipid particle metabolism can cause pathological states such as atherosclerosis. Here, elevated low-density lipoprotein (LDL) accumulation leads to fat-filled lesions or plaques in arterial walls. In this chapter, we provide a detailed set of protocols for the rapid and safe purification of lipid particles from human blood using high-speed ultracentrifugation. We provide a detailed set of assays for further analysis of the biochemical and cellular properties of these lipid particles. By combining these assays, we can better understand the complex roles of different lipid particles in normal physiology and disease pathology.
    Keywords:  Gradient purification; HDL; Human embryonic kidney 293T (HEK293T) cells; Human umbilical vein endothelial cells (HUVECs); LDL; Lipid particles; SDS-PAGE; VLDL
    DOI:  https://doi.org/10.1007/978-1-0716-1924-7_12
  12. 3 Biotech. 2022 Mar;12(3): 80
      The well-known secondary metabolite-producing bacterium Streptomyces coelicolor is a natural choice for the development of super-hosts optimized for the heterologous expression of antibiotic biosynthetic gene clusters (BGCs). In this study, we used S. coelicolor M145 and its derivative strain M1146 where all active BGCs have been deleted and generated high-resolution quantitative time series metabolite profiles under two cultivation conditions (phosphate and nitrogen limitation to cease growth and trigger secondary metabolism). Five targeted LC-MS/MS-based methods were used to quantify intracellular primary metabolites covering phosphorylated metabolites, amino acids, organic acids, (deoxy) nucleoside/sugar phosphates, Nicotinamide adenine dinucleotide (NAD), and Coenzyme A (CoA). The nitrogen limitation resulted in a sharp decline in respiration and an immediate drop in the cell mass concentration. Intracellularly, a reduction in the level of the metabolites next to α-ketoglutarate in the tricarboxylic acid cycle and a decrease in the NADH pool were among the most prominent adaptation to this nutrient limitation. Phosphate limitation evoked a different adaptation of the metabolite pools as most of the phosphorylated metabolite pools except 6-phosphogluconic acid (6PG) pool were downregulated. 13C-isotope-labeling experiments revealed the simultaneous activity of both glycolysis and gluconeogenesis during the co-utilization of glucose and glutamate. The S. coelicolor M1146 strain had similar time-series metabolite profile dynamics as the parent M145 strain, except for a visibly increased 6PG pool in the stationary phase. In general, the nutrient limitation had a larger effect on the metabolite pool levels than the absence of secondary metabolite production in M1146. This study provides new insight into the primary carbon metabolism and its link to the secondary metabolism which is needed for further optimization of both super-host genotype and cultivation conditions.
    Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03146-x.
    Keywords:  Antibiotics; Bioreactor; LC–MS/MS; Metabolomics; Secondary metabolism; Targeted metabolite profiling
    DOI:  https://doi.org/10.1007/s13205-022-03146-x