bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2021‒10‒31
thirteen papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh

  1. Nat Methods. 2021 Oct 28.
      Liquid chromatography-high-resolution mass spectrometry (LC-MS)-based metabolomics aims to identify and quantify all metabolites, but most LC-MS peaks remain unidentified. Here we present a global network optimization approach, NetID, to annotate untargeted LC-MS metabolomics data. The approach aims to generate, for all experimentally observed ion peaks, annotations that match the measured masses, retention times and (when available) tandem mass spectrometry fragmentation patterns. Peaks are connected based on mass differences reflecting adduction, fragmentation, isotopes, or feasible biochemical transformations. Global optimization generates a single network linking most observed ion peaks, enhances peak assignment accuracy, and produces chemically informative peak-peak relationships, including for peaks lacking tandem mass spectrometry spectra. Applying this approach to yeast and mouse data, we identified five previously unrecognized metabolites (thiamine derivatives and N-glucosyl-taurine). Isotope tracer studies indicate active flux through these metabolites. Thus, NetID applies existing metabolomic knowledge and global optimization to substantially improve annotation coverage and accuracy in untargeted metabolomics datasets, facilitating metabolite discovery.
  2. J Biol Chem. 2021 Oct 22. pii: S0021-9258(21)01147-9. [Epub ahead of print] 101341
      Adipose tissue-resident macrophages (ATMs) maintain metabolic homeostasis but also contribute to obesity-induced adipose tissue inflammation and metabolic dysfunction. Central to these contrasting effects of ATMs on metabolic homeostasis is the interaction of macrophages with fatty acids. Fatty acid levels are increased within adipose tissue in various pathological and physiological conditions, but appear to initiate inflammatory responses only upon interaction with particular macrophage subsets within obese adipose tissue. The molecular basis underlying these divergent outcomes is likely due to phenotypic differences between ATM subsets, although how macrophage polarization state influences the metabolism of exogenous fatty acids is relatively unknown. Herein, using stable isotope-labelled and non-labelled fatty acids in combination with mass spectrometry lipidomics, we show marked differences in the utilisation of exogenous fatty acids within inflammatory macrophages (M1 macrophaghes) and macrophages involved in tissue homeostasis (M2 macrophages). Specifically, the accumulation of exogenous fatty acids within triacylglycerols and cholesterol esters is significantly higher in M1 macrophages, while there is an increased enrichment of exogenous fatty acids within glycerophospholipids, ether lipids, and sphingolipids in M2 macrophages. Finally, we show that functionally distinct ATM populations in vivo have distinct lipid compositions. Collectively, this study identifies new aspects of the metabolic reprogramming that occur in distinct macrophage polarization states. The channelling of exogenous fatty acids into particular lipid synthetic pathways may contribute to the sensitivity/resistance of macrophage subsets to the inflammatory effects of increased environmental fatty acid levels.
    Keywords:  cell metabolism; fatty acid metabolism; macrophage; phospholipid metabolism; sphingolipid
  3. Bioanalysis. 2021 Oct 26.
      Metabolite profiling is an indispensable part of drug discovery and development, enabling a comprehensive understanding of the drug's metabolic behavior. Liquid chromatography-mass spectrometry facilitates metabolite profiling by reducing sample complexity and providing high sensitivity. This review discusses the in vivo metabolite profiling involving LC-MS/MS and the utilization of QTOF, QQQ mass analyzers with a particular emphasis on a mass filter. Further, a summary of sample extraction procedures in biological matrices such as plasma, urine, feces, serum and hair as in vivo samples are outlined. toward the end, we present 15 case studies in biological matrices and their LC-MS/MS conditions to understand the metabolic disposition.
    Keywords:  LC-MS; biotransformation; metabolite profiling; plasma; urine
  4. J Lipid Res. 2021 Oct 25. pii: S0022-2275(21)00125-5. [Epub ahead of print] 100143
      Free fatty acids (FFAs) display pleiotropic functions in human diseases. Short-, medium-, and long-chain fatty acids (SCFAs, MCFAs, and LCFAs) are derived from different origins, and precise quantification of these FFAs is critical for revealing their roles in biological processes. However, accessing stable isotope-labeled internal standards (SIL-IS) is difficult, and different chain lengths of FFAs challenge the chromatographic coverage. Here, we developed a metabolomics strategy to analyze FFAs based on isotope-free liquid chromatography-mass spectrometry-multiple reaction monitoring (LC-MS-MRM) integrated with dual derivatization. Samples and dual derivatization internal standards (DD-ISs) were synthesized using 2-dimethylaminoethylamine (DMED) or dansylhydrazine (Dns-Hz) as a "light" label under mild and efficient reaction conditions, and N, N-diethyl ethylene diamine (DEEA) or N, N-diethyldansulfonyl hydrazide (Dens-HZ) as a "heavy" label. General MRM parameters were designed to analyze these FFAs. The limit of detection (LOD) of SCFAs varied from 0.5 to 3 nM. Furthermore, we show this approach exhibits good linearity (R2=0.99374 to 0.99929), there is no serious substrate interference, and no quench steps are required, confirming the feasibility and reliability of the method. Using this method, we successfully quantified 15 types of SCFAs in fecal samples from hepatocellular carcinoma (HCC) patients and healthy individuals; among these, propionate, butyrate, isobutyrate, and 2-methylbutyrate were significantly decreased in the HCC group compared to the healthy control group. These results indicate that the integrated LC-MS metabolomics with isotope-free and dual derivatization is an efficient approach for quantifying FFAs, and may be useful for identifying lipid biomarkers of cancer.
    Keywords:  2-dimethylaminoethylamine (DMED); Free fatty acids (FFAs); N, N-diethyl ethylene diamine (DEEA); N, N-diethyldansulfonyl hydrazide (Dens-HZ); dansylhydrazine (Dns-Hz); derivatization; hepatocellular carcinoma (HCC); isotope-free; liquid chromatography-mass spectrometry (LC-MS); short chain fatty acids (SCFAs)
  5. Anal Bioanal Chem. 2021 Oct 27.
      Stable isotope labelling in combination with high-resolution mass spectrometry approaches are increasingly used to analyze both metabolite and protein modification dynamics. To enable correct estimation of the resulting dynamics, it is critical to correct the measured values for naturally occurring stable isotopes, a process commonly called isotopologue correction or deconvolution. While the importance of isotopologue correction is well recognized in metabolomics, it has received far less attention in proteomics approaches. Although several tools exist that enable isotopologue correction of mass spectrometry data, the majority is tailored for the analysis of low molecular weight metabolites. We here present PICor which has been developed for isotopologue correction of complex isotope labelling experiments in proteomics or metabolomics and demonstrate the importance of appropriate correction for accurate determination of protein modifications dynamics, using histone acetylation as an example.
    Keywords:  Isotopologue correction; Mass spectrometry; Protein modifications; Stable isotope labelling
  6. Nat Protoc. 2021 Oct 27.
      Protein lipidation is one of the most widespread post-translational modifications (PTMs) found in nature, regulating protein function, structure and subcellular localization. Lipid transferases and their substrate proteins are also attracting increasing interest as drug targets because of their dysregulation in many disease states. However, the inherent hydrophobicity and potential dynamic nature of lipid modifications makes them notoriously challenging to detect by many analytical methods. Chemical proteomics provides a powerful approach to identify and quantify these diverse protein modifications by combining bespoke chemical tools for lipidated protein enrichment with quantitative mass spectrometry-based proteomics. Here, we report a robust and proteome-wide approach for the exploration of five major classes of protein lipidation in living cells, through the use of specific chemical probes for each lipid PTM. In-cell labeling of lipidated proteins is achieved by the metabolic incorporation of a lipid probe that mimics the specific natural lipid, concomitantly wielding an alkyne as a bio-orthogonal labeling tag. After incorporation, the chemically tagged proteins can be coupled to multifunctional 'capture reagents' by using click chemistry, allowing in-gel fluorescence visualization or enrichment via affinity handles for quantitative chemical proteomics based on label-free quantification (LFQ) or tandem mass-tag (TMT) approaches. In this protocol, we describe the application of lipid probes for N-myristoylation, N- and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylation in multiple cell lines to illustrate both the workflow and data obtained in these experiments. We provide detailed workflows for method optimization, sample preparation for chemical proteomics and data processing. A properly trained researcher (e.g., technician, graduate student or postdoc) can complete all steps from optimizing metabolic labeling to data processing within 3 weeks. This protocol enables sensitive and quantitative analysis of lipidated proteins at a proteome-wide scale at native expression levels, which is critical to understanding the role of lipid PTMs in health and disease.
  7. Cancer Biol Ther. 2021 Oct 25. 1-20
      Tumor metastasis is the leading cause of cancer mortality, often characterized by abnormal cell growth and invasion to distant organs. The cancer invasion due to epithelial to mesenchymal transition is affected by metabolic and oxygen availability in the tumor-associated micro-environment. A precise alteration in oxygen and metabolic signaling between healthy and metastatic cells is a substantial probe for understanding tumor progression and metastasis. Molecular heterogeneity in the tumor microenvironment help to sustain the metastatic cell growth during their survival shift from low to high metabolic-oxygen-rich sites and reinforces the metastatic events. This review highlighted the crucial role of oxygen and metabolites in metastatic progression and exemplified the role of metabolic rewiring and oxygen availability in cancer cell adaptation. Furthermore, we have also addressed potential applications of altered oxygen and metabolic networking with tumor type that could be a signature pattern to assess tumor growth and chemotherapeutics efficacy in managing cancer metastasis.
    Keywords:  Cancer metabolism; angeogenesis; cancer recurrence; dormancy; lymphogenesis; metabolic reprogramming; metastasis
  8. Cell Rep. 2021 Oct 26. pii: S2211-1247(21)01373-5. [Epub ahead of print]37(4): 109903
      Sleep is crucial to restore body functions and metabolism across nearly all tissues and cells, and sleep restriction is linked to various metabolic dysfunctions in humans. Using exhaled breath analysis by secondary electrospray ionization high-resolution mass spectrometry, we measured the human exhaled metabolome at 10-s resolution across a night of sleep in combination with conventional polysomnography. Our subsequent analysis of almost 2,000 metabolite features demonstrates rapid, reversible control of major metabolic pathways by the individual vigilance states. Within this framework, whereas a switch to wake reduces fatty acid oxidation, a switch to slow-wave sleep increases it, and the transition to rapid eye movement sleep results in elevation of tricarboxylic acid (TCA) cycle intermediates. Thus, in addition to daily regulation of metabolism, there exists a surprising and complex underlying orchestration across sleep and wake. Both likely play an important role in optimizing metabolic circuits for human performance and health.
    Keywords:  breath analysis; high-resolution mass spectrometry; metabolomics; secondary electrospray ionization; sleep
  9. Cell Rep. 2021 Oct 26. pii: S2211-1247(21)01368-1. [Epub ahead of print]37(4): 109898
      After demyelinating injury of the central nervous system, resolution of the mounting acute inflammation is crucial for the initiation of a regenerative response. Here, we aim to identify fatty acids and lipid mediators that govern the balance of inflammatory reactions within demyelinating lesions. Using lipidomics, we identify bioactive lipids in the resolution phase of inflammation with markedly elevated levels of n-3 polyunsaturated fatty acids. Using fat-1 transgenic mice, which convert n-6 fatty acids to n-3 fatty acids, we find that reduction of the n-6/n-3 ratio decreases the phagocytic infiltrate. In addition, we observe accelerated decline of microglia/macrophages and enhanced generation of oligodendrocytes in aged mice when n-3 fatty acids are shuttled to the brain. Thus, n-3 fatty acids enhance lesion recovery and may, therefore, provide the basis for pro-regenerative medicines of demyelinating diseases in the central nervous system.
    Keywords:  demyelination; inflammation resolution; lipids; microglia; myelin; remyelination
  10. Nat Commun. 2021 Oct 26. 12(1): 6176
      Serine is a non-essential amino acid that is critical for tumour proliferation and depletion of circulating serine results in reduced tumour growth and increased survival in various cancer models. While many cancer cells cultured in a standard tissue culture medium depend on exogenous serine for optimal growth, here we report that these cells are less sensitive to serine/glycine depletion in medium containing physiological levels of metabolites. The lower requirement for exogenous serine under these culture conditions reflects both increased de novo serine synthesis and the use of hypoxanthine (not present in the standard medium) to support purine synthesis. Limiting serine availability leads to increased uptake of extracellular hypoxanthine, sparing available serine for other pathways such as glutathione synthesis. Taken together these results improve our understanding of serine metabolism in physiologically relevant nutrient conditions and allow us to predict interventions that may enhance the therapeutic response to dietary serine/glycine limitation.
  11. Anal Biochem. 2021 Oct 21. pii: S0003-2697(21)00331-6. [Epub ahead of print] 114430
      This study describes LC-ESI-MS/MS method that covers the analysis of various cellular acyl-CoA in a single injection. The method is based on a quick extraction step eliminating LLE/SPE clean up. Method performance characteristics were determined after spiking acyl-CoA standards in different concentrations into a surrogate matrix. The extensive matrix effect for most acyl-CoA except for palmitoyl-CoA was compensated by using isotopically labeled internal standard and matrix-matched calibration. As a result of the high matrix effect, the accuracy for palmitoyl-CoA at the low concentration deviated from the target range of ±20%. The developed method was applied to identify twenty-one cellular acyl-CoA in SK-HEP-1 cells and screening for alterations in acyl-CoA levels post Mito Q antioxidant intervention.
    Keywords:  Acyl-CoA; Liquid chromatography tandem mass spectrometry
  12. J Proteome Res. 2021 Oct 28.
      Mass spectrometry-based proteomics is a high-throughput technology generating ever-larger amounts of data per project. However, storing, processing, and interpreting these data can be a challenge. A key element in simplifying this process is the development of interactive frameworks focusing on visualization that can greatly simplify both the interpretation of data and the generation of new knowledge. Here we present PeptideShaker Online, a user-friendly web-based framework for the identification of mass spectrometry-based proteomics data, from raw file conversion to interactive visualization of the resulting data. Storage and processing of the data are performed via the versatile Galaxy platform (through SearchGUI, PeptideShaker, and moFF), while the interaction with the results happens via a locally installed web server, thus enabling researchers to process and interpret their own data without requiring advanced bioinformatics skills or direct access to compute-intensive infrastructures. The source code, additional documentation, and a fully functional demo is available at
    Keywords:  Galaxy; data processing; interaction; mass spectrometry; visualization
  13. Nat Protoc. 2021 Oct 29.
      Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.