bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2024–11–10
fourteen papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz



  1. Methods Enzymol. 2024 ;pii: S0076-6879(24)00404-X. [Epub ahead of print]707 101-152
      The multiple functions of mitochondria are governed by their proteome comprising 1000-1500 proteins depending on the organism. However, only few proteins are synthesized inside mitochondria, whereas most are "born" outside mitochondria. To reach their destined location, these mitochondrial proteins follow specific import routes established by a mitochondrial translocase network. A detailed understanding of the role and interplay of the different translocases is imperative to understand mitochondrial biology and how mitochondria are integrated into the cellular network. Mass spectrometry (MS) proved to be effective to study the translocase network regarding composition, functions, interplay, and cellular responses evoked by dysfunction. In this chapter, we provide protocols tailored to MS-enabled functional analysis of mutants and interactomes of mitochondrial translocation proteins. In the first part, we exemplify the MS-based proteomics analysis of translocation mutants for delineating the human mitochondrial importome following depletion of the central translocation protein TOMM40. The protocol comprises metabolic stable isotope labeling, TOMM40 knockdown, preparation of mitochondrial fractions, and sample preparation for liquid chromatography (LC)-MS. For deep MS analysis, prefractionation of peptide mixtures by high pH reversed-phase LC is described. In the second part, we outline an affinity purification MS approach to reveal the association of an orphaned protein with the translocase TIM23. The protocol covers FLAG-tag affinity purification of protein complexes from mitochondrial fractions and downstream sample preparation for interactome analysis. In the last unifying part, we describe methods for LC-MS, data processing, statistical analysis and visualization of quantitative MS data, and provide a Python code for effective, customizable analysis.
    Keywords:  Affinity-purification mass spectrometry; Data analysis; Importome; Interactome; Mass spectrometry; Mitochondria; Protein quantification; Proteomics; SILAC; Translocation mutants
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.059
  2. Brief Bioinform. 2024 Sep 23. pii: bbae553. [Epub ahead of print]25(6):
      Mass spectrometry (MS)-based proteomics has become instrumental in comprehensively investigating complex biological systems. Data-independent acquisition (DIA)-MS, utilizing hybrid spectral library search strategies, allows for the simultaneous quantification of thousands of proteins, showing promise in enhancing protein identification and quantification precision. However, low-quality profiles can considerably undermine quantitative precision, resulting in inaccurate protein quantification. To tackle this challenge, we introduced STAVER, a novel algorithm that leverages standardized benchmark datasets to reduce non-biological variation in large-scale DIA-MS analyses. By eliminating unwanted noise in MS signals, STAVER significantly improved protein quantification precision, especially in hybrid spectral library searches. Moreover, we validated STAVER's robustness and applicability across multiple large-scale DIA datasets, demonstrating significantly enhanced precision and reproducibility of protein quantification. STAVER offers an innovative and effective approach for enhancing the quality of large-scale DIA proteomic data, facilitating cross-platform and cross-laboratory comparative analyses. This advancement significantly enhances the consistency and reliability of findings in clinical research. The complete package is available at https://github.com/Ran485/STAVER.
    Keywords:  STAVER algorithm; bioinformatics; data-independent acquisition; non-biological noise; proteomics analysis; quantitative proteomics
    DOI:  https://doi.org/10.1093/bib/bbae553
  3. J Lipid Res. 2024 Oct 25. pii: S0022-2275(24)00182-2. [Epub ahead of print] 100677
      Compound lipids comprise a diverse group of metabolites present in living systems, and metabolic- and environmentally-driven structural distinctions across this family is increasingly linked to biological function. However, methods for deconvoluting these often isobaric lipid species are lacking or require specialized instrumentation. Notably, acyl-chain diversity within cells may be influenced by nutritional states, metabolic dysregulation, or genetic alterations. Therefore, a reliable, validated method of quantifying structurally similar even-, odd-, and branched-chain acyl groups within intact compound lipids will be invaluable for gaining molecular insights into their biological functions. Here we demonstrate the chromatographic resolution of isobaric lipids containing distinct combinations of straight-chain and branched-chain acyl groups via ultra-high-pressure liquid chromatography (UHPLC)-mass spectrometry (MS) using a C30 liquid chromatography column. Using metabolically-engineered adipocytes lacking branched-keto acid dehydrogenase A (Bckdha), we validate this approach through a combination of fatty acid supplementation and metabolic tracing using monomethyl branched-chain fatty acids and valine. We observe resolution of numerous isobaric triacylglycerols and other compound lipids, demonstrating the resolving utility of this method. This approach adds to the toolbox for laboratories to quantify and characterize acyl chain diversity across the lipidome.
    Keywords:  BCKDH; Branched-chain fatty acids; C30 chromatography; stable isotope tracing
    DOI:  https://doi.org/10.1016/j.jlr.2024.100677
  4. Cold Spring Harb Perspect Med. 2024 Nov 05. pii: a041814. [Epub ahead of print]
      Cancer cells undergo changes in metabolism that distinguish them from non-malignant tissue. These may provide a growth advantage by promoting oncogenic signaling and redirecting intermediates to anabolic pathways that provide building blocks for new cellular components. Cancer metabolism is far from uniform, however, and recent work has shed light on its heterogenity within and between tumors. This work is also revealing how cancer metabolism adapts to the tumor microenvironment, as well as ways in which we may capitalize on metabolic changes in cancer cells to create new therapies.
    DOI:  https://doi.org/10.1101/cshperspect.a041814
  5. Anal Chem. 2024 Nov 06.
      Spatial metabolomics has emerged as a powerful tool capable of revealing metabolic gradients throughout complex heterogeneous tissues. While mass spectrometry imaging (MSI) technologies designed to generate spatial metabolomic data have improved significantly over time, metabolite coverage is still a significant limitation. It is possible to achieve deeper metabolite coverage by imaging in positive and negative polarities or imaging several serial sections with different targeted biomolecular classes. However, this significantly increases the number of tissue samples required for biological studies and reduces the capacity for larger sample cohorts. Herein, we introduce lithium-doped nanospray desorption electrospray ionization (nano-DESI) as a simple and robust method to increase spatial metabolomics coverage, which is achieved through enhancements to ionization efficiencies in positive ion mode for metabolites and lipids lacking basic moieties, and improved structurally diagnostic tandem mass spectra for [M + Li]+ adducts. Specifically, signal intensities were found to be enhanced by 10-1000× for 96 compounds including small molecule metabolites, fatty acids, neutral lipids (e.g., diacylglycerols, DAG), and phospholipids when lithium was added to the ESI solvent. In addition, proof-of-principle results reveal that lithium-doped nano-DESI MSI was able to comprehensively visualize metabolites and lipids in the prostaglandin (PG) biosynthetic pathway with PG isomeric resolution in an ovarian tumor section. These data show colocalization of fatty acid (FA) 20:4 containing DAGs, FA 20:4 monoacylglycerols (MAGs), and FA 20:4 with PGE2 and disparate localizations of PGD2. Overall, this study describes a simple and powerful approach to more comprehensively probe the spatial metabolome with MSI.
    DOI:  https://doi.org/10.1021/acs.analchem.4c03553
  6. Eur J Surg Oncol. 2024 Oct 24. pii: S0748-7983(24)00851-5. [Epub ahead of print] 108783
      Endometrial cancer (EC) is increasing incidence among women, and it constitutes a health problem for women globally. An important aspect of EC management involves the use of protein biomarkers for early detection and monitoring. Protein biomarkers allow the identification of high-risk patients, the detection of the disease in its early stages, and the assessment of treatment responses. Mass spectrometry (MS)-based proteomics offers robust analytical techniques and a comprehensive understanding of proteins. Proteomics methods allow scientists to investigate both the quantities and functions of proteins. Thus, it provides valuable insights into how proteins are altered under different conditions. This review summarizes recent advances in MS-based proteomic biomarker discovery for EC, focusing on different sample types and MS-based techniques used in clinical studies. The review emphasized in detail the most commonly used key sources such as blood, urine, vaginal fluids and tissue. Furthermore, MS-based proteomics techniques such as untargeted, targeted, sequential window acquisition of all theoretical mass spectra (SWATH-MS) and mass spectrometry imaging used in the discovery and validation/validation phases were evaluated. This review highlights the importance of biomarker discovery and clinical translation to improve diagnostic and therapeutic outcomes in EC. It aims to provide a comprehensive overview of MS-based proteomics in EC, guiding future research and clinical applications.
    Keywords:  Biomarkers; Endometrial cancer; Mass spectrometry; Proteomics
    DOI:  https://doi.org/10.1016/j.ejso.2024.108783
  7. Mol Genet Metab. 2023 Oct 20. pii: S1096-7192(23)00341-4. [Epub ahead of print] 107711
      Fatty acid oxidation disorders (FAOD) are inborn errors of metabolism that occur due to deficiency of specific enzyme activities and transporter proteins involved in the mitochondrial metabolism of fatty acids, causing a deficiency in ATP production. The identification of suitable biomarkers plays a crucial role in predicting the future risk of disease and monitoring responses to therapies. Acyl-CoAs are directly involved in the steps of fatty acid oxidation and are the primary biomarkers associated with FAOD. However, acyl-CoAs are not used as diagnostic biomarkers in hospitals and clinics as they are present intracellularly with low endogenous levels. Additionally, the analytical method development of acyl-CoAs is quite challenging due to diverse physicochemical properties and instability. Hence, secondary biomarkers such as acylcarnitines are used for the identification of FAOD. In this review, the focus is on the analytical techniques that have evolved over the years for the identification and quantitation of acyl-CoAs. Among these techniques, liquid chromatography-mass spectrometry clearly has an advantage in terms of sensitivity and selectivity. Stable isotope labeling by essential nutrients in cell culture (SILEC) enables the generation of labeled internal standards. Each acyl-CoA species has a distinct pattern of instability and degradation, and the use of appropriately matched internal standards can compensate for such issues. Although significant progress has been made in measuring acyl-CoAs, more efforts are needed for bringing these technical advancements to hospitals and clinics. This review also highlights the difficulties involved in the routine use of acyl-CoAs as a diagnostic biomarker and some of the measures that can be adopted by clinics and hospitals for overcoming these limitations.
    Keywords:  Acyl-CoA; Biomarkers; Fatty acid oxidation disorders; LC-MS; Newborn screening; SILEC
    DOI:  https://doi.org/10.1016/j.ymgme.2023.107711
  8. Am J Respir Cell Mol Biol. 2024 Nov 05.
      Changes in metabolic activity are key regulators of macrophage activity. Pro-inflammatory macrophages upregulate glycolysis, which promotes an inflammatory phenotype, whereas pro-repair macrophages rely upon oxidative metabolism and glutaminolysis to support their activity. Work to understand how metabolism regulates macrophage phenotype has been done primarily in macrophage cell lines and bone marrow-derived macrophages (BMDM). Our study sought to understand changes in metabolic activity of murine tissue-resident alveolar macrophages (AM) in response to LPS stimulation and to contrast them to BMDM. These studies also determined the contribution of glutamine metabolism using the glutamine inhibitor, DON. We found that compared to BMDM, AM have higher rates of oxygen consumption and contain a higher concentration of intracellular metabolites involved in fatty acid oxidation. In response to LPS, BMDM but not AM increased rates of glycolysis. Inhibition of glutamine metabolism using DON altered the metabolic activity of AM but not BMDM. Within AM, glutamine inhibition led to increases in intracellular metabolites involved in glycolysis, the TCA cycle, fatty acid oxidation, and amino acid metabolism. Glutamine inhibition also altered the metabolic response to LPS within AM but not BMDM. Our data reveal striking differences in the metabolic activity of AM and BMDM.
    Keywords:  glutamine; glycolysis; lipids; macrophage; metabolomics
    DOI:  https://doi.org/10.1165/rcmb.2023-0249OC
  9. J Sep Sci. 2024 Nov;47(21): e70014
      Lipid extraction of complex biological samples is essential for high-quality data in liquid chromatography-mass spectrometry (LC-MS)-based lipidomics. This study introduces a three-phase liquid extraction (3PLE)-ultra-high-performance LC coupled with quadrupole time-of-flight tandem MS method. This method was successfully applied to lipidomics analysis of breast cancer cells, including highly metastatic MDA-MB-231 and slightly metastatic MCF7 cells. The 3PLE method employed an n-hexane/methyl tert-butyl ether/acetonitrile/water solvent system that formed one aqueous and two organic phases. Neutral and polar lipids were enriched in the upper and middle organic phases, respectively, and combined for detection, thereby reducing analysis time. Compared with the Bligh and Dyer method, 3PLE achieved higher sensitivity and detected more features, with over a 50% increase in the relative abundance of nearly 50% of the differential lipids. In total, 21 differential lipids were identified in the MDA-MB-231 group and 22 in the MCF7 group compared to normal breast epithelial cells (MCF10A). Pathway analysis suggested that lipid changes in breast cancer cells were associated with glycerophospholipid metabolism, arachidonic acid metabolism, sphingolipid metabolism, and linoleic acid metabolism. The study presents a highly efficient lipidomics method, providing a scientific foundation for understanding breast cancer pathogenesis and aiding in diagnosis.
    Keywords:  breast cancer cells | lipidomics | liquid chromatography | mass spectrometry | three‐phase liquid extraction
    DOI:  https://doi.org/10.1002/jssc.70014
  10. J Chromatogr A. 2024 Oct 26. pii: S0021-9673(24)00849-5. [Epub ahead of print]1738 465475
      The biosynthesis and homeostasis of cholesterol are essential for cellular function. Cholesterol is a major lipid with multiple roles in membrane stability, signaling, or as a precursor for other molecules. Because of the structural similarity of the sterols involved in the biosynthesis, their accurate identification and quantification is still challenging. Moreover, the huge difference in the concentration of cholesterol and its precursors can cause interferences during the detection. To overcome these problems, a heart-cut liquid chromatographic method was developed by evaluating 38 different columns to achieve optimal separation. The method efficiently separates all sterol biosynthesis intermediates, with detection limits in the low nmol/L-range and an upper limit of quantification of 9 mmol/L for cholesterol by using triple quadrupole mass spectrometric detection. Investigation of lung carcinoma cells treated with statins demonstrated the capability to detect a biological response, showing inhibition of sterol synthesis. This technique offers a robust tool for studying cholesterol biosynthesis and its role in disease.
    Keywords:  2D-LC; Cancer; Lipidomics; Statins; Sterols
    DOI:  https://doi.org/10.1016/j.chroma.2024.465475
  11. Antioxid Redox Signal. 2024 Nov 04.
      Significance: Glucose-induced lipid metabolism is essential for preserving functional β-cells, and its disruption is linked to type 2 diabetes (T2D) development. Lipids are an integral part of the cells playing an indispensable role as structural components, energy storage molecules, and signals. Recent Advances: Glucose presence significantly impacts lipid metabolism in β-cells, where fatty acids are primarily synthesized de novo and/or are transported from the bloodstream. This process is regulated by the glycerolipid/free fatty acid cycle, which includes lipogenic and lipolytic reactions producing metabolic coupling factors crucial for insulin secretion. Disrupted lipid metabolism involving oxidative stress and inflammation is a hallmark of T2D. Critical Issues: Lipid metabolism in β-cells is complex involving multiple simultaneous processes. Exact compartmentalization and quantification of lipid metabolism and its intermediates, especially in response to glucose or chronic hyperglycemia, are essential. Current research often uses non-physiological conditions, which may not accurately reflect in vivo situations. Future Directions: Identifying and quantifying individual steps and their signaling, including redox, within the complex fatty acid and lipid metabolic pathways as well as the metabolites formed during acute versus chronic glucose stimulation, will uncover the detailed mechanisms of glucose-stimulated insulin secretion. This knowledge is crucial for understanding T2D pathogenesis and identifying pharmacological targets to prevent this disease. Antioxid. Redox Signal. 00, 000-000.
    Keywords:  diabetes; fatty acids; glucose-induced insulin secretion; glycerolipid/free fatty acid cycle; lipid metabolism
    DOI:  https://doi.org/10.1089/ars.2024.0724
  12. J Lipid Res. 2024 Nov 04. pii: S0022-2275(24)00199-8. [Epub ahead of print] 100694
      Several oxylipins are regulators of inflammation. They are formed by enzymes such as lipoxygenases or cyclooxygenases, but also stereorandomly by autoxidation. Reversed-phase liquid chromatography-tandem-mass-spectrometry (LC-MS/MS) methods for oxylipin quantification do not separate enantiomers. Here, we combine sensitive and selective oxylipin analysis with chiral separation using two-dimensional (2D)-LC-MS/MS. By multiple heart-cutting, the oxylipin peaks are transferred onto a chiral column. 45 enantiomeric pairs of (di-)hydroxy-fatty acids are separated with full gradient elution within 1.80min, yielding lower limits of quantification <1pg on column. Concentrations as well as enantiomeric fractions of oxylipins can be determined, even at low concentrations or at high enantiomeric excess of one isomer. The developed achiral-chiral multiple heart-cutting 2D-LC-MS/MS method offers unprecedented selectivity, enabling a better understanding of the formation route of these lipid mediators. This is demonstrated by distinguishing the formation of hydroxy-fatty acids by (acetylated) cyclooxygenase-2 and radical-mediated autoxidation. Applying the method to human M2-like-macrophages, we show that the so-called specialized pro-resolving mediators (SPM) 5,15-DiHEPE and 7,17-DiHDHA as well as 5,15-DiHETE were present as (S,S)-enantiomers, supporting their enzymatic formation. In contrast, at least eight isomers (including protectin DX but not neutroprotectin D1) of 10,17-DiHDHA are present in immune cells, indicating formation by autoxidation. In human plasma of healthy subjects, none of these dihydroxy-fatty acids are not present. However, we demonstrate that all four isomers quickly form via autoxidation if the samples are stored improperly. Thus, dihydroxy-FA should only be reported as SPM, such as resolvin D5 or resolvin E4, if an enantioselective analysis has been carried out.
    Keywords:  arachidonic acid; autoxidation; cyclooxygenase; enantioselective analysis; enzymatic oxidation; lipid oxidation; lipidomics; lipoxygenase; resolvins; sample storage
    DOI:  https://doi.org/10.1016/j.jlr.2024.100694
  13. Mol Cell Proteomics. 2024 Oct 30. pii: S1535-9476(24)00161-0. [Epub ahead of print] 100871
      Mass-spectrometry-based proteomics allows the quantification of thousands of proteins, protein variants, and their modifications, in many biological samples. These are derived from the measurement of peptide relative quantities, and it is not always possible to distinguish proteins with similar sequences due to the absence of protein-specific peptides. In such cases, peptide signals are reported in protein groups that can correspond to several genes. Here, we show that multi-gene protein groups have a limited impact on GO-term enrichment, but selecting only one gene per group affects network analysis. We thus present the Cytoscape app Proteo Visualizer (https://apps.cytoscape.org/apps/ProteoVisualizer) that is designed for retrieving protein interaction networks from STRING using protein groups as input and thus allows visualisation and network analysis of bottom-up MS-based proteomics data sets.
    DOI:  https://doi.org/10.1016/j.mcpro.2024.100871
  14. J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Oct 31. pii: S1570-0232(24)00368-4. [Epub ahead of print]1248 124359
      Methylmalonic acid (MMA) is a reverse biomarker of vitamin B12 that is increasingly utilized in clinical practice. However, its low sensitivity and susceptibility to strong interference from isomer present chromatographic challenges. We have developed a rapid derivatization method for plasma MMA at room temperature, converting it to the corresponding 2,2,2-trifluoroethylamide derivative using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 2,2,2-trifluoroethylamine hydrochloride (TFEA). Amidization was completed within 10 min, followed by protein precipitation extraction of the amides with trichloroacetic acid for Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This technique notably enhanced the signal-to-noise ratio of MMA in chromatography. The derivatized MMA exhibited excellent linearity within a concentration range of 42.4-2711.9 nmol/L, with a correlation coefficient (R2) of 0.9990. The intraday and interday precision of replicate measurements ranged from 2.4 % to 4.4 % and 2.6 % to 2.8 %, respectively, while the recovery fell between 97.9 % and 100.1 %.
    Keywords:  Derivatization; Liquid chromatography-tandem mass; Methylmalonic acid; Vitamin B12
    DOI:  https://doi.org/10.1016/j.jchromb.2024.124359