bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2020‒02‒02
twelve papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research

  1. Methods Mol Biol. 2020 ;2110 47-58
      Protocols are described for preparation of various types of samples for differential protein expression proteomics using nano liquid chromatography-tandem mass spectrometry (nLCMS/MS). Fresh frozen tissue, formalin-fixed paraffin-embedded (FFPE) tissue, cells, pull-downs, immunoprecipitations, and plasma or serum can be processed. For transplantation studies, it should be noted that samples must be comparable; thus, similar cells or organ systems from the same species can be compared when only a minority of the proteins change expression levels. For xenotransplantation studies, caution should be taken, as different species possess proteins with differences in primary structure. The sample preparation technique is based on spin filter devices using sodium deoxycholate (SDC) as detergent for extraction of proteins. The detergent is removed using phase extraction with ethyl acetate, and samples can be used for label-free quantification (LFQ) analysis or peptides can be labeled with tandem mass tags (TMT).
    Keywords:  Cells; Differential protein expression analysis; FFPE; Label-free quantification; Plasma; Proteomics; Pull-downs; Serum; Spin filter device-based preparation; TMT labeling; Tissue; nLC-MS/MS
  2. J Proteome Res. 2020 Jan 27.
      Vendor-independent software tools for quantification of small molecules and metabolites are lacking, especially for targeted analysis workflows. Skyline is a freely available, open-source software tool for targeted quantitative mass spectrometry method development and data processing with a ten-year history supporting 6 major instrument vendors. Designed initially for proteomic analysis, we describe the expansion of Skyline to data for small molecule analysis, including selected reaction monitoring (SRM), high-resolution mass spectrometry (HRMS), and calibrated quantification. This fundamental expansion of Skyline from a peptide-sequence centric tool to a molecule-centric tool makes it agnostic to the source of the molecule while retaining Skyline features critical for workflows in both peptide and more general biomolecular research. The data visualization and interrogation features already available in Skyline - such as peak picking, chromatographic alignment, and transition selection - have been adapted to support small molecule data, including metabolomics. Herein, we explain the conceptual workflow for small molecule analysis using Skyline, demonstrate Skyline performance benchmarked against a comparable instrument vendor software tool, and present additional real-world applications. Further, we include step-by-step instructions on using Skyline for small molecule quantitative method development and data analysis on data acquired with a variety of mass spectrometers from multiple instrument vendors.
  3. Int J Cancer. 2020 Jan 29.
      In noncancerous tissues, neighboring cells coexist in metabolic harmony. This metabolic harmony is disrupted in cancerous tissues, often accompanied by genetic mutations. Tumor cells fundamentally change the metabolite profiles in the tumor microenvironment to favor their own growth. In this review, we will discuss several examples in which genetic mutations reprogram tumor cell metabolic pathways, leading to the consumption of essential nutrients in the tumor microenvironment, production of toxic byproducts, and suppression of antitumor immune cell metabolic fitness and tumor-killing function. Finally, we will briefly discuss how immune checkpoint blockade overcomes the metabolic suppression of tumor-infiltrating immune cells. This article is protected by copyright. All rights reserved.
    Keywords:  T cell metabolism; immunometabolism; oncogenic mutations
  4. Oncogene. 2020 Jan 31.
      Epithelial to mesenchymal transition (EMT) is a dynamic process that drives cancer cell plasticity and is thought to play a major role in metastasis. Here we show, using MDA-MB-231 cells as a model, that the plasticity of at least some metastatic breast cancer cells is dependent on the transcriptional co-regulator CBFβ. We demonstrate that CBFβ is essential to maintain the mesenchymal phenotype of triple-negative breast cancer cells and that CBFβ-depleted cells undergo a mesenchymal to epithelial transition (MET) and re-organise into acini-like structures, reminiscent of those formed by epithelial breast cells. We subsequently show, using an inducible CBFβ system, that the MET can be reversed, thus demonstrating the plasticity of CBFβ-mediated EMT. Moreover, the MET can be reversed by expression of the EMT transcription factor Slug whose expression is dependent on CBFβ. Finally, we demonstrate that loss of CBFβ inhibits the ability of metastatic breast cancer cells to invade bone cell cultures and suppresses their ability to form bone metastases in vivo. Together our findings demonstrate that CBFβ can determine the plasticity of the metastatic cancer cell phenotype, suggesting that its regulation in different micro-environments may play a key role in the establishment of metastatic tumours.
  5. Cell Metab. 2020 Jan 17. pii: S1550-4131(20)30001-2. [Epub ahead of print]
      Continual efferocytic clearance of apoptotic cells (ACs) by macrophages prevents necrosis and promotes injury resolution. How continual efferocytosis is promoted is not clear. Here, we show that the process is optimized by linking the metabolism of engulfed cargo from initial efferocytic events to subsequent rounds. We found that continual efferocytosis is enhanced by the metabolism of AC-derived arginine and ornithine to putrescine by macrophage arginase 1 (Arg1) and ornithine decarboxylase (ODC). Putrescine augments HuR-mediated stabilization of the mRNA encoding the GTP-exchange factor Dbl, which activates actin-regulating Rac1 to facilitate subsequent rounds of AC internalization. Inhibition of any step along this pathway after first-AC uptake suppresses second-AC internalization, whereas putrescine addition rescues this defect. Mice lacking myeloid Arg1 or ODC have defects in efferocytosis in vivo and in atherosclerosis regression, while treatment with putrescine promotes atherosclerosis resolution. Thus, macrophage metabolism of AC-derived metabolites allows for optimal continual efferocytosis and resolution of injury.
    Keywords:  arginase; arginine; atherosclerosis; atherosclerosis regression; efferocytosis; inflammation resolution; intracellular metabolism; macrophage; polyamines; putrescine
  6. Trends Cell Biol. 2020 Jan 23. pii: S0962-8924(19)30219-3. [Epub ahead of print]
      Increasing evidence implicates metabolic pathways as key regulators of cell fate and function. Although the metabolism of glucose, amino acids, and fatty acids is essential to maintain overall energy homeostasis, the choice of a given metabolic pathway and the levels of particular substrates and intermediates increasingly appear to modulate specific cellular activities. This connection is likely related to the growing appreciation that molecules such as acetyl-CoA act as a shared currency between metabolic flux and chromatin modification. We review recent evidence for a role of metabolism in modulating cellular function in four distinct contexts. These areas include the immune system, the tumor microenvironment, the fibrotic response, and stem cell function. Together, these examples suggest that metabolic pathways do not simply provide the fuel that powers cellular activities but instead help to shape and determine cellular identity.
  7. Talanta. 2020 Apr 01. pii: S0039-9140(19)31273-1. [Epub ahead of print]210 120640
      A notable change in the body fluids nucleosides of cancer patients has been actively highlighted in searches for new biomarkers to early cancer detection. For this reason, improvements of bioanalytical methods for these compounds focused on a noninvasive sampling trend are of great importance. Therefore, this work aimed firstly to develop efficient methods for nucleoside analysis in urine and serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS), applying different strategies to quantify nine nucleosides, and further identify other untargeted nucleosides. Sample preparation was based on protein precipitation and affinity-solid phase extraction (SPE), whereas quantification was performed using a triple quadrupole (QqQ) mass analyzer operating in the selected reaction monitoring (SRM) mode. Surrogates matrices were proposed as an alternative to standard addition calibration. Specifically, to quantitate creatinine, a simple LC-MS/MS method was validated and used for normalization of urinary metabolites quantitation. To identify the other nucleosides, LC methods using different MS scans modes were evaluated on a quadrupole-time of flight (Q-TOF) or a hybrid triple quadrupole linear ion trap (Q-trap). Validation was performed for nucleosides quantification using the synthetic matrices of urine and serum, and selectivity, linearity, accuracy, reproducibility, matrix effect, LOD's and LOQ's were accessed, providing trustworthy results for bioanalysis purposes. Both LC-Q-Trap/MS and LC-Q-TOF/MS methods showed proper sensitivity for structural characterization on assays with urine and serum samples from healthy volunteers and could also be used in the identification of untargeted nucleosides. The investigated approaches delivered in-depth results and seem promising for future applications on urine and serum samples analyses aiming to validate nucleosides as cancer biomarkers.
    Keywords:  Blood serum; LC-MS/MS; Method development; Method validation; Nucleosides; Urine
  8. Anal Chim Acta. 2020 Mar 01. pii: S0003-2670(19)31352-2. [Epub ahead of print]1100 66-74
      Short-chain fatty acids (SCFAs) and hydroxylated short-chain fatty acids (OH-SCFAs) are crucial intermediates related to a variety of diseases, such as bowel disease, cardiovascular disease, renal disease and cancer. A global profiling method to screen SCFAs and OH-SCFAs was developed by tagging these analytes with d0/d6-N, N-dimethyl-6,7-dihydro-5H-pyrrolo [3,4-d] pyrimidine-2-amine (d0/d6-DHPP) and using ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-MS/MS) in parallel reaction monitoring (PRM) mode. The derivatization procedure was simple and rapid. The targeted compounds could be derivatized within 3 min under mild condition and analyzed without the need of further purification. The derivatization significantly improved the chromatographic performance and mass spectrometry response. The d6-DHPP tagged standards were used as internal standards, which remarkably reduced the matrix effects. The use of high resolution PRM mode made it possible to locate unknown SCFA and OH-SCFA species, and greatly reduced the false positive identification results. The developed method was successfully applied to the analysis of mouse fecal, serum, and liver tissue samples harvested from the breast cancer nude mice that had been exposed with 2,2',4,4'-tetrabromodiphenyl ether (BDE-47). Results showed that 40 analytes (10 SCFAs and 30 OH-SCFAs) were characterized. Semi-quantitative analysis indicated that the exposure of BDE-47 to the mice altered the SCFA and OH-SCFA metabolism, especially in the high dose group. This study provides a high-throughput method to characterize SCFAs and OH-SCFAs in mouse samples.
    Keywords:  2,2′,4,4′-Tetrabromodiphenyl ether; Derivatization; Hydroxylated short-chain fatty acids; Parallel reaction monitoring; Short-chain fatty acids
  9. J Lipid Res. 2020 Jan 27. pii: jlr.RA120000583. [Epub ahead of print]
      Cholesteryl ester transfer protein (CETP) exists as full-length (FL) and exon 9 (E9)-deleted isoforms. The function of E9-deleted CETP is poorly understood. Here, we investigated the role of E9-deleted CETP in regulating the secretion of FL-CETP by cells, and explored its possible role in intracellular lipid metabolism. CETP overexpression in cells that naturally express CETP confirmed that E9-deleted CETP is not secreted, and show that cellular FL- and E9-deleted CETP form an isolatable complex. Co-expression of CETP isoforms lowered cellular levels of both proteins, and impaired FL-CETP secretion. These effects were due to reduced synthesis of both isoforms, however the predominate consequence of FL- and E9-deleted CETP co-expression is impaired FL-CETP synthesis. We reported before that reducing both CETP isoforms or overexpressing FL-CETP impairs cellular triglyceride storage. To investigate this further, E9-deleted CETP was expressed in SW872 cells that naturally synthesize CETP and in mouse 3T3-L1 cells that do not. E9-deleted CETP overexpression stimulated SW872 triglyceride synthesis and increased stored triglyceride 2-fold. Expression of E9-deleted CETP in mouse 3T3-L1 cells produced a similar lipid phenotype. In vitro, FL-CETP promotes the transfer of triglyceride from endoplasmic reticulum-enriched membranes to lipid droplets. E9-deleted CETP also promoted this transfer, although less effectively, and it inhibited the transfer driven by FL-CETP. We conclude that FL- and E9-deleted CETP isoforms interact to mutually decrease their intracellular levels and impair FL-CETP secretion by reducing CETP biosynthesis. E9-deleted CETP, like FL-CETP, alters cellular triglyceride metabolism and storage but in a contrary manner.
    Keywords:  Lipid droplets; Lipid transfer proteins; Lipids; Secretion; Triglycerides; alternative splicing; cholesteryl ester transfer protein; lipid metabolism
  10. Nat Metab. 2020 Jan;2(1): 41-49
      Central to cellular metabolism and cell proliferation are highly conserved signalling pathways controlled by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK)1,2, dysregulation of which are implicated in pathogenesis of major human diseases such as cancer and type 2 diabetes. AMPK pathways leading to reduced cell proliferation are well established and, in part, act through inhibition of TOR complex-1 (TORC1) activity. Here we demonstrate reciprocal regulation, specifically that TORC1 directly down-regulates AMPK signalling by phosphorylating the evolutionarily conserved residue Ser367 in the fission yeast AMPK catalytic subunit Ssp2, and AMPK α1Ser347/α2Ser345 in the mammalian homologs, which is associated with reduced phosphorylation of activation loop Thr172. Genetic or pharmacological inhibition of TORC1 signalling led to AMPK activation in the absence of increased AMP:ATP ratios; under nutrient stress conditions this was associated with growth limitation in both yeast and human cell cultures. Our findings reveal fundamental, bi-directional regulation between two major metabolic signalling networks and uncover new opportunity for cancer treatment strategies aimed at suppressing cell proliferation in the nutrient-poor tumor microenvironment.
  11. Anal Chem. 2020 Jan 28.
      Current methods for liquid-liquid extractions generally require microliter to milliliter volumes of solvents and sample, long equilibration times, and manual procedures. Extraction methods for samples in microfluidic systems have been limited as this tool is difficult to implement on the nanoliter or smaller scale. We have developed slug-flow nanoextraction (SFNE), a method based on droplet microfluidics that allows multiple liquid-liquid extractions to be performed simultaneously in a capillary tube, using only 5 nL of sample and extraction solvent per extraction. Each two-phase slug is segmented from the others by immiscible carrier fluid. We found rapid extractions (< 5 s), partition coefficients matching those achieved at larger scale extractions, and potential to preconcentrate samples through volume manipulation. This method was used to accurately and rapidly determine octanol-water partition coefficients (Kow), determining identical Kow as the shake-flask method for acetaminophen, Kow = 2.48 ± 0.02. The measurement, along with calibration and 12 replicates, was complete within 5 min, consuming under 150 nL of solvent and sample. The method was also applied to extract analytes from complex biological samples prior to electrospray ionization-tandem mass spectrometry (ESI-MS/MS) at a rate of 6 s per sample, allowing for simultaneous determination of five different drugs spiked into human plasma, synthetic urine (SU), and artificial cerebral spinal fluid (aCSF) using ethyl acetate as the extraction phase. The signal-to-noise (S/N) for analytes improved up to19-fold compared to direct ESI-MS of single-phase droplets (aqueous plugs segmented by carrier fluid), with limits of detection down to 7 nM (35 amol).
  12. Bioanalysis. 2020 Jan 27.
      Aim: Characterization of phosphatidylcholines (PCs) and lysophosphatidylcholine in human plasma using LC-IT-MSn. The characterization approach was based on trapping the eluted positive ions and applying low voltage for fragmentation to MS2 and further fragmentation of the most abundant two peaks to obtain MS3. This approach allowed linking the MS3 data to MS2 and precursor ion. Methodology: The fatty acid part, at sn-1 and sn-2 of the glycerol backbone, could be identified based on the favored cleavage pathway. Conclusion: The dysregulated PCs and lysophosphatidylcholines in human plasma obtained from acute coronary syndrome cases, and Type 2 diabetes patients suffering no coronary syndromes were estimated and matched versus healthy volunteers. An epoxide form of 16:0-18:2 PC was confirmed, m/z 774.6.
    Keywords:  Type 2 diabetes; acute coronary syndrome; biomarker; fragmentation pathway; high-performance liquid chromatography; ion trap mass spectrometry; lysophosphatidylcholine; oxidized phosphatidylcholine; phosphatidylcholines; triple quad mass spectrometry