bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2021‒07‒18
28 papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh

  1. Nat Rev Cancer. 2021 Jul 16.
      Tumour initiation and progression requires the metabolic reprogramming of cancer cells. Cancer cells autonomously alter their flux through various metabolic pathways in order to meet the increased bioenergetic and biosynthetic demand as well as mitigate oxidative stress required for cancer cell proliferation and survival. Cancer driver mutations coupled with environmental nutrient availability control flux through these metabolic pathways. Metabolites, when aberrantly accumulated, can also promote tumorigenesis. The development and application of new technologies over the last few decades has not only revealed the heterogeneity and plasticity of tumours but also allowed us to uncover new metabolic pathways involved in supporting tumour growth. The tumour microenvironment (TME), which can be depleted of certain nutrients, forces cancer cells to adapt by inducing nutrient scavenging mechanisms to sustain cancer cell proliferation. There is growing appreciation that the metabolism of cell types other than cancer cells within the TME, including endothelial cells, fibroblasts and immune cells, can modulate tumour progression. Because metastases are a major cause of death of patients with cancer, efforts are underway to understand how metabolism is harnessed by metastatic cells. Additionally, there is a new interest in exploiting cancer genetic analysis for patient stratification and/or dietary interventions in combination with therapies that target metabolism. In this Perspective, we highlight these main themes that are currently under investigation in the context of in vivo tumour metabolism, specifically emphasizing questions that remain unanswered.
  2. Mol Metab. 2021 Jul 10. pii: S2212-8778(21)00139-3. [Epub ahead of print] 101294
      BACKGROUND: There is growing interest in the analysis of tumor metabolism to identify cancer-specific metabolic vulnerabilities and therapeutic targets. The identification of such candidate metabolic pathways mainly relies on the highly sensitive identification and quantitation of numerous metabolites and metabolic fluxes using metabolomics and isotope tracing analyses. However, nutritional requirements and metabolic routes used by cancer cells cultivated in vitro do not always reflect the metabolic demands of malignant cells within the tumor milieu. Therefore, to be able to understand how the metabolism of a tumor cell in its physiological environment differs from that of normal cells, these analyses must be performed in vivo.SCOPE OF REVIEW: This review covers the physiological impact of the exogenous administration of a stable isotope tracer into cancer animal models. We discuss specific aspects of in vivo isotope tracing protocols based on discrete bolus injections of a labeled metabolite: the tracer administration per se and the fasting period prior to tracer administration. In addition, we illustrate the complex physiological scenarios that arise when studying tumor metabolism by isotopic labeling in animal models fed with a diet restricted in a specific amino acid. Finally, we provide strategies to minimize those limitations.
    MAJOR CONCLUSIONS: There is a growing evidence that metabolic dependencies in cancers are influenced by tissue environments, cancer lineage, and genetic events. More and more studies are describing discrepancies in tumor metabolic dependencies when studied in in vitro settings or in in vivo models, including cancer patients. Therefore, in depth in vivo profiling of tumor metabolic routes within the appropriate patho-physiological environment will be key to identifying relevant alterations that contribute to cancer onset and progression.
    Keywords:  Fasting; Inter-organ exchange; Stable isotope tracing; Tracer administration; Tumor metabolism
  3. Front Oncol. 2021 ;11 646779
      Background: Laryngeal cancer (LaC) remains one of the most common tumors of the respiratory tract with higher incidence in men than in women. The larynx is a small but vital organ on the neck. The dysfunction of the larynx can cause serious health problems such as hoarseness, respiratory distress, and dysphonia. Many lipids (e.g. phospholipid, cholesterol, fatty acid) have been recognized as a crucial role in tumorigenesis. However, the lipid biomarkers are lacking and the lipid molecular pathogenesis of LaC is still unclear.Methods: This study aims to identify new LaC-related lipid biomarkers used for the diagnosis or early diagnosis of LaC and to uncover their molecular characteristics. Thus, we conducted serum and tissue nontargeted lipidomics study from LaC patients (n = 29) and normal controls (NC) (n = 36) via ultra-high performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry (HRMS). Multivariate and univariate statistics analyses were used to discriminate LaC patients from NC.
    Results: As expected, a lipid panel including LPC (16:0) and PE (18:0p_20:4) was defined to distinguish the LaC patients from healthy individuals with very high diagnosis performance (area under the curve (AUC) value = 1.000, sensitivity value = 1.000, and specificity value = 1.000). In addition, the levels of Cer, CerG1, SM, PC, PC-O, PE, PI, PS, and ChE in the LaC group significantly increased as compared with the NC group. However, the levels of LPC, LPC-O, LPE, LPE-p, and DG in the LaC group significantly deceased when the one was compared with the NC group. Among significantly changed lipid species, lysophospholipids containing a palmitoyl chain or an arachidonic acid acyl chain remarkably decreased and phospholipids including a palmitoyl chain or an arachidonic acid acyl chain increased in the LaC patients.
    Conclusion: Our results not only indicate that lipidomics is powerful tool to explore abnormal lipid metabolism for the laC, but suggest that lysophospholipids and phospholipids may serve as potential biomarkers for diagnosis of LaC.
    Keywords:  UHPLC-mass spectrometry; biomarker; laryngeal cancer; lipid metabolic abnormality; lipidomics
  4. Anal Bioanal Chem. 2021 Jul 09.
      Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method was developed with the aim to unambiguously identify a large number of lipid species from multiple lipid classes in human plasma. The optimized RP-UHPLC/MS method employed the C18 column with sub-2-μm particles with the total run time of 25 min. The chromatographic resolution was investigated with 42 standards from 18 lipid classes. The UHPLC system was coupled to high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) measuring full-scan and tandem mass spectra (MS/MS) in positive- and negative-ion modes with high mass accuracy. Our identification approach was based on m/z values measured with mass accuracy within 5 ppm tolerance in the full-scan mode, characteristic fragment ions in MS/MS, and regularity in chromatographic retention dependences for individual lipid species, which provides the highest level of confidence for reported identifications of lipid species including regioisomeric and other isobaric forms. The graphs of dependences of retention times on the carbon number or on the number of double bond(s) in fatty acyl chains were constructed to support the identification of lipid species in homologous lipid series. Our list of identified lipid species is also compared with previous publications investigating human blood samples by various MS-based approaches. In total, we have reported more than 500 lipid species representing 26 polar and nonpolar lipid classes detected in NIST Standard reference material 1950 human plasma.
    Keywords:  Human plasma; Lipidomics; Lipids; Mass spectrometry; Retention behavior; Reversed-phase; Ultrahigh-performance liquid chromatography
  5. Anal Chem. 2021 Jul 16.
      Metabolomics is a powerful and essential technology for profiling metabolic phenotypes and exploring metabolic reprogramming, which enables the identification of biomarkers and provides mechanistic insights into physiology and disease. However, its applications are still limited by the technical challenges particularly in its detection sensitivity for the analysis of biological samples with limited amount, necessitating the development of highly sensitive approaches. Here, we developed a highly sensitive liquid chromatography tandem mass spectrometry method based on a 3-nitrophenylhydrazine (3-NPH) derivatization strategy that simultaneously targets carbonyl, carboxyl, and phosphoryl groups for targeted metabolomic analysis (HSDccp-TM) in biological samples. By testing 130 endogenous metabolites including organic acids, amino acids, carbohydrates, nucleotides, carnitines, and vitamins, we showed that the derivatization strategy resulted in significantly improved detection sensitivity and chromatographic separation capability. Metabolic profiling of merely 60 oocytes and 5000 hematopoietic stem cells primarily isolated from mice demonstrated that this method enabled routine metabolomic analysis in trace amounts of biospecimens. Moreover, the derivatization strategy bypassed the tediousness of inferring the MS fragmentation patterns and simplified the complexity of monitoring ion pairs of metabolites, which greatly facilitated the metabolic flux analysis (MFA) for glycolysis, the tricarboxylic acid (TCA) cycle, and pentose phosphate pathway (PPP) in cell cultures. In summary, the novel 3-NPH derivatization-based method with high sensitivity, good chromatographic separation, and broad coverage showed great potential in promoting metabolomics and MFA, especially in trace amounts of biospecimens.
  6. Clin Chem Lab Med. 2021 Jul 09.
      OBJECTIVES: Lipid mediators are bioactive lipids which help regulate inflammation. We aimed to develop an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify 58 pro-inflammatory and pro-resolving lipid mediators in plasma, determine preliminary reference ranges for adolescents, and investigate how total parenteral nutrition (TPN) containing omega-3 polyunsaturated fatty acid (n-3 PUFA) or n-6 PUFA based lipid emulsions influence lipid mediator concentrations in plasma.METHODS: Lipid mediators were extracted from plasma using SPE and measured using UHPLC-MS/MS. EDTA plasma was collected from healthy adolescents between 13 and 17 years of age to determine preliminary reference ranges and from mice given intravenous TPN for seven days containing either an n-3 PUFA or n-6 PUFA based lipid emulsion.
    RESULTS: We successfully quantified 43 lipid mediators in human plasma with good precision and recovery including several leukotrienes, prostaglandins, resolvins, protectins, maresins, and lipoxins. We found that the addition of methanol to human plasma after blood separation reduces post blood draw increases in 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE), 12S-hydroxyeicosatrienoic acid (12S-HETrE), 14-hydroxydocosahexaenoic acid (14-HDHA) and thromboxane B2 (TXB2). Compared to the n-6 PUFA based TPN, the n-3 PUFA based TPN increased specialized pro-resolving mediators such as maresin 1 (MaR1), MaR2, protectin D1 (PD1), PDX, and resolvin D5 (RvD5), and decreased inflammatory lipid mediators such as leukotriene B4 (LTB4) and prostaglandin D2 (PGD2).
    CONCLUSIONS: Our method provides an accurate and sensitive quantification of 58 lipid mediators from plasma samples, which we used to establish a preliminary reference range for lipid mediators in plasma samples of adolescents; and to show that n-3 PUFA, compared to n-6 PUFA rich TPN, leads to a less inflammatory lipid mediator profile in mice.
    Keywords:  AA; AEA; ALOX12; COX; CYP; D-series resolvin; DGLA; DHA; DHEA; DPA; DiHETE; EPA; EPEA; EpDPA; EpEDE; EpETE; EpOME; HDHA; HEPE; HETE; HETrE; HODE; LA; LC-MS/MS; LOX; LTB4; LXA4; LXB4; MaR; PD1; PDX; PGD2; PGE2; PGF1α; PUFA; RvD; SPE; SPM; TPN; TXB2; UHPLC-MS/MS; arachidonate 12-lipoxygenase; arachidonic acid; arachidonoyl ethanolamide; cyclooxygenase; cytochrome P450 monooxygenase; dihomo-γ linoleic acid; dihydroxyeicosatetraenoic acid; docosahexaenoic acid; docosahexaenoyl ethanolamide; docosapentaenoic acid; eicosanoids; eicosapentaenoic acid; eicosapentaenoyl ethanolamide; epoxydocosapentaenoic acid; epoxyeicosadienoic acid; epoxyeicosatetraenoic acid; epoxyoctadecenoic acid; hydroxydocosahexaenoic acid; hydroxyeicosapentaenoic acid; hydroxyeicosatetraenoic acid; hydroxyeicosatrienoic acid; hydroxyoctadecadienoic acid; leukotriene B4; linoleic acid; lipid mediators; lipoxin A4; lipoxin B4; lipoxygenase; maresin; oxylipins; polyunsaturated fatty acid; prostaglandin D2; prostaglandin E2; prostaglandin F1α; protectin D1; protectin DX; reference value; solid phase extraction; specialized pro-resolving mediator; thromboxane B2; total parenteral nutrition; ultra-high-performance liquid chromatography-tandem mass spectrometry; α-linolenic acid; αLA
  7. J Biol Chem. 2021 Jul 09. pii: S0021-9258(21)00750-X. [Epub ahead of print] 100950
      Mammalian cells synthesize H2S from sulfur containing amino acids and are also exposed to exogenous sources of this signaling molecule, notably from gut microbes. As an inhibitor of complex IV in the electron transport chain, H2S can have a profound impact on metabolism, suggesting the hypothesis that metabolic reprogramming is a primary mechanism by which H2S signals. In this study, we report that H2S increases lipogenesis in many cell types, using carbon derived from glutamine rather than from glucose. H2S-stimulated lipid synthesis is sensitive to the mitochondrial NAD(P)H pools and is enabled by reductive carboxylation of α-ketoglutarate. Lipidomics analysis revealed that H2S elicits time-dependent changes across several lipid classes, e.g., upregulating triglycerides while down regulating phosphatidylcholine. Direct analysis of triglyceride concentration revealed that H2S induces a net increase in the size of this lipid pool. These results provide a mechanistic framework for understanding the effects of H2S on increasing lipid droplets in adipocytes and population studies that have pointed to a positive correlation between cysteine (a substrate for H2S synthesis) and fat mass.
  8. Anal Bioanal Chem. 2021 Jul 12.
      Detailed molecular analysis is of increasing importance in research into the regulation of biochemical pathways, organismal growth and disease. Lipidomics in particular is increasingly sought after as it provides insight into molecular species involved in energy storage, signalling and fundamental cellular structures. This has led to the use of a range of tools and techniques to acquire lipidomics data. 31P NMR for lipidomics offers well-resolved head group/lipid class analysis, structural data that can be used to inform and strengthen interpretation of mass spectrometry data and part of a priori structural determination. In the present study, we codify the use of 31P NMR for lipidomics studies to make the technique more accessible to new users and more useful for a wider range of questions. The technique can be used in isolation (phospholipidomics) or as a part of determining lipid composition (lipidomics). We describe the process from sample extraction to data processing and analysis. This pipeline is important because it allows greater thoroughness in lipidomics studies and increases scope for answering scientific questions about lipid-containing systems.
    Keywords:  31P NMR; Lipid profiling; Lipidomics
  9. Cell Rep. 2021 Jul 13. pii: S2211-1247(21)00721-X. [Epub ahead of print]36(2): 109345
      Upon nutrient stimulation, pre-adipocytes undergo differentiation to transform into mature adipocytes capable of storing nutrients as fat. We profiled cellular metabolite consumption to identify early metabolic drivers of adipocyte differentiation. We find that adipocyte differentiation raises the uptake and consumption of numerous amino acids. In particular, branched-chain amino acid (BCAA) catabolism precedes and promotes peroxisome proliferator-activated receptor gamma (PPARγ), a key regulator of adipogenesis. In early adipogenesis, the mitochondrial sirtuin SIRT4 elevates BCAA catabolism through the activation of methylcrotonyl-coenzyme A (CoA) carboxylase (MCCC). MCCC supports leucine oxidation by catalyzing the carboxylation of 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA. Sirtuin 4 (SIRT4) expression is decreased in adipose tissue of numerous diabetic mouse models, and its expression is most correlated with BCAA enzymes, suggesting a potential role for SIRT4 in adipose pathology through the alteration of BCAA metabolism. In summary, this work provides a temporal analysis of adipocyte differentiation and uncovers early metabolic events that stimulate transcriptional reprogramming.
    Keywords:  BCAA catabolism; MCCC; PPARg; SIRT4; adipogenesis; amino acids; differentiation; sirtuin
  10. Anal Chem. 2021 Jul 16.
      Studies of cellular metabolism can provide profound insights into the underlying molecular mechanisms and metabolic function. To date, the majority of cellular metabolism studies based on chromatography-mass spectrometry (MS) require population cells to obtain informative metabolome. These methods are not only time-consuming but also not suitable for amount-limited cell samples such as circulating tumor cells, stem cells, and neurons. Therefore, it is extremely essential to develop analytical methods enabling to detect metabolome from tens of cells in a high-throughput and high-sensitivity way. In this work, a novel platform for rapid and sensitive detection of lipidome in 20 mammalian cells was proposed using capillary microsampling combined with high-resolution spectral stitching nanoelectrospray ionization direct-infusion MS. It can be used to collect cells rapidly and accurately via the capillary microprobe, extract lipids directly in a 96-well plate using a spray solvent, and detect more than 500 lipids covering 19 lipid subclasses within 3 min. This novel platform was successfully applied to study the lipid features of different cancer cell types and subtypes as well as target cells from tissue samples. This study provides a strategy for determining the lipid species with rich information in tens of cells and demonstrates great potential for clinical applications.
  11. Bioinformatics. 2021 07 12. 37(Suppl_1): i434-i442
      MOTIVATION: Tandem mass spectrometry data acquired using data independent acquisition (DIA) is challenging to interpret because the data exhibits complex structure along both the mass-to-charge (m/z) and time axes. The most common approach to analyzing this type of data makes use of a library of previously observed DIA data patterns (a 'spectral library'), but this approach is expensive because the libraries do not typically generalize well across laboratories.RESULTS: Here, we propose DIAmeter, a search engine that detects peptides in DIA data using only a peptide sequence database. Although some existing library-free DIA analysis methods (i) support data generated using both wide and narrow isolation windows, (ii) detect peptides containing post-translational modifications, (iii) analyze data from a variety of instrument platforms and (iv) are capable of detecting peptides even in the absence of detectable signal in the survey (MS1) scan, DIAmeter is the only method that offers all four capabilities in a single tool.
    AVAILABILITY AND IMPLEMENTATION: The open source, Apache licensed source code is available as part of the Crux mass spectrometry analysis toolkit (
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
  12. Anal Chem. 2021 Jul 16.
      In-source fragmentation (ISF) is a naturally occurring phenomenon during electrospray ionization (ESI) in liquid chromatography-mass spectrometry (LC-MS) analysis. ISF leads to false metabolite annotation in untargeted metabolomics, prompting misinterpretation of the underlying biological mechanisms. Conventional metabolomic data cleaning mainly focuses on the annotation of adducts and isotopes, and the recognition of ISF features is mainly based on common neutral losses and the LC coelution pattern. In this work, we recognized three increasingly important patterns of ISF features, including (1) coeluting with their precursor ions, (2) being in the tandem MS (MS2) spectra of their precursor ions, and (3) sharing similar MS2 fragmentation patterns with their precursor ions. Based on these patterns, we developed an R package, ISFrag, to comprehensively recognize all possible ISF features from LC-MS data generated from full-scan, data-dependent acquisition, and data-independent acquisition modes without the assistance of common neutral loss information or MS2 spectral library. Tested using metabolite standards, we achieved a 100% correct recognition of level 1 ISF features and over 80% correct recognition for level 2 ISF features. Further application of ISFrag on untargeted metabolomics data allows us to identify ISF features that can potentially cause false metabolite annotation at an omics-scale. With the help of ISFrag, we performed a systematic investigation of how ISF features are influenced by different MS parameters, including capillary voltage, end plate offset, ion energy, and "collision energy". Our results show that while increasing energies can increase the number of real metabolic features and ISF features, the percentage of ISF features might not necessarily increase. Finally, using ISFrag, we created an ISF pathway to visualize the relationships between multiple ISF features that belong to the same precursor ion. ISFrag is freely available on GitHub (
  13. EMBO Rep. 2021 Jul 14. e51981
      Glutaminolysis is known to correlate with ovarian cancer aggressiveness and invasion. However, how this affects the tumor microenvironment is elusive. Here, we show that ovarian cancer cells become addicted to extracellular glutamine when silenced for glutamine synthetase (GS), similar to naturally occurring GS-low, glutaminolysis-high ovarian cancer cells. Glutamine addiction elicits a crosstalk mechanism whereby cancer cells release N-acetylaspartate (NAA) which, through the inhibition of the NMDA receptor, and synergistically with IL-10, enforces GS expression in macrophages. In turn, GS-high macrophages acquire M2-like, tumorigenic features. Supporting this in␣vitro model, in silico data and the analysis of ascitic fluid isolated from ovarian cancer patients prove that an M2-like macrophage phenotype, IL-10 release, and NAA levels positively correlate with disease stage. Our study uncovers the unprecedented role of glutamine metabolism in modulating macrophage polarization in highly invasive ovarian cancer and highlights the anti-inflammatory, protumoral function of NAA.
    Keywords:  IL-10; N-acetylaspartate; TAMs; metabolism; ovarian cancer
  14. JCI Insight. 2021 Jul 15. pii: 140288. [Epub ahead of print]
      The alpha ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is a Hypoxia-Inducible Factor (HIF) target that uses molecular oxygen to hydroxylate peptidyl prolyl residues. While PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about effects of this highly conserved enzyme in insulin-secreting β-cells in vivo. Here, we show that deletion of PHD3 specifically in β-cells (βPHD3KO) is associated with impaired glucose homeostasis in mice fed high fat diet. In the early stages of dietary fat excess, βPHD3KO islets energetically rewire, leading to defects in the management of pyruvate fate and a shift from glycolysis to increased fatty acid oxidation (FAO). However, under more prolonged metabolic stress, this switch to preferential FAO in βPHD3KO islets is associated with impaired glucose-stimulated ATP/ADP rises, Ca2+ fluxes and insulin secretion. Thus, PHD3 might be a pivotal component of the β-cell glucose metabolism machinery in mice by suppressing the use of fatty acids as a primary fuel source during the early phases of metabolic stress.
    Keywords:  Beta cells; Bioenergetics; Endocrinology; Insulin; Metabolism
  15. Front Cell Dev Biol. 2021 ;9 675617
      Exposure of tumor cells to ionizing radiation (IR) alters the microenvironment, particularly the fatty acid (FA) profile and activity. Moreover, abnormal FA metabolism, either catabolism or anabolism, is essential for synthesizing biological membranes and delivering molecular signals to induce ferroptotic cell death. The current review focuses on the bistable regulation characteristics of FA metabolism and explains how FA catabolism and anabolism pathway crosstalk harmonize different ionizing radiation-regulated ferroptosis responses, resulting in pivotal cell fate decisions. In summary, targeting key molecules involved in lipid metabolism and ferroptosis may amplify the tumor response to IR.
    Keywords:  cancer; cell death; fatty acid metabolism; ferroptosis; irradiation
  16. Cancer Prev Res (Phila). 2021 Jul 12. pii: canprevres.0612.2021. [Epub ahead of print]
      Novel biomarkers for HCC surveillance in cirrhotic patients are urgently needed. Exosomes and their lipid content in particular, represent potentially valuable noninvasive diagnostic biomarkers. We isolated exosomes from plasma of 72 cirrhotic patients, including 31 with HCC. Exosomes and unfractionated plasma were processed for untargeted lipidomics using ultra-high-resolution mass spectrometry. A total of 2,864 lipid species, belonging to 52 classes, were identified. Both exosome fractionation and HCC diagnosis had significant impact on the lipid profiles. Ten lipid classes were enriched in HCC exosomes compared to non-HCC exosomes. Dilysocardiolipins were detected in 35% of the HCC exosomes but in none of the non-HCC exosomes (p<0.001). Cardiolipins and sphingosines had the highest differential effects (fold change of 133.08, q=0.001 and 38.57, q<0.001, respectively). In logistic regression analysis, high abundances of exosomal sphingosines, dilysocardiolipins, lysophosphatidylserines and (O-acyl)-1-hydroxy fatty acids were strongly associated with HCC (OR [95% CI]: 271.1 [14.0-5251.9], p<0.001; 46.5 [2.3-939.9], p=0.012; 14.9 [4.3-51.2), p<0.001; 10.3 [3.2-33.1], p<0.001). Four lipid classes were depleted in HCC exosomes compared to non-HCC exosomes. In logistic regression analysis, lack of detection of sulfatides and acylGlcSitosterol esters was strongly associated with HCC (OR [95% CI]: 215.5 [11.5-4035.9], p<0.001; 26.7 [1.4-528.4], p=0.031). These HCC-associated changes in lipid composition of exosomes reflected alterations in glycerophospholipid metabolism, retrograde endocannabinoid signaling and ferroptosis. In conclusion, this study identified candidate biomarkers for early detection of HCC as well as altered pathways in exosomes that may contribute to tumor development and progression.
  17. Cell Death Dis. 2021 Jul 13. 12(7): 698
      Sorafenib, a protein kinase inhibitor approved for the treatment of hepatocellular carcinoma and advanced renal cell carcinoma, has been repeatedly reported to induce ferroptosis by possibly involving inhibition of the cystine/glutamate antiporter, known as system xc-. Using a combination of well-defined genetically engineered tumor cell lines and canonical small molecule ferroptosis inhibitors, we now provide unequivocal evidence that sorafenib does not induce ferroptosis in a series of tumor cell lines unlike the cognate system xc- inhibitors sulfasalazine and erastin. We further show that only a subset of tumor cells dies by ferroptosis upon sulfasalazine and erastin treatment, implying that certain cell lines appear to be resistant to system xc- inhibition, while others undergo ferroptosis-independent cell death. From these findings, we conclude that sorafenib does not qualify as a bona fide ferroptosis inducer and that ferroptosis induced by system xc- inhibitors can only be achieved in a fraction of tumor cell lines despite robust expression of SLC7A11, the substrate-specific subunit of system xc-.
  18. Methods Mol Biol. 2021 ;2358 221-228
      Phosphoproteomics has drawn great attention of biologist since phosphorylation is proven to play an important role in regulation of proteins. Mass spectrometry technology has helped with the development of phosphoproteomics due to its ability in generating large amount of detailed information after analyzing the phosphoproteome samples. However, interpreting the phosphoproteome data deprived from mass spectrometry can be time-consuming. Here, we introduced a free R language-based platform which can be used in accelerating phosphoproteome data analysis. This platform has integrated different functions and methods which are popularly used in phosphoproteome data analysis, so users can customize their analysis according to their demands.
    Keywords:  Mass spectrometry; Phosphoproteomics data analysis; R language; cRacker
  19. Toxicol Appl Pharmacol. 2021 Jul 07. pii: S0041-008X(21)00244-1. [Epub ahead of print]426 115640
      Benzo[a]pyrene (BaP) is a strong carcinogen for lung cancer, and forkhead-box A1 (FOXA1) plays an oncogenic role in BaP-transformed cell THBEc1. To explore the remodeling of metabolic pattern caused by BaP-induced transformation and the possible role FOXA1 might play in it, we compared metabolic patterns between THBEc1 cells and control using untargeted metabolomics and lipidomics analysis, and determined the effects of FOXA1 knockout on the metabolic pattern of THBEc1 cells. Metabolomics and lipidomics identified a total of 15 and 46 differential metabolites and lipids between THBEc1 and 16HBE cells, respectively, and a total of 4 and 1 differential metabolites and lipids between FOXA1 knockout cell THBEc1-ΔFOXA1-c34 and control cell THBEc1-ctrl, respectively. Analysis results of metabolites and metabolic pathways indicated the metabolic pattern remodeling may be related to the alteration in glucose metabolism during BaP-induced transformation. Western blotting revealed the up-regulation of enolase-2 (ENO2), pyruvate carboxylase (PCB), aconitase-2 (ACO2) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) (Thr202/Tyr204), the down-regulation of succinate dehydrogenase complex subunit A (SDHA) and phosphoenolpyruvate carboxykinase 2 (PCK2) in THBEc1 cells. The detection results of metabolites related to glucose metabolism demonstrated the decreasing of lactic acid content in cells, lactic acid production in culture medium and citric acid content in mitochondria, and the increasing of ATP production of THBEc1 cells. FOXA1 knockout partially reversed the changes of ENO2, SDHA, PCK2 and p-ERK1/2 (Thr202/Tyr204) levels, lactic acid release, citric acid content in mitochondria of THBEc1 cells. In conclusion, FOXA1 knockout partially reversed the remodeling of glucose metabolism caused by BaP-induced malignant transformation. Our findings provide a clue for the possible role of FOXA1 in glucose metabolism regulation.
    Keywords:  Benzo[a]Pyrene; FOXA1; Lipidomics; Lung Cancer; Metabolomics; THBEc1 cells
  20. J Biol Chem. 2021 Jul 13. pii: S0021-9258(21)00754-7. [Epub ahead of print] 100954
      PPARδ is a nuclear receptor transcription factor that plays an important role in the regulation of metabolism, inflammation, and cancer. In addition, the nutrient-sensing kinase AMPK is a critical regulator of cellular energy in coordination with PPARδ. However, the molecular mechanism of AMPK/PPARδ pathway on cancer progression is still unclear. Here we found that activated AMPK induced PPARδ-S50 phosphorylation in cancer cells, while the PPARδ/S50A (non-phosphorylation mimic) mutant reversed this event. Further analysis showed that the PPARδ/S50E (phosphorylation mimic) but not the PPARδ/S50A mutant increased PPARδ protein stability, which led to reduced p62/SQSTM1-mediated degradation of misfolded PPARδ. Furthermore, PPARδ-S50 phosphorylation decreased PPARδ transcription activity and alleviated PPARδ-mediated uptake of glucose and glutamine in cancer cells. Soft agar and xenograft tumor model analysis showed that the PPARδ/S50E mutant but not the PPARδ/S50A mutant inhibited colon cancer cell proliferation and tumor growth, which was associated with inhibition of Glut1 and SLC1A5 transporter protein expression. These findings reveal a new mechanism of AMPK-induced PPARδ-S50 phosphorylation, accumulation of misfolded PPARδ protein, and inhibition of PPARδ transcription activity contributing to the suppression of colon tumor formation.
    Keywords:  AMPK; PPARδ; cancer; misfolded protein; phosphorylation
  21. Front Chem. 2021 ;9 702260
      The modification on proteins with O-linked N-acetyl-β-D-glucosamine (O-GlcNAcylation) is essential for normal cell physiology. Dysregulation of O-GlcNAcylation leads to many human diseases, such as cancer, diabetes and neurodegenerative diseases. Recently, the functional role of O-GlcNAcylation in different physiological states has been elucidated due to the booming detection technologies. Chemical approaches for the enrichment of O-GlcNAcylated proteins combined with mass spectrometry-based proteomics enable the profiling of protein O-GlcNAcylation in a system-wide level. In this review, we summarize recent progresses on the enrichment and proteomic profiling of protein O-GlcNAcylation.
    Keywords:  O-GlcNAcylation; chemical tools; enrichment strategies; mass spectrometry; quantitative proteomics
  22. Bio Protoc. 2021 Jun 20. 11(12): e4066
      Over the last decade, lipids have emerged as possessing an ever-increasing number of key functions, especially in membrane trafficking. For instance, phosphatidic acid (PA) has been proposed to play a critical role in different steps along the secretory pathway or during phagocytosis. To further investigate in detail the precise nature of PA activities, we need to identify the organelles in which PA is synthesized and the PA subspecies involved in these biological functions. Indeed, PA, like all phospholipids, has a large variety based on its fatty acid composition. The recent development of PA sensors has helped us to follow intracellular PA dynamics but has failed to provide information on individual PA species. Here, we describe a method for the subcellular fractionation of RAW264.7 macrophages that allows us to obtain membrane fractions enriched in specific organelles based on their density. Lipids from these membrane fractions are precipitated and subsequently processed by advanced mass spectrometry-based lipidomics analysis to measure the levels of different PA species based on their fatty acyl chain composition. This approach revealed the presence of up to 50 different species of PA in cellular membranes, opening up the possibility that a single class of phospholipid could play multiple functions in any given organelle. This protocol can be adapted or modified and used for the evaluation of other intracellular membrane compartments or cell types of interest.
    Keywords:  Lipidomics; Macrophages; Mass spectrometry; Phosphatidic acid; RAW264.7; Subcellular membrane fractions
  23. J Proteome Res. 2021 Jul 15.
      Annotating product ion peaks in tandem mass spectra is essential for evaluating spectral quality and validating peptide identification. This task is more complex for glycopeptides and is crucial for the confident determination of glycosylation sites in glycoproteins. MS_Piano (Mass Spectrum Peptide Annotation) software was developed for reliable annotation of peaks in collision induced dissociation (CID) tandem mass spectra of peptides or N-glycopeptides for given peptide sequences, charge states, and optional modifications. The program annotates each peak in high or low resolution spectra with possible product ion(s) and the mass difference between the measured and theoretical m/z values. Spectral quality is measured by two major parameters: the ratio between the sum of unannotated vs all peak intensities in the top 20 peaks, and the intensity of the highest unannotated peak. The product ions of peptides, glycans, and glycopeptides in spectra are labeled in different class-type colors to facilitate interpretation. MS_Piano assists validating peptide and N-glycopeptide identification from database and library searches and provides quality control and optimizes search reliability in custom developed peptide mass spectral libraries. The software is freely available in .exe and .dll formats for the Windows operating system.
    Keywords:  glycopeptide fragmentation; glycopeptide identification; glycoproteomics; mass spectrometry; peak annotation; peptide fragmentation; peptide identification; proteomics; software
  24. FEBS J. 2021 Jul 16.
      Autophagy is a catabolic process that captures cellular waste and degrades them in the lysosome. The main function of autophagy is quality control of cytosolic proteins and organelles, and intracellular recycling of nutrients in order to maintain cellular homeostasis. Autophagy is upregulated in many cancers to promote cell survival, proliferation and metastasis. Both cell-autonomous autophagy (also known as tumor autophagy) and non-cell autonomous autophagy (also known as host autophagy) supports tumorigenesis through different mechanisms, including inhibition of p53 activation, sustaining redox homeostasis, maintenance of essential amino acids levels in order to support energy production and biosynthesis, and inhibition of anti-tumor immune responses. Therefore, autophagy may serve as a tumor-specific vulnerability and targeting autophagy could be a novel strategy in cancer treatment.
    Keywords:  Autophagy; Cancer; Cancer Metabolism; Immune Response; Metastasis; p53
  25. J Proteome Res. 2021 Jul 14.
      Chromatographic separation is often an important part of mass-spectrometry-based proteomic analysis. It reduces the complexity of the initial samples before they are introduced to mass-spectrometric detection and chromatographic characteristics (such as retention time) add analytical features to the analyte. The acquisition and analysis of chromatographic data are thus of great importance, and specialized software is used for the extraction of quantitative information in an efficient and optimized manner. However, occasionally, automatic peak picking and correct peak boundary setting is challenged by, for instance, aberration of peak shape, peak truncation, and peak tailing, and a manual review of a large number of peaks is frequently required. To support this part of the analysis, we present here a software tool, Peakfit, that fits acquired chromatographic data to the log-normal peak equation and reports the calculated peak parameters. The program is written in R and can easily be integrated into Skyline, a popular software packages that is frequently used for proteomic parallel reaction monitoring applications. The program is capable of processing large data sets (>10 000 peaks) and detecting sporadic outliers in peak boundary selection performed, for instance, in Skyline. In an example data set, available via ProteomeXchange with identifier PXD026875, we demonstrated the capability of the program to characterize chromatographic peaks and showed an example of its ability to objectively and reproducibly detect and solve problematic peak-picking situations.
    Keywords:  data processing; parallel reaction monitoring; peak fitting; peak modeling; software tool
  26. J Proteome Res. 2021 Jul 16.
      Improvements in both liquid chromatography (LC) and mass spectrometry (MS) instrumentation have greatly enhanced proteomic and small molecule metabolomic analysis in recent years. Less focus has been on the improved capability to detect and quantify small bioactive peptides, even though the exact sequences of the peptide species produced can have important biological consequences. Endogenous bioactive peptide hormones, for example, are generated by the targeted and regulated cleavage of peptides from their prohormone sequence. This process may include organ specific variants, as proglucagon is converted to glucagon in the pancreas but glucagon-like peptide-1 (GLP-1) in the small intestine, with glucagon raising, whereas GLP-1, as an incretin, lowering blood glucose. Therefore, peptidomics workflows must preserve the structure of the processed peptide products to prevent the misidentification of ambiguous peptide species. The poor in vivo and in vitro stability of peptides in biological matrices is a major factor that needs to be considered when developing methods to study them. The bioinformatic analysis of peptidomics data sets requires the inclusion of specific post-translational modifications, which are critical for the function of many bioactive peptides. This review aims to discuss and contrast the various extraction, analytical, and bioinformatics approaches used for human peptidomics studies in a multitude of matrices.
    Keywords:  clinical analysis; mass spectrometry; peptidomics
  27. Adv Exp Med Biol. 2021 ;1332 17-33
      Amino acids are the main building blocks for life. Aside from their roles in composing proteins, functional amino acids and their metabolites play regulatory roles in key metabolic cascades, gene expressions, and cell-to-cell communication via a variety of cell signaling pathways. These metabolic networks are necessary for maintenance, growth, reproduction, and immunity in humans and animals. These amino acids include, but are not limited to, arginine, glutamine, glutamate, glycine, leucine, proline, and tryptophan. We will discuss these functional amino acids in cell signaling pathways in mammals with a particular emphasis on mTORC1, AMPK, and MAPK pathways for protein synthesis, nutrient sensing, and anti-inflammatory responses, as well as cell survival, growth, and development.
    Keywords:  AMPK; Functional amino acids; MAPK; Sensor; Transceptor; mTORC1