bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2021–08–01
27 papers selected by
Giovanny Rodríguez Blanco, University of Edinburgh
  1. In vivo isotope tracing reveals the versatility of glucose as a brown adipose tissue substrate.
  2. Improved identification and quantification of peptides in mass spectrometry data via chemical and random additive noise elimination (CRANE).
  3. Current Challenges and Recent Developments in Mass Spectrometry-Based Metabolomics.
  4. Proteomics for Low Cell Numbers: How to Optimize the Sample Preparation Workflow for Mass Spectrometry Analysis.
  5. Single Quadrupole Multiple Fragment Ion Monitoring Quantitative Mass Spectrometry.
  6. Spatial epi-proteomics enabled by histone post-translational modification analysis from low-abundance clinical samples.
  7. Advances in chemical proteomic evaluation of lipid kinases-DAG kinases as a case study.
  8. NAD+ metabolism and cardiometabolic health: the human evidence.
  9. Hepatic mTORC1 signaling activates ATF4 as part of its metabolic response to feeding and insulin.
  10. Metabolism in the Tumour-Bone Microenvironment.
  11. Multidimensional Separations of Intact Phase II Steroid Metabolites Utilizing LC-Ion Mobility-HRMS.
  12. Identification of covalent modifications regulating immune signaling complex composition and phenotype.
  13. Precise Quantitation of PTEN by Immuno-MRM: A Tool To Resolve the Breast Cancer Biomarker Controversy.
  14. FIGS: Featured Ion-Guided Stoichiometry for Data-Independent Proteomics through Dynamic Deconvolution.
  15. Monitoring autophagy-dependent ferroptosis.
  16. Ferroptosis in the tumor microenvironment: perspectives for immunotherapy.
  17. PRMT1-dependent regulation of RNA metabolism and DNA damage response sustains pancreatic ductal adenocarcinoma.
  18. PrSM-Level Side-by-Side Comparison of Online LC-MS Methods with Intact Histone H3 and H4 Proteoforms.
  19. STING suppresses bone cancer pain via immune and neuronal modulation.
  20. Acoustic Ejection Mass Spectrometry for High-Throughput Analysis.
  21. Non-targeted LC-MS and CE-MS for biomarker discovery in bioreactors: Influence of separation, mass spectrometry and data processing tools.
  22. Lipid metabolism, inflammation, and foam cell formation in health and metabolic disorders: targeting mTORC1.
  23. Statistical analysis in metabolic phenotyping.
  24. Metabolic hallmarks of liver regeneration.
  25. Review: Multiplexed profiling of biomarkers in extracellular vesicles for cancer diagnosis and therapy monitoring.
  26. Comparison of Unit Resolution Versus High-Resolution Accurate Mass for Parallel Reaction Monitoring.
  27. DDASSQ: an open-source, multiple peptide sequencing strategy for label free quantification based on an OpenMS pipeline in the KNIME analytics platform.
  1. Cell Rep. 2021 Jul 27. pii: S2211-1247(21)00882-2. [Epub ahead of print]36(4): 109459
    In vivo isotope tracing reveals the versatility of glucose as a brown adipose tissue substrate.
    Su Myung Jung, Will G Doxsey, Johnny Le, John A Haley, Lorena Mazuecos, Amelia K Luciano, Huawei Li, Cholsoon Jang, David A Guertin.
      Active brown adipose tissue (BAT) consumes copious amounts of glucose, yet how glucose metabolism supports thermogenesis is unclear. By combining transcriptomics, metabolomics, and stable isotope tracing in vivo, we systematically analyze BAT glucose utilization in mice during acute and chronic cold exposure. Metabolite profiling reveals extensive temperature-dependent changes in the BAT metabolome and transcriptome upon cold adaptation, discovering unexpected metabolite markers of thermogenesis, including increased N-acetyl-amino acid production. Time-course stable isotope tracing further reveals rapid incorporation of glucose carbons into glycolysis and TCA cycle, as well as several auxiliary pathways, including NADPH, nucleotide, and phospholipid synthesis pathways. Gene expression differences inconsistently predict glucose fluxes, indicating that posttranscriptional mechanisms also govern glucose utilization. Surprisingly, BAT swiftly generates fatty acids and acyl-carnitines from glucose, suggesting that lipids are rapidly synthesized and immediately oxidized. These data reveal versatility in BAT glucose utilization, highlighting the value of an integrative-omics approach to understanding organ metabolism.
    Keywords:  BAT; brown adipocyte; brown adipose tissue; brown fat; glucose metabolism; lipid metabolism; metabolomics; stable isotope tracing; temperature acclimation; thermogenesis
    DOI:  https://doi.org/10.1016/j.celrep.2021.109459
  2. Bioinformatics. 2021 Jul 29. pii: btab563. [Epub ahead of print]
    Improved identification and quantification of peptides in mass spectrometry data via chemical and random additive noise elimination (CRANE).
    Akila J Seneviratne, Sean Peters, David Clarke, Michael Dausmann, Michael Hecker, Brett Tully, Peter G Hains, Qing Zhong.
       MOTIVATION: The output of electrospray ionisation - liquid chromatography mass spectrometry (ESI-LC-MS) is influenced by multiple sources of noise and major contributors can be broadly categorised as baseline, random and chemical noise. Noise has a negative impact on the identification and quantification of peptides, which influences the reliability and reproducibility of MS-based proteomics data. Most attempts at denoising have been made on either spectra or chromatograms independently, thus important two-dimensional information is lost because the mass-to-charge ratio and retention time dimensions are not considered jointly.
    RESULTS: This paper presents a novel technique for denoising raw ESI-LC-MS data via two-dimensional undecimated wavelet transform, which is applied to proteomics data acquired by data-independent acquisition MS (DIA-MS). We demonstrate that denoising DIA-MS data results in the improvement of peptide identification and quantification in complex biological samples.
    AVAILABILITY: The software is available on Github (https://github.com/CMRI-ProCan/CRANE). The datasets were obtained from ProteomeXchange (Identifiers-PXD002952 and PXD008651). Preliminary data and intermediate files are available via ProteomeXchange (Identifiers-PXD020529 and PXD025103).
    SUPPLEMENTARY INFORMATION: Supplementary information is available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btab563
  3. Annu Rev Anal Chem (Palo Alto Calif). 2021 07 27. 14(1): 467-487
    Current Challenges and Recent Developments in Mass Spectrometry-Based Metabolomics.
    Stephanie L Collins, Imhoi Koo, Jeffrey M Peters, Philip B Smith, Andrew D Patterson.
      High-resolution mass spectrometry (MS) has advanced the study of metabolism in living systems by allowing many metabolites to be measured in a single experiment. Although improvements in mass detector sensitivity have facilitated the detection of greater numbers of analytes, compound identification strategies, feature reduction software, and data sharing have not kept up with the influx of MS data. Here, we discuss the ongoing challenges with MS-based metabolomics, including de novo metabolite identification from mass spectra, differentiation of metabolites from environmental contamination, chromatographic separation of isomers, and incomplete MS databases. Because of their popularity and sensitive detection of small molecules, this review focuses on the challenges of liquid chromatography-mass spectrometry-based methods. We then highlight important instrumentational, experimental, and computational tools that have been created to address these challenges and how they have enabled the advancement of metabolomics research.
    Keywords:  chromatography; isotope tracing; mass spectrometry; metabolomics; multi-stage mass spectrometry; retention indices
    DOI:  https://doi.org/10.1146/annurev-anchem-091620-015205
  4. J Proteome Res. 2021 Jul 30.
    Proteomics for Low Cell Numbers: How to Optimize the Sample Preparation Workflow for Mass Spectrometry Analysis.
    Sara Kassem, Kyra van der Pan, Anniek L de Jager, Brigitta A E Naber, Inge F de Laat, Alesha Louis, Jacques J M van Dongen, Cristina Teodosio, Paula Díez.
      Nowadays, massive genomics and transcriptomics data can be generated at the single-cell level. However, proteomics in this setting is still a big challenge. Despite the great improvements in sensitivity and performance of mass spectrometry instruments and the better knowledge on sample preparation processing, it is widely acknowledged that multistep proteomics workflows may lead to substantial sample loss, especially when working with paucicellular samples. Still, in clinical fields, frequently limited sample amounts are available for downstream analysis, thereby hampering comprehensive characterization at protein level. To aim at better protein and peptide recoveries, we compare existing and novel approaches in the multistep sample preparation protocols for mass spectrometry studies, from sample collection, cell lysis, protein quantification, and electrophoresis/staining to protein digestion, peptide recovery, and LC-MS/MS instruments. From this critical evaluation, we conclude that the recent innovations and technologies, together with high quality management of samples, make proteomics on paucicellular samples possible, which will have immediate impact for the proteomics community.
    Keywords:  mass spectrometry; paucicellular sample; sample preparation; single-cell proteomics
    DOI:  https://doi.org/10.1021/acs.jproteome.1c00321
  5. Anal Chem. 2021 Jul 27.
    Single Quadrupole Multiple Fragment Ion Monitoring Quantitative Mass Spectrometry.
    Jingchuan Xue, Rico J E Derks, Bill Webb, Elizabeth M Billings, Aries Aisporna, Martin Giera, Gary Siuzdak.
      Single quadrupole mass spectrometry (MS) with enhanced in-source multiple fragment ion monitoring was designed to perform high sensitivity quantitative mass analyses. Enhanced in-source fragmentation amplifies fragmentation from traditional soft electrospray ionization producing fragment ions that have been found to be identical to those generated in tandem MS. We have combined enhanced in-source fragmentation data with criteria established by the European Union Commission Directive 2002/657/EC for electron ionization single quadrupole quantitative analysis to perform quantitative analyses. These experiments were performed on multiple types of complex samples that included a mixture of 50 standards, as well as cell and plasma extracts. The dynamic range for these quantitative analyses was comparable to triple quadrupole multiple reaction monitoring (MRM) analyses at up to 5 orders of magnitude with the cell and plasma extracts showing similar matrix effects across both platforms. Amino acid and fatty acid measurements performed from certified NIST 1950 plasma with isotopically labeled standards demonstrated accuracy in the range of 91-110% for the amino acids, 76-129% for the fatty acids, and good precision (coefficient of variation <10%). To enhance specificity, a newly developed correlated ion monitoring algorithm was designed to facilitate these analyses. This algorithm autonomously processes, aligns, filters, and compiles multiple ions within one chromatogram enabling both precursor and in-source fragment ions to be correlated within a single chromatogram, also enabling the detection of coeluting species based on precursor and fragment ion ratios. Single quadrupole instrumentation can provide MRM level quantitative performance by monitoring/correlating precursor and fragment ions facilitating high sensitivity analysis on existing single quadrupole instrumentation that are generally inexpensive, easy to operate, and technically less complex.
    DOI:  https://doi.org/10.1021/acs.analchem.1c01246
  6. Clin Epigenetics. 2021 Jul 28. 13(1): 145
    Spatial epi-proteomics enabled by histone post-translational modification analysis from low-abundance clinical samples.
    Roberta Noberini, Evelyn Oliva Savoia, Stefania Brandini, Francesco Greco, Francesca Marra, Giovanni Bertalot, Giancarlo Pruneri, Liam A McDonnell, Tiziana Bonaldi.
       BACKGROUND: Increasing evidence linking epigenetic mechanisms and different diseases, including cancer, has prompted in the last 15 years the investigation of histone post-translational modifications (PTMs) in clinical samples. Methods allowing the isolation of histones from patient samples followed by the accurate and comprehensive quantification of their PTMs by mass spectrometry (MS) have been developed. However, the applicability of these methods is limited by the requirement for substantial amounts of material.
    RESULTS: To address this issue, in this study we streamlined the protein extraction procedure from low-amount clinical samples and tested and implemented different in-gel digestion strategies, obtaining a protocol that allows the MS-based analysis of the most common histone PTMs from laser microdissected tissue areas containing as low as 1000 cells, an amount approximately 500 times lower than what is required by available methods. We then applied this protocol to breast cancer patient laser microdissected tissues in two proof-of-concept experiments, identifying differences in histone marks in heterogeneous regions selected by either morphological evaluation or MALDI MS imaging.
    CONCLUSIONS: These results demonstrate that analyzing histone PTMs from very small tissue areas and detecting differences from adjacent tumor regions is technically feasible. Our method opens the way for spatial epi-proteomics, namely the investigation of epigenetic features in the context of tissue and tumor heterogeneity, which will be instrumental for the identification of novel epigenetic biomarkers and aberrant epigenetic mechanisms.
    Keywords:  Epigenetics; Histone post-translational modifications; Laser microdissection; MALDI-MSI ; Mass spectrometry; Proteomics
    DOI:  https://doi.org/10.1186/s13148-021-01120-7
  7. Curr Opin Chem Biol. 2021 Jul 23. pii: S1367-5931(21)00094-6. [Epub ahead of print]65 101-108
    Advances in chemical proteomic evaluation of lipid kinases-DAG kinases as a case study.
    Timothy B Ware, Ku-Lung Hsu.
      Advancements in chemical proteomics and mass spectrometry lipidomics are providing new opportunities to understand lipid kinase activity, specificity, and regulation on a global cellular scale. Here, we describe recent developments in chemical biology of lipid kinases with a focus on those members that phosphorylate diacylglycerols. We further discuss future implications of how these mass spectrometry-based approaches can be adapted for studies of additional lipid kinase members with the aim of bridging the gap between protein and lipid kinase-focused investigations.
    Keywords:  Activity-based protein profiling; Chemical proteomics; Diacylglycerol; Kinase; Kinome; Lipid; Lipidomics; Mass spectrometry; Phosphatidic acid; Phosphoinositide; Phosphorylation; Signaling; SuFEx; SuTEx; Sulfonyl fluorides; Sulfonyl triazoles
    DOI:  https://doi.org/10.1016/j.cbpa.2021.06.007
  8. Cardiovasc Res. 2021 Jul 27. 117(9): e106-e109
    NAD+ metabolism and cardiometabolic health: the human evidence.
    Mahmoud Abdellatif, Joseph A Baur.
      
    Keywords:  Ageing; Cardiovascular disease; Diabetes; NMN; NR; Niacin; Nicotinamide; Obesity
    DOI:  https://doi.org/10.1093/cvr/cvab212
  9. Mol Metab. 2021 Jul 22. pii: S2212-8778(21)00156-3. [Epub ahead of print] 101309
    Hepatic mTORC1 signaling activates ATF4 as part of its metabolic response to feeding and insulin.
    Vanessa Byles, Yann Cormerais, Krystle Kalafut, Victor Barrera, James E Hughes Hallett, Shannan Ho Sui, John M Asara, Christopher M Adams, Gerta Hoxhaj, Issam Ben-Sahra, Brendan D Manning.
       OBJECTIVE: The mechanistic target of rapamycin complex 1 (mTORC1) is dynamically regulated by fasting and feeding cycles in the liver to promote protein and lipid synthesis while suppressing autophagy. However, beyond these functions, the metabolic response of the liver to feeding and insulin signaling orchestrated by mTORC1 remains poorly defined. Here, we determine whether ATF4, a stress responsive transcription factor recently found to be independently regulated by mTORC1 signaling in proliferating cells, is responsive to hepatic mTORC1 signaling to alter hepatocyte metabolism.
    METHODS: ATF4 protein levels and expression of canonical gene targets were analyzed in the liver following fasting and physiological feeding in the presence or absence of the mTORC1 inhibitor rapamycin. Primary hepatocytes from wild-type or liver-specific Atf4 knockout (LAtf4KO) mice were used to characterize the effects of insulin-stimulated mTORC1-ATF4 function on hepatocyte gene expression and metabolism. Both unbiased steady-state metabolomics and stable-isotope tracing methods were employed to define mTORC1 and ATF4-dependent metabolic changes. RNA-sequencing was used to determine global changes in feeding-induced transcripts in the livers of wild-type versus LAtf4KO mice.
    RESULTS: We demonstrate that ATF4 and its metabolic gene targets are stimulated by mTORC1 signaling in the liver in response to feeding and in a hepatocyte-intrinsic manner by insulin. While we demonstrate that de novo purine and pyrimidine synthesis is stimulated by insulin through mTORC1 signaling in primary hepatocytes, this regulation was independent of ATF4. Metabolomics and metabolite tracing studies revealed that insulin-mTORC1-ATF4 signaling stimulates pathways of non-essential amino acid synthesis in primary hepatocytes, including those of alanine, aspartate, methionine, and cysteine, but not serine.
    CONCLUSION: The results demonstrate that ATF4 is a novel metabolic effector of mTORC1 in liver, extending the molecular consequences of feeding and insulin-induced mTORC1 signaling in this key metabolic tissue to the control of amino acid metabolism.
    Keywords:  ATF4; feeding; insulin; liver; mTORC1; methionine metabolism
    DOI:  https://doi.org/10.1016/j.molmet.2021.101309
  10. Curr Osteoporos Rep. 2021 Jul 28.
    Metabolism in the Tumour-Bone Microenvironment.
    Jessica Whitburn, Claire M Edwards.
       PURPOSE OF REVIEW: For solid tumours such as breast and prostate cancer, and haematological malignancies such as myeloma, bone represents a supportive home, where the cellular crosstalk is known to underlie both tumour growth and survival, and the development of the associated bone disease. The importance of metabolic reprogramming is becoming increasingly recognised, particularly within cancer biology, enabling tumours to adapt to changing environments and pressures. This review will discuss our current understanding of metabolic requirements and adaptations within the tumour-bone microenvironment.
    RECENT FINDINGS: The bone provides a unique metabolic microenvironment, home to highly energy-intensive processes such as bone resorption and bone formation, both of which are dysregulated in the presence of cancer. Approaches such as metabolomics demonstrate metabolic plasticity in patients with advanced disease. Metabolic crosstalk between tumour cells and surrounding stroma supports disease pathogenesis. There is increasing evidence for a key role for metabolic reprogramming within the tumour-bone microenvironment to drive disease progression. As such, understanding these metabolic adaptations should reveal new therapeutic targets and approaches.
    Keywords:  Bone; Cancer; Glycolysis; Metabolism; Metastasis; Microenvironment
    DOI:  https://doi.org/10.1007/s11914-021-00695-7
  11. Anal Chem. 2021 Jul 28.
    Multidimensional Separations of Intact Phase II Steroid Metabolites Utilizing LC-Ion Mobility-HRMS.
    Don E Davis, Katrina L Leaptrot, David C Koomen, Jody C May, Gustavo de A Cavalcanti, Monica C Padilha, Henrique M G Pereira, John A McLean.
      The detection and unambiguous identification of anabolic-androgenic steroid metabolites are essential in clinical, forensic, and antidoping analyses. Recently, sulfate phase II steroid metabolites have received increased attention in steroid metabolism and drug testing. In large part, this is because phase II steroid metabolites are excreted for an extended time, making them a potential long-term chemical marker of choice for tracking steroid misuse in sports. Comprehensive analytical methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been used to detect and identify glucuronide and sulfate steroids in human urine with high sensitivity and reliability. However, LC-MS/MS identification strategies can be hindered by the fact that phase II steroid metabolites generate nonselective ion fragments across the different metabolite markers, limiting the confidence in metabolite identifications that rely on exact mass measurement and MS/MS information. Additionally, liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is sometimes insufficient at fully resolving the analyte peaks from the sample matrix (commonly urine) chemical noise, further complicating accurate identification efforts. Therefore, we developed a liquid chromatography-ion mobility-high resolution mass spectrometry (LC-IM-HRMS) method to increase the peak capacity and utilize the IM-derived collision cross section (CCS) values as an additional molecular descriptor for increased selectivity and to improve identifications of intact steroid analyses at low concentrations.
    DOI:  https://doi.org/10.1021/acs.analchem.1c02163
  12. Mol Syst Biol. 2021 Jul;17(7): e10125
    Identification of covalent modifications regulating immune signaling complex composition and phenotype.
    Annika Frauenstein, Stefan Ebner, Fynn M Hansen, Ankit Sinha, Kshiti Phulphagar, Kirby Swatek, Daniel Hornburg, Matthias Mann, Felix Meissner.
      Cells signal through rearrangements of protein communities governed by covalent modifications and reversible interactions of distinct sets of proteins. A method that identifies those post-transcriptional modifications regulating signaling complex composition and functional phenotypes in one experimental setup would facilitate an efficient identification of novel molecular signaling checkpoints. Here, we devised modifications, interactions and phenotypes by affinity purification mass spectrometry (MIP-APMS), comprising the streamlined cloning and transduction of tagged proteins into functionalized reporter cells as well as affinity chromatography, followed by MS-based quantification. We report the time-resolved interplay of more than 50 previously undescribed modification and hundreds of protein-protein interactions of 19 immune protein complexes in monocytes. Validation of interdependencies between covalent, reversible, and functional protein complex regulations by knockout or site-specific mutation revealed ISGylation and phosphorylation of TRAF2 as well as ARHGEF18 interaction in Toll-like receptor 2 signaling. Moreover, we identify distinct mechanisms of action for small molecule inhibitors of p38 (MAPK14). Our method provides a fast and cost-effective pipeline for the molecular interrogation of protein communities in diverse biological systems and primary cells.
    Keywords:  mass spectrometry; posttranslational modifications; protein interactions; proteomics; signaling networks
    DOI:  https://doi.org/10.15252/msb.202010125
  13. Anal Chem. 2021 Jul 29.
    Precise Quantitation of PTEN by Immuno-MRM: A Tool To Resolve the Breast Cancer Biomarker Controversy.
    Sahar Ibrahim, Cathy Lan, Catherine Chabot, Georgia Mitsa, Marguerite Buchanan, Adriana Aguilar-Mahecha, Mounib Elchebly, Oliver Poetz, Alan Spatz, Mark Basik, Gerald Batist, René P Zahedi, Christoph H Borchers.
      The tumor suppressor PTEN is the main negative regulator of PI3K/AKT/mTOR signaling and is commonly found downregulated in breast cancer (BC). Conflicting data from conventional immunoassays such as immunohistochemistry (IHC) has sparked controversy about PTEN's role as a prognostic and predictive biomarker in BC, which can be largely attributed to the lack of specificity, sensitivity, and interlaboratory standardization. Here, we present a fully standardized, highly sensitive, robust microflow immuno-MRM (iMRM) assay that enables precise quantitation of PTEN concentrations in cells and fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissues, down to 0.1 fmol/10 μg of extracted protein, with high interday and intraday precision (CV 6.3%). PTEN protein levels in BC PDX samples that were determined by iMRM correlate well with semiquantitative IHC and WB data. iMRM, however, allowed the precise quantitation of PTEN-even in samples that were deemed to be PTEN negative by IHC or western blot (WB)-while requiring substantially less tumor tissue than WB. This is particularly relevant because the extent of PTEN downregulation in tumors has been shown to correlate with severity. Our standardized and robust workflow includes an 11 min microflow LC-MRM analysis on a triple-quadrupole MS and thus provides a much needed tool for the study of PTEN as a potential biomarker for BC.
    DOI:  https://doi.org/10.1021/acs.analchem.1c00975
  14. J Proteome Res. 2021 Jul 26.
    FIGS: Featured Ion-Guided Stoichiometry for Data-Independent Proteomics through Dynamic Deconvolution.
    Yan Fang, Qing-Run Li, Zhen-Hang Zhang, Jia-Rui Wu, Hao-Ran Zheng, Rong Zeng.
      Data-independent acquisition (DIA) has significant advantages for mass spectrometry (MS)-based peptide quantification, while mixed spectra remain challenging for precise stoichiometry. We here choose to analyze the library spectra in specific sets preferentially and locally. Accordingly, the featured ions are defined as the fragment ions uniquely assigned to corresponding precursors in a given spectrum set, which are generated by dynamic deconvolution of the mixed mass spectra. Then, we present featured ion-guided stoichiometry (FIGS), a universal method for accurate and robust peptide quantification for the DIA-MS data. We validate the high performance on the quantification sensitivity, accuracy, and efficiency of FIGS. Notably, our FIGS dramatically improves the quantification accuracy for the full dynamic range, especially for low-abundance peptides.
    Keywords:  data-independent acquisition; dynamic spectral deconvolution; mass spectrometry; peptide quantification
    DOI:  https://doi.org/10.1021/acs.jproteome.1c00438
  15. Methods Cell Biol. 2021 ;pii: S0091-679X(20)30195-3. [Epub ahead of print]165 163-176
    Monitoring autophagy-dependent ferroptosis.
    Jingbo Li, Rui Kang, Daolin Tang.
      Ferroptosis is an iron-dependent form of regulated cell death, driven by the accumulation of lipid peroxidation. Autophagy is a lysosome-dependent degradation process that can be used to remove and recover intracellular components, such as dysfunctional proteins and damaged organelles. By regulating iron storage and oxidative stress, excessive autophagy is involved in the induction and execution of ferroptosis. In particular, several types of selective autophagy (e.g., ferritinophagy, lipophagy, clockophagy, and chaperone-mediated autophagy) increase the susceptibility to ferroptotic cell death by degrading anti-ferroptotic regulators (e.g., ferritin, GPX4, ARNTL, and lipid droplets). These two integrated biological processes play a pathological role in the occurrence and development of human diseases, such as cancer, neurodegenerative disorders, ischemia and reperfusion injury. Therefore, it is important to develop reliable methods to evaluate the kinetics of autophagosome formation, iron accumulation, and lipid peroxidation. Here, we introduce some protocols (such as western blotting, lipid peroxidation assay kits and probes, and iron probes) to monitor the process of autophagy-dependent ferroptosis.
    Keywords:  Autophagy; Cell death; Ferroptosis; Lipid peroxidation; Metabolism
    DOI:  https://doi.org/10.1016/bs.mcb.2020.10.012
  16. Trends Mol Med. 2021 Jul 23. pii: S1471-4914(21)00181-7. [Epub ahead of print]
    Ferroptosis in the tumor microenvironment: perspectives for immunotherapy.
    Huanji Xu, Di Ye, Meiling Ren, Hongying Zhang, Feng Bi.
      Targeting ferroptosis, which provokes lipid peroxidation in cancer cells, presents potentially new avenues for anticancer therapy. Recent studies have begun to explore how immune cells in the tumor microenvironment (TME) respond and adapt to lethal lipid peroxides (LPOs). A better understanding of this process in the TME is likely to uncover another side of ferroptosis in cancer immunity and promote the development of ferroptosis-targeted therapy. This Opinion article overviews the main metabolic processes in ferroptosis, summarizes the emerging roles of ferroptosis not only in immune cells in the TME but also in the crosstalk between tumor cells and immune cells, and presents a perspective on the targeting of ferroptosis in cancer immunotherapy.
    Keywords:  ferroptosis; immune cells; immunotherapy; tumor microenvironment
    DOI:  https://doi.org/10.1016/j.molmed.2021.06.014
  17. Nat Commun. 2021 Jul 30. 12(1): 4626
    PRMT1-dependent regulation of RNA metabolism and DNA damage response sustains pancreatic ductal adenocarcinoma.
    Virginia Giuliani, Meredith A Miller, Chiu-Yi Liu, Stella R Hartono, Caleb A Class, Christopher A Bristow, Erika Suzuki, Lionel A Sanz, Guang Gao, Jason P Gay, Ningping Feng, Johnathon L Rose, Hideo Tomihara, Joseph R Daniele, Michael D Peoples, Jennifer P Bardenhagen, Mary K Geck Do, Qing E Chang, Bhavatarini Vangamudi, Christopher Vellano, Haoqiang Ying, Angela K Deem, Kim-Anh Do, Giannicola Genovese, Joseph R Marszalek, Jeffrey J Kovacs, Michael Kim, Jason B Fleming, Ernesto Guccione, Andrea Viale, Anirban Maitra, M Emilia Di Francesco, Timothy A Yap, Philip Jones, Giulio Draetta, Alessandro Carugo, Frederic Chedin, Timothy P Heffernan.
      Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has remained clinically challenging to manage. Here we employ an RNAi-based in vivo functional genomics platform to determine epigenetic vulnerabilities across a panel of patient-derived PDAC models. Through this, we identify protein arginine methyltransferase 1 (PRMT1) as a critical dependency required for PDAC maintenance. Genetic and pharmacological studies validate the role of PRMT1 in maintaining PDAC growth. Mechanistically, using proteomic and transcriptomic analyses, we demonstrate that global inhibition of asymmetric arginine methylation impairs RNA metabolism, which includes RNA splicing, alternative polyadenylation, and transcription termination. This triggers a robust downregulation of multiple pathways involved in the DNA damage response, thereby promoting genomic instability and inhibiting tumor growth. Taken together, our data support PRMT1 as a compelling target in PDAC and informs a mechanism-based translational strategy for future therapeutic development.Statement of significancePDAC is a highly lethal cancer with limited therapeutic options. This study identified and characterized PRMT1-dependent regulation of RNA metabolism and coordination of key cellular processes required for PDAC tumor growth, defining a mechanism-based translational hypothesis for PRMT1 inhibitors.
    DOI:  https://doi.org/10.1038/s41467-021-24798-y
  18. J Proteome Res. 2021 Jul 30.
    PrSM-Level Side-by-Side Comparison of Online LC-MS Methods with Intact Histone H3 and H4 Proteoforms.
    Kin-Wing Lui, Sai-Ming Ngai.
      The heterogeneity of histone H3 proteoforms makes histone H3 top-down analysis challenging. To enhance the detection coverage of the proteoforms, performing liquid chromatography (LC) front-end to mass spectrometry (MS) detection is recommended. Here, using optimized electron-transfer/high-energy collision dissociation (EThcD) parameters, we have conducted a proteoform-spectrum match (PrSM)-level side-by-side comparison of reversed-phase LC-MS (RPLC-MS), "dual-gradient" weak cation-exchange/hydrophilic interaction LC-MS (dual-gradient WCX/HILIC-MS), and "organic-rich" WCX/HILIC-MS on the top-down analyses of H3.1, H3.2, and H4 proteins extracted from a HeLa cell culture. While both dual-gradient WCX/HILIC and organic-rich WCX/HILIC could resolve intact H3 and H4 proteoforms by the number of acetylations, the organic-rich method could enhance the separations of different trimethyl/acetyl near-isobaric H3 proteoforms. In comparison with RPLC-MS, both of the WCX/HILIC-MS methods enhanced the qualities of the H3 PrSMs and remarkably improved the range, reproducibility, and confidence in the identifications of H3 proteoforms.
    Keywords:  ESI-compatible WCX/HILIC; RPLC; histones; online LC-MS; post-translation modifications; top-down proteomics
    DOI:  https://doi.org/10.1021/acs.jproteome.1c00308
  19. Nat Commun. 2021 07 27. 12(1): 4558
    STING suppresses bone cancer pain via immune and neuronal modulation.
    Kaiyuan Wang, Christopher R Donnelly, Changyu Jiang, Yihan Liao, Xin Luo, Xueshu Tao, Sangsu Bang, Aidan McGinnis, Michael Lee, Matthew J Hilton, Ru-Rong Ji.
      Patients with advanced stage cancers frequently suffer from severe pain as a result of bone metastasis and bone destruction, for which there is no efficacious treatment. Here, using multiple mouse models of bone cancer, we report that agonists of the immune regulator STING (stimulator of interferon genes) confer remarkable protection against cancer pain, bone destruction, and local tumor burden. Repeated systemic administration of STING agonists robustly attenuates bone cancer-induced pain and improves locomotor function. Interestingly, STING agonists produce acute pain relief through direct neuronal modulation. Additionally, STING agonists protect against local bone destruction and reduce local tumor burden through modulation of osteoclast and immune cell function in the tumor microenvironment, providing long-term cancer pain relief. Finally, these in vivo effects are dependent on host-intrinsic STING and IFN-I signaling. Overall, STING activation provides unique advantages in controlling bone cancer pain through distinct and synergistic actions on nociceptors, immune cells, and osteoclasts.
    DOI:  https://doi.org/10.1038/s41467-021-24867-2
  20. Anal Chem. 2021 Jul 28.
    Acoustic Ejection Mass Spectrometry for High-Throughput Analysis.
    Hui Zhang, Chang Liu, Wenyi Hua, Lucien P Ghislain, Jianhua Liu, Lisa Aschenbrenner, Stephen Noell, Kenneth J Dirico, Lorraine F Lanyon, Claire M Steppan, Mike West, Don W Arnold, Thomas R Covey, Sammy S Datwani, Matthew D Troutman.
      We describe a mass spectrometry (MS) analytical platform resulting from the novel integration of acoustic droplet ejection (ADE) technology, an open-port interface (OPI), and electrospray ionization (ESI)-MS that creates a transformative system enabling high-speed sampling and label-free analysis. The ADE technology delivers nanoliter droplets in a touchless manner with high speed, precision, and accuracy. Subsequent sample dilution within the OPI, in concert with the capabilities of modern ESI-MS, eliminates the laborious sample preparation and method development required in current approaches. This platform is applied to a variety of experiments, including high-throughput (HT) pharmacology screening, label-free in situ enzyme kinetics, in vitro absorption, distribution, metabolism, elimination, pharmacokinetic and biomarker analysis, and HT parallel medicinal chemistry.
    DOI:  https://doi.org/10.1021/acs.analchem.1c01137
  21. Sci Total Environ. 2021 Jul 17. pii: S0048-9697(21)04084-5. [Epub ahead of print]798 149012
    Non-targeted LC-MS and CE-MS for biomarker discovery in bioreactors: Influence of separation, mass spectrometry and data processing tools.
    Oliver Höcker, Dirk Flottmann, Torsten C Schmidt, Christian Neusüß.
      Liquid separation coupled to mass spectrometry is often used for non-targeted analyses in various fields, such as metabolomics. However, the combination of non-standardized methods, various mass spectrometers (MS) and processing tools for data evaluation affect biomarker discovery potentially. Here, we present a comprehensive study of these factors based on non-targeted liquid chromatography coupled to time-of-flight (TOF) and Orbitrap MS and capillary zone electrophoresis to Orbitrap analyses of the same bioreactor samples, describing the correlation of its gas yield with changing feature signal intensity. The three datasets were processed with MZmine 2 and XCMS online and subsequential Partial Least Square Regression (PLSR) with Variable Importance in Projection (VIP) ranking for feature prioritization. The six feature tables were compared to evaluate their overlap of shared features and the influence of the processing software and MS instrument on the VIP values and fold changes. The overlaps, defined as a fraction of one feature table found in the comparative table, were from 27% to 57% for the comparison of MZmine and XCMS and from 15% to 50% between Orbitrap and TOF data sets, respectively. Considering the most relevant features only (VIP >1.5), the overlaps were increased significantly in all cases from 26% to 95%. For the same data set, both VIP values and fold changes were well correlated, however, varied significantly between TOF and Orbitrap. CE-MS showed higher total feature numbers compared to LC-MS, most likely due to its more appropriate selectivity, different sample preparation, and/or the sensitive nano-ESI interface. Since only less than 10% of MS/MS data overlapped, CE-MS provided complementary information to LC-MS. Overall, our systematic study proves the benefits of using different separation techniques and processing tools but also indicates a significant influence of mass spectrometry on comprehensive biomarker discovery.
    Keywords:  Biogas plant; Bioreactor; Capillary electrophoresis; Liquid chromatography; Mass spectrometry; Non-targeted analysis
    DOI:  https://doi.org/10.1016/j.scitotenv.2021.149012
  22. J Mol Med (Berl). 2021 Jul 26.
    Lipid metabolism, inflammation, and foam cell formation in health and metabolic disorders: targeting mTORC1.
    So Yeong Cheon, KyoungJoo Cho.
      Metabolic homeostasis is important for maintaining a healthy lifespan. Lipid metabolism is particularly necessary for the maintenance of metabolic energy sources and their storage, and the structure and function of cell membranes, as well as for the regulation of nutrition through lipogenesis, lipolysis, and lipophagy. Dysfunctional lipid metabolism leads to the development of metabolic disorders, such as atherosclerosis, diabetes mellitus, and non-alcoholic fatty liver disease (NAFLD). Furthermore, dyslipidaemia causes inflammatory responses and foam cell formation. Mechanistic target of rapamycin (mTOR) signalling is a key regulator of diverse cellular processes, including cell metabolism and cell fate. mTOR complex 1 (mTORC1) is involved in lipid metabolism and immune responses in the body. Therefore, the mTORC1 signalling pathway has been suggested as a potential therapeutic target for the treatment of metabolic disorders. In this review, we focus on the roles of mTORC1 in lipid metabolism and inflammation, and present current evidence on its involvement in the development and progression of metabolic disorders.
    Keywords:  Foam cells; Inflammation; Lipid metabolism; Mechanistic target of rapamycin (mTOR); Metabolic disorders
    DOI:  https://doi.org/10.1007/s00109-021-02117-8
  23. Nat Protoc. 2021 Jul 28.
    Statistical analysis in metabolic phenotyping.
    Benjamin J Blaise, Gonçalo D S Correia, Gordon A Haggart, Izabella Surowiec, Caroline Sands, Matthew R Lewis, Jake T M Pearce, Johan Trygg, Jeremy K Nicholson, Elaine Holmes, Timothy M D Ebbels.
      Metabolic phenotyping is an important tool in translational biomedical research. The advanced analytical technologies commonly used for phenotyping, including mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy, generate complex data requiring tailored statistical analysis methods. Detailed protocols have been published for data acquisition by liquid NMR, solid-state NMR, ultra-performance liquid chromatography (LC-)MS and gas chromatography (GC-)MS on biofluids or tissues and their preprocessing. Here we propose an efficient protocol (guidelines and software) for statistical analysis of metabolic data generated by these methods. Code for all steps is provided, and no prior coding skill is necessary. We offer efficient solutions for the different steps required within the complete phenotyping data analytics workflow: scaling, normalization, outlier detection, multivariate analysis to explore and model study-related effects, selection of candidate biomarkers, validation, multiple testing correction and performance evaluation of statistical models. We also provide a statistical power calculation algorithm and safeguards to ensure robust and meaningful experimental designs that deliver reliable results. We exemplify the protocol with a two-group classification study and data from an epidemiological cohort; however, the protocol can be easily modified to cover a wider range of experimental designs or incorporate different modeling approaches. This protocol describes a minimal set of analyses needed to rigorously investigate typical datasets encountered in metabolic phenotyping.
    DOI:  https://doi.org/10.1038/s41596-021-00579-1
  24. Trends Endocrinol Metab. 2021 Jul 22. pii: S1043-2760(21)00132-6. [Epub ahead of print]
    Metabolic hallmarks of liver regeneration.
    Roya Solhi, Majid Lotfinia, Roberto Gramignoli, Mustapha Najimi, Massoud Vosough.
      Despite the crucial role of cell metabolism in biological processes, particularly cell division, metabolic aspects of liver regeneration are not well defined. Better understanding of the metabolic activity governing division of liver cells will provide powerful insights into mechanisms of physiological and pathological liver regeneration. Recent studies have provided evidence that metabolic response to liver failure might be a proximal signal to initiate cell proliferation in liver regeneration. In this review, we highlight how lipids, carbohydrates, and proteins dynamically change and orchestrate liver regeneration. In addition, we discuss translational studies in which metabolic intervention has been used to treat chronic liver diseases (CLDs).
    Keywords:  AMPK; hepatocyte proliferation; hypoglycemia; lipolysis; liver regeneration
    DOI:  https://doi.org/10.1016/j.tem.2021.06.002
  25. Anal Chim Acta. 2021 Aug 29. pii: S0003-2670(21)00459-1. [Epub ahead of print]1175 338633
    Review: Multiplexed profiling of biomarkers in extracellular vesicles for cancer diagnosis and therapy monitoring.
    Sisi Zhou, Yao Yang, Yafeng Wu, Songqin Liu.
      Extracellular vesicles (EVs) are nanoscale vesicles secreted by normal and pathological cells. The types and levels of surface proteins and internal nucleic acids in EVs are closely related to their original cells, tumor occurrence, and development. Thus, the sensitive and accurate detection of EV biomarkers is a reliable approach for noninvasive disease diagnosis and treatment response monitoring. However, the purification and molecular profiling of these EVs are technically challenging. Much effort has been dedicated to developing new methods for the detection of multiple EV biomarkers. In this review, we summarize the recent progress in EV protein and nucleic acid biomarker analysis. Additionally, we systematically discuss the advantages of multiplexed EV biomarker detection for accurate cancer diagnosis, therapy monitoring, and cancer screening. This article aims to present an overview of all kinds of analytical technologies for assessing EVs and their applications in clinical settings.
    Keywords:  Biosensors; Cancer diagnosis; Extracellular vesicles; Multiplex analysis; Therapy monitoring
    DOI:  https://doi.org/10.1016/j.aca.2021.338633
  26. J Proteome Res. 2021 Jul 28.
    Comparison of Unit Resolution Versus High-Resolution Accurate Mass for Parallel Reaction Monitoring.
    Lilian R Heil, Philip M Remes, Michael J MacCoss.
      Parallel reaction monitoring (PRM) is an increasingly popular alternative to selected reaction monitoring (SRM) for targeted proteomics. PRM's strengths over SRM are that it monitors all product ions in a single spectrum, thus eliminating the need to select interference-free product ions prior to data acquisition, and that it is most frequently performed on high-resolution instruments, such as quadrupole-orbitrap and quadrupole-time-of-flight instruments. Here, we show that the primary advantage of PRM is the ability to monitor all transitions in parallel and that high-resolution data are not necessary to obtain high-quality quantitative data. We run the same scheduled PRM assay, measuring 432 peptides from 126 plasma proteins, multiple times on an Orbitrap Eclipse Tribrid mass spectrometer, alternating separate liquid chromatography-tandem mass spectrometry runs between the high-resolution Orbitrap and the unit resolution linear ion trap for PRM. We find that both mass analyzers have similar technical precision and that the linear ion trap's superior sensitivity gives it better lower limits of quantitation for over 62% of peptides in the assay.
    Keywords:  parallel reaction monitoring; plasma; targeted proteomics
    DOI:  https://doi.org/10.1021/acs.jproteome.1c00377
  27. Proteomics. 2021 Jul 27. e2000319
    DDASSQ: an open-source, multiple peptide sequencing strategy for label free quantification based on an OpenMS pipeline in the KNIME analytics platform.
    Monika Svecla, Giulia Garrone, Fiorenza Faré, Giacomo Aletti, Giuseppe Danilo Norata, Giangiacomo Beretta.
      In this study we investigated the performance of a computational pipeline for protein identification and label free quantification of LC-MS/MS data sets from experimental animal tissue samples, as well as the impact of its specific peptide search combinatorial approach. The full pipeline workflow was composed of peptide search engine adapters based on different identification algorithms, in the frame of the open-source OpenMS software running within the KNIME analytics platform. Two different in silico tryptic digestion, database-search assisted approaches (X!Tandem and MS-GF+), de novo peptide sequencing based on Novor and consensus library search (SpectraST), were tested for the processing of LC-MS/MS raw datafiles obtained from proteomic LC-MS experiments done on proteolytic extracts from mouse ex-vivo liver samples. The results from proteomic LFQ were compared to those based on the application of the two software tools MaxQuant® and Proteome DiscovererTM for protein inference and label-free data analysis in shotgun proteomics. Data are available via ProteomeXchange with identifier PXD025097. This article is protected by copyright. All rights reserved.
    Keywords:  LFQ; proteomics; search engine; workflow
    DOI:  https://doi.org/10.1002/pmic.202000319
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