bims-mascan 2024-04-14 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2024–04–14
fiveteen papers selected by
Giovanny Rodriguez Blanco, University of Edinburgh

Inserting Pre-analytical Chromatographic Priming Runs Significantly Improves Targeted Pathway Proteomics with Sample Multiplexing.
Determination of fatty acid uptake and desaturase activity in mammalian cells by NMR-based stable isotope tracing.
A CRISPRi/a screening platform to study cellular nutrient transport in diverse microenvironments.
Bayesian functional analysis for untargeted metabolomics data with matching uncertainty and small sample sizes.
Metabolic Signaling in Cancer Metastasis.
MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation.
Purine salvage promotes treatment resistance in H3K27M-mutant diffuse midline glioma.
A guide to ferroptosis in cancer.
Functionalizing tandem mass tags for streamlining click-based quantitative chemoproteomics.
A lipid atlas of human and mouse immune cells provides insights into ferroptosis susceptibility.
HIF1α-dependent uncoupling of glycolysis suppresses tumor cell proliferation.
Removal of isobaric interference using pseudo-multiple reaction monitoring and energy-resolved mass spectrometry for the isotope dilution quantification of a tryptic peptide.
Glutaminase deficiency in rod photoreceptors disrupts nonessential amino acid levels to activate the integrated stress response and induce rapid degeneration.
Targeting Fatty Acid Desaturase I Inhibits Renal Cancer Growth Via ATF3-mediated ER Stress Response.
Systematic evaluation of benzoylation for liquid chromatography-mass spectrometry analysis of different analyte classes.

J Proteome Res. 2024 Apr 09.

Inserting Pre-analytical Chromatographic Priming Runs Significantly Improves Targeted Pathway Proteomics with Sample Multiplexing.

Steven R Shuken, Qing Yu, Steven P Gygi.

  GoDig, a platform for targeted pathway proteomics without the need for manual assay scheduling or synthetic standards, is a powerful, flexible, and easy-to-use method that uses tandem mass tags to increase sample throughput up to 18-fold relative to label-free methods. Though the protein-level success rates of GoDig are high, the peptide-level success rates are more limited, hampering assays of harder-to-quantify proteins and site-specific phenomena. To guide the optimization of GoDig assays as well as improvements to the GoDig platform, we created GoDigViewer, a new stand-alone software that provides detailed visualizations of GoDig runs. GoDigViewer guided the implementation of "priming runs," an acquisition mode with significantly higher success rates. In this mode, two or more chromatographic priming runs are automatically performed to improve the accuracy and precision of target elution orders, followed by analytical runs which quantify targets. Using priming runs, success rates exceeded 97% for a list of 400 peptide targets and 95% for a list of 200 targets that are usually not quantified using untargeted mass spectrometry. We used priming runs to establish a quantitative assay of 125 macroautophagy proteins that had a >95% success rate and revealed differences in macroautophagy expression profiles across four human cell lines.

Keywords:  chromatography; instrument API; intelligent data acquisition; isobaric labeling; macroautophagy; pathway proteomics; quantitative mass spectrometry; real-time search; tandem mass tags; targeted proteomics

DOI:  https://doi.org/10.1021/acs.jproteome.4c00096
Anal Chim Acta. 2024 May 15. pii: S0003-2670(24)00312-X. [Epub ahead of print]1303 342511

Determination of fatty acid uptake and desaturase activity in mammalian cells by NMR-based stable isotope tracing.

Penghui Lin, James Sledziona, Kubra B Akkaya-Colak, Maria M Mihaylova, Andrew N Lane.

   BACKGROUND: Mammalian cells both import exogenous fatty acids and synthesize them de novo. Palmitate, the end product of fatty acid synthase (FASN) is a substrate for stearoyl-CoA desaturases (Δ-9 desaturases) that introduce a single double bond into fatty acyl-CoA substrates such as palmitoyl-CoA and stearoyl-CoA. This process is particularly upregulated in lipogenic tissues and cancer cells. Tracer methodology is needed to determine uptake versus de novo synthesis of lipids and subsequent chain elongation and desaturation. Here we describe an NMR method to determine the uptake of 13C-palmitate from the medium into HCT116 human colorectal cancer cells, and the subsequent desaturation and incorporation into complex lipids.
RESULTS: Exogenous 13C16-palmitate was absorbed from the medium by HCT116 cells and incorporated primarily into complex glycerol lipids. Desaturase activity was determined from the quantification of double bonds in acyl chains, which was greatly reduced by ablation of the major desaturase SCD1.
SIGNIFICANCE: The NMR approach requires minimal sample preparation, is non-destructive, and provides direct information about the level of saturation and incorporation of fatty acids into complex lipids.

Keywords:  Desaturase activity; Fatty acid uptake; NMR

DOI:  https://doi.org/10.1016/j.aca.2024.342511
Nat Cell Biol. 2024 Apr 11.

A CRISPRi/a screening platform to study cellular nutrient transport in diverse microenvironments.

Christopher Chidley, Alicia M Darnell, Benjamin L Gaudio, Evan C Lien, Anna M Barbeau, Matthew G Vander Heiden, Peter K Sorger.

Blocking the import of nutrients essential for cancer cell proliferation represents a therapeutic opportunity, but it is unclear which transporters to target. Here we report a CRISPR interference/activation screening platform to systematically interrogate the contribution of nutrient transporters to support cancer cell proliferation in environments ranging from standard culture media to tumours. We applied this platform to identify the transporters of amino acids in leukaemia cells and found that amino acid transport involves high bidirectional flux dependent on the microenvironment composition. While investigating the role of transporters in cystine starved cells, we uncovered a role for serotonin uptake in preventing ferroptosis. Finally, we identified transporters essential for cell proliferation in subcutaneous tumours and found that levels of glucose and amino acids can restrain proliferation in that environment. This study establishes a framework for systematically identifying critical cellular nutrient transporters, characterizing their function and exploring how the tumour microenvironment impacts cancer metabolism.

DOI: https://doi.org/10.1038/s41556-024-01402-1
Brief Bioinform. 2024 Mar 27. pii: bbae141. [Epub ahead of print]25(3):

Bayesian functional analysis for untargeted metabolomics data with matching uncertainty and small sample sizes.

Guoxuan Ma, Jian Kang, Tianwei Yu.

  Untargeted metabolomics based on liquid chromatography-mass spectrometry technology is quickly gaining widespread application, given its ability to depict the global metabolic pattern in biological samples. However, the data are noisy and plagued by the lack of clear identity of data features measured from samples. Multiple potential matchings exist between data features and known metabolites, while the truth can only be one-to-one matches. Some existing methods attempt to reduce the matching uncertainty, but are far from being able to remove the uncertainty for most features. The existence of the uncertainty causes major difficulty in downstream functional analysis. To address these issues, we develop a novel approach for Bayesian Analysis of Untargeted Metabolomics data (BAUM) to integrate previously separate tasks into a single framework, including matching uncertainty inference, metabolite selection and functional analysis. By incorporating the knowledge graph between variables and using relatively simple assumptions, BAUM can analyze datasets with small sample sizes. By allowing different confidence levels of feature-metabolite matching, the method is applicable to datasets in which feature identities are partially known. Simulation studies demonstrate that, compared with other existing methods, BAUM achieves better accuracy in selecting important metabolites that tend to be functionally consistent and assigning confidence scores to feature-metabolite matches. We analyze a COVID-19 metabolomics dataset and a mouse brain metabolomics dataset using BAUM. Even with a very small sample size of 16 mice per group, BAUM is robust and stable. It finds pathways that conform to existing knowledge, as well as novel pathways that are biologically plausible.

Keywords:  Bayesian latent factor model; matching uncertainty; metabolite network analysis

DOI:  https://doi.org/10.1093/bib/bbae141
Cancer Discov. 2024 Mar 29. OF1-OF19

Metabolic Signaling in Cancer Metastasis.

Sarah Krieg, Sara Isabel Fernandes, Constantinos Kolliopoulos, Ming Liu, Sarah-Maria Fendt.

Metastases, which are the leading cause of death in patients with cancer, have metabolic vulnerabilities. Alterations in metabolism fuel the energy and biosynthetic needs of metastases but are also needed to activate cell state switches in cells leading to invasion, migration, colonization, and outgrowth in distant organs. Specifically, metabolites can activate protein kinases as well as receptors and they are crucial substrates for posttranslational modifications on histone and nonhistone proteins. Moreover, metabolic enzymes can have moonlighting functions by acting catalytically, mainly as protein kinases, or noncatalytically through protein-protein interactions. Here, we summarize the current knowledge on metabolic signaling in cancer metastasis.
SIGNIFICANCE: Effective drugs for the prevention and treatment of metastases will have an immediate impact on patient survival. To overcome the current lack of such drugs, a better understanding of the molecular processes that are an Achilles heel in metastasizing cancer cells is needed. One emerging opportunity is the metabolic changes cancer cells need to undergo to successfully metastasize and grow in distant organs. Mechanistically, these metabolic changes not only fulfill energy and biomass demands, which are often in common between cancer and normal but fast proliferating cells, but also metabolic signaling which enables the cell state changes that are particularly important for the metastasizing cancer cells.

DOI: https://doi.org/10.1158/2159-8290.CD-24-0174
Nucleic Acids Res. 2024 Apr 08. pii: gkae253. [Epub ahead of print]

MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation.

Zhiqiang Pang, Yao Lu, Guangyan Zhou, Fiona Hui, Lei Xu, Charles Viau, Aliya F Spigelman, Patrick E MacDonald, David S Wishart, Shuzhao Li, Jianguo Xia.

We introduce MetaboAnalyst version 6.0 as a unified platform for processing, analyzing, and interpreting data from targeted as well as untargeted metabolomics studies using liquid chromatography - mass spectrometry (LC-MS). The two main objectives in developing version 6.0 are to support tandem MS (MS2) data processing and annotation, as well as to support the analysis of data from exposomics studies and related experiments. Key features of MetaboAnalyst 6.0 include: (i) a significantly enhanced Spectra Processing module with support for MS2 data and the asari algorithm; (ii) a MS2 Peak Annotation module based on comprehensive MS2 reference databases with fragment-level annotation; (iii) a new Statistical Analysis module dedicated for handling complex study design with multiple factors or phenotypic descriptors; (iv) a Causal Analysis module for estimating metabolite - phenotype causal relations based on two-sample Mendelian randomization, and (v) a Dose-Response Analysis module for benchmark dose calculations. In addition, we have also improved MetaboAnalyst's visualization functions, updated its compound database and metabolite sets, and significantly expanded its pathway analysis support to around 130 species. MetaboAnalyst 6.0 is freely available at https://www.metaboanalyst.ca.

DOI: https://doi.org/10.1093/nar/gkae253
Cancer Metab. 2024 Apr 09. 12(1): 11

Purine salvage promotes treatment resistance in H3K27M-mutant diffuse midline glioma.

Erik R Peterson, Peter Sajjakulnukit, Andrew J Scott, Caleb Heaslip, Anthony Andren, Kari Wilder-Romans, Weihua Zhou, Sravya Palavalasa, Navyateja Korimerla, Angelica Lin, Alexandra O'Brien, Ayesha Kothari, Zitong Zhao, Li Zhang, Meredith A Morgan, Sriram Venneti, Carl Koschmann, Nada Jabado, Costas A Lyssiotis, Maria G Castro, Daniel R Wahl.

   BACKGROUND: Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are a fatal form of brain cancer. These tumors often carry a driver mutation on histone H3 converting lysine 27 to methionine (H3K27M). DMG-H3K27M are characterized by altered metabolism and resistance to standard of care radiation (RT) but how the H3K27M mediates the metabolic response to radiation and consequent treatment resistance is uncertain.
METHODS: We performed metabolomics on irradiated and untreated H3K27M isogenic DMG cell lines and observed an H3K27M-specific enrichment for purine synthesis pathways. We profiled the expression of purine synthesis enzymes in publicly available patient data and our models, quantified purine synthesis using stable isotope tracing, and characterized the in vitro and in vivo response to de novo and salvage purine synthesis inhibition in combination with RT.
RESULTS: DMG-H3K27M cells activate purine metabolism in an H3K27M-specific fashion. In the absence of genotoxic treatment, H3K27M-expressing cells have higher relative activity of de novo synthesis and apparent lower activity of purine salvage demonstrated via stable isotope tracing of key metabolites in purine synthesis and by lower expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the rate-limiting enzyme of purine salvage into IMP and GMP. Inhibition of de novo guanylate synthesis radiosensitized DMG-H3K27M cells in vitro and in vivo. Irradiated H3K27M cells upregulated HGPRT expression and hypoxanthine-derived guanylate salvage but maintained high levels of guanine-derived salvage. Exogenous guanine supplementation decreased radiosensitization in cells treated with combination RT and de novo purine synthesis inhibition. Silencing HGPRT combined with RT markedly suppressed DMG-H3K27M tumor growth in vivo.
CONCLUSIONS: Our results indicate that DMG-H3K27M cells rely on highly active purine synthesis, both from the de novo and salvage synthesis pathways. However, highly active salvage of free purine bases into mature guanylates can bypass inhibition of the de novo synthetic pathway. We conclude that inhibiting purine salvage may be a promising strategy to overcome treatment resistance in DMG-H3K27M tumors.

Keywords:  Diffuse midline glioma; H3K27M; Purine metabolism; Radiation therapy resistance

DOI:  https://doi.org/10.1186/s40170-024-00341-7
Mol Oncol. 2024 Apr 08.

A guide to ferroptosis in cancer.

Fatma Isil Yapici, Christina M Bebber, Silvia von Karstedt.

  Ferroptosis is a newly identified iron-dependent type of regulated cell death that can also be regarded as death caused by the specific collapse of the lipid antioxidant defence machinery. Ferroptosis has gained increasing attention as a potential therapeutic strategy for therapy-resistant cancer types. However, many ferroptosis-inducing small molecules do not reach the pharmacokinetic requirements for their effective clinical use yet. Nevertheless, their clinical optimization is under development. In this review, we summarize the current understanding of molecular pathways regulating ferroptosis, how cells protect themselves from the induction of ferroptotic cell death, and how a better understanding of cancer cell metabolism can represent vulnerabilities for ferroptosis-based therapies. Lastly, we discuss the context-dependent effect of ferroptosis on various cell types within the tumor microenvironment and address controversies on how tissue ferroptosis might impact systemic cancer immunity in a paracrine manner.

Keywords:  cancer biology; cancer metabolism; cell death; ferroptosis; inflammation; iron

DOI:  https://doi.org/10.1002/1878-0261.13649
Commun Chem. 2024 Apr 10. 7(1): 80

Functionalizing tandem mass tags for streamlining click-based quantitative chemoproteomics.

Nikolas R Burton, Keriann M Backus.

Mapping the ligandability or potential druggability of all proteins in the human proteome is a central goal of mass spectrometry-based covalent chemoproteomics. Achieving this ambitious objective requires high throughput and high coverage sample preparation and liquid chromatography-tandem mass spectrometry analysis for hundreds to thousands of reactive compounds and chemical probes. Conducting chemoproteomic screens at this scale benefits from technical innovations that achieve increased sample throughput. Here we realize this vision by establishing the silane-based cleavable linkers for isotopically-labeled proteomics-tandem mass tag (sCIP-TMT) proteomic platform, which is distinguished by early sample pooling that increases sample preparation throughput. sCIP-TMT pairs a custom click-compatible sCIP capture reagent that is readily functionalized in high yield with commercially available TMT reagents. Synthesis and benchmarking of a 10-plex set of sCIP-TMT reveal a substantial decrease in sample preparation time together with high coverage and high accuracy quantification. By screening a focused set of four cysteine-reactive electrophiles, we demonstrate the utility of sCIP-TMT for chemoproteomic target hunting, identifying 789 total liganded cysteines. Distinguished by its compatibility with established enrichment and quantification protocols, we expect sCIP-TMT will readily translate to a wide range of covalent chemoproteomic applications.

DOI: https://doi.org/10.1038/s42004-024-01162-x
Nat Cell Biol. 2024 Apr 08.

A lipid atlas of human and mouse immune cells provides insights into ferroptosis susceptibility.

Pooranee K Morgan, Gerard Pernes, Kevin Huynh, Corey Giles, Sudip Paul, Adam Alexander T Smith, Natalie A Mellett, Amy Liang, Tilly van Buuren-Milne, Camilla Bertuzzo Veiga, Thomas J C Collins, Yangsong Xu, Man K S Lee, T Michael De Silva, Peter J Meikle, Graeme I Lancaster, Andrew J Murphy.

The cellular lipidome comprises thousands of unique lipid species. Here, using mass spectrometry-based targeted lipidomics, we characterize the lipid landscape of human and mouse immune cells ( www.cellularlipidatlas.com ). Using this resource, we show that immune cells have unique lipidomic signatures and that processes such as activation, maturation and development impact immune cell lipid composition. To demonstrate the potential of this resource to provide insights into immune cell biology, we determine how a cell-specific lipid trait-differences in the abundance of polyunsaturated fatty acid-containing glycerophospholipids (PUFA-PLs)-influences immune cell biology. First, we show that differences in PUFA-PL content underpin the differential susceptibility of immune cells to ferroptosis. Second, we show that low PUFA-PL content promotes resistance to ferroptosis in activated neutrophils. In summary, we show that the lipid landscape is a defining feature of immune cell identity and that cell-specific lipid phenotypes underpin aspects of immune cell physiology.

DOI: https://doi.org/10.1038/s41556-024-01377-z
Cell Rep. 2024 Apr 11. pii: S2211-1247(24)00431-5. [Epub ahead of print]43(4): 114103

HIF1α-dependent uncoupling of glycolysis suppresses tumor cell proliferation.

Andrés A Urrutia, Claudia Mesa-Ciller, Andrea Guajardo-Grence, H Furkan Alkan, Inés Soro-Arnáiz, Anke Vandekeere, Ana Margarida Ferreira Campos, Sebastian Igelmann, Lucía Fernández-Arroyo, Gianmarco Rinaldi, Doriane Lorendeau, Katrien De Bock, Sarah-Maria Fendt, Julián Aragonés.

  Hypoxia-inducible factor-1α (HIF1α) attenuates mitochondrial activity while promoting glycolysis. However, lower glycolysis is compromised in human clear cell renal cell carcinomas, in which HIF1α acts as a tumor suppressor by inhibiting cell-autonomous proliferation. Here, we find that, unexpectedly, HIF1α suppresses lower glycolysis after the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step, leading to reduced lactate secretion in different tumor cell types when cells encounter a limited pyruvate supply such as that typically found in the tumor microenvironment in vivo. This is because HIF1α-dependent attenuation of mitochondrial oxygen consumption increases the NADH/NAD+ ratio that suppresses the activity of the NADH-sensitive GAPDH glycolytic enzyme. This is manifested when pyruvate supply is limited, since pyruvate acts as an electron acceptor that prevents the increment of the NADH/NAD+ ratio. Furthermore, this anti-glycolytic function provides a molecular basis to explain how HIF1α can suppress tumor cell proliferation by increasing the NADH/NAD+ ratio.

Keywords:  CP: Cancer; CP: Metabolism; HIF1α; NADH; glycolysis; hypoxia; mitochondria; oxygen; renal cell carcinoma; tumor

DOI:  https://doi.org/10.1016/j.celrep.2024.114103
J Mass Spectrom. 2024 May;59(5): e5025

Removal of isobaric interference using pseudo-multiple reaction monitoring and energy-resolved mass spectrometry for the isotope dilution quantification of a tryptic peptide.

Alicia Maroto, Dany Jeanne Dit Fouque, Rémy Lartia, Antony Memboeuf.

  Energy-resolved mass spectrometry (ERMS) and an isotopically labelled internal standard were successfully combined to accurately quantify a tryptic peptide despite the presence of an isobaric interference. For this purpose, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) experiments were conducted into an ion trap instrument using an unconventional 8 m/z broadband isolation window, which encompassed both the tryptic peptide and its internal standard. Interference removal was assessed by determining an excitation voltage that was high enough to maintain a constant value for the analyte/internal standard peaks intensity ratio, thus ensuring accurate quantification even in the presence of isobaric contamination. Pseudo-multiple reaction monitoring (MRM) was employed above this excitation voltage to quantify the trypic peptide. The internal standard calibration model showed no lack of fit and exhibited a linear dynamic range from 0.5 μM up to 2.5 μM. The detection limit was 0.08 μM. The accuracy of the method was evaluated by quantifying the tryptic peptide of three reference samples intentionally contaminated with the isobaric interference. All the reference samples were accurately quantified with ∼1% deviation despite the isobaric contamination. Furthermore, we have demonstrated that this methodology can also be applied to quantify the isobaric peptide by standard additions down to 0.2 μM. Finally, liquid chromatography ERMS (LC ERMS) experiments yielded similar results, suggesting the potential of the proposed methodology for analysing complex samples.

Keywords:  energy‐resolved mass spectrometry; gas phase collisional purification; isobaric interference; peptide quantification; pseudo‐MRM; survival yield

DOI:  https://doi.org/10.1002/jms.5025
bioRxiv. 2024 Mar 27. pii: 2024.03.26.582525. [Epub ahead of print]

Glutaminase deficiency in rod photoreceptors disrupts nonessential amino acid levels to activate the integrated stress response and induce rapid degeneration.

Moloy T Goswami, Eric Weh, Shubha Subramanya, Katherine M Weh, Hima Bindu Durumutla, Heather Hager, Nicholas Miller, Sraboni Chaudhury, Anthony Andren, Peter Sajjakulnukit, Cagri G Besirli, Costas A Lyssiotis, Thomas J Wubben.

The bioenergetic demand of photoreceptors rivals that of cancer cells, and numerous metabolic similarities exist between these cells. Glutamine (Gln) anaplerosis via the tricarboxylic acid (TCA) cycle provides biosynthetic intermediates and is a hallmark of cancer metabolism. In this process, Gln is first converted to glutamate via glutaminase (GLS), which is a crucial pathway in many cancer cells. To date, no study has been undertaken to examine the role of Gln metabolism in vivo in photoreceptors. Here, mice lacking GLS in rod photoreceptors were generated. Animals lacking GLS experienced rapid photoreceptor degeneration with concomitant functional loss. Gln has multiple roles in metabolism including redox balance, biosynthesis of nucleotides and amino acids, and supplementing the TCA cycle. Few alterations were noted in redox balance. Unlabeled targeted metabolomics demonstrated few changes in glycolytic and TCA cycle intermediates, which corresponded with a lack of significant changes in mitochondrial function. GLS deficiency in rod photoreceptors did decrease the fractional labelling of TCA cycle intermediates when provided uniformly labeled 13 C-Gln in vivo . However, supplementation with alpha-ketoglutarate provided only marginal rescue of photoreceptor degeneration. Nonessential amino acids, glutamate and aspartate, were decreased in the retina of mice lacking GLS in rod photoreceptors. In accordance with this amino acid deprivation, the integrated stress response (ISR) was found to be activated with decreased global protein synthesis. Importantly, supplementation with asparagine delayed photoreceptor degeneration to a greater degree than alpha-ketoglutarate. These data show that GLS-mediated Gln catabolism is essential for rod photoreceptor amino acid biosynthesis, function, and survival.
Significance Statement: Glucose has been central in the study of photoreceptor cell metabolism. Recently, it was shown that fuel sources besides glucose can meet the metabolic needs of photoreceptors. Glutamine (Gln) is the most abundant circulating amino acid and has many biosynthetic and bioenergetic roles in cells. Glutaminolysis is the process by which Gln is metabolized into tricarboxylic acid cycle intermediates to provide biosynthetic precursors. Here, Gln is first converted to glutamate via the enzyme glutaminase (GLS). This research demonstrates that deletion of GLS in rod photoreceptors alters retinal metabolism, activates the integrated stress response (ISR), and results in rapid photoreceptor degeneration. As such, Gln is a critical fuel source that supports photoreceptor cell biomass, redox balance, and survival.

DOI: https://doi.org/10.1101/2024.03.26.582525
bioRxiv. 2024 Mar 27. pii: 2024.03.23.586426. [Epub ahead of print]

Targeting Fatty Acid Desaturase I Inhibits Renal Cancer Growth Via ATF3-mediated ER Stress Response.

Gioia Heravi, Zhenjie Liu, Mackenzie Herroon, Alexis Wilson, Yang-Yi Fan, Yang Jiang, Nivisa Vakeesan, Li Tao, Zheyun Peng, Kezhong Zhang, Jing Li, Robert S Chapkin, Izabela Podgorski, Wanqing Liu.

Monounsaturated fatty acids (MUFAs) play a pivotal role in maintaining endoplasmic reticulum (ER) homeostasis, an emerging hallmark of cancer. However, the role of polyunsaturated fatty acid (PUFAs) desaturation in persistent ER stress driven by oncogenic abnormalities remains elusive. Fatty Acid Desaturase 1 (FADS1) is a rate-limiting enzyme controlling the bioproduction of long-chain PUFAs. Our previous research has demonstrated the significant role of FADS1 in cancer survival, especially in kidney cancers. We explored the underlying mechanism in this study. We found that pharmacological inhibition or knockdown of the expression of FADS1 effectively inhibits renal cancer cell proliferation and induces cell cycle arrest. The stable knockdown of FADS1 also significantly inhibits tumor formation in vivo . Mechanistically, we show that while FADS1 inhibition induces ER stress, its expression is also augmented by ER-stress inducers. Notably, FADS1-inhibition sensitized cellular response to ER stress inducers, providing evidence of FADS1's role in modulating the ER stress response in cancer cells. We show that, while FADS1 inhibition-induced ER stress leads to activation of ATF3, ATF3-knockdown rescues the FADS1 inhibition-induced ER stress and cell growth suppression. In addition, FADS1 inhibition results in the impaired biosynthesis of nucleotides and decreases the level of UPD-N-Acetylglucosamine, a critical mediator of the unfolded protein response. Our findings suggest that PUFA desaturation is crucial for rescuing cancer cells from persistent ER stress, supporting FADS1 as a new therapeutic target.

DOI: https://doi.org/10.1101/2024.03.23.586426
J Chromatogr A. 2024 Apr 04. pii: S0021-9673(24)00246-2. [Epub ahead of print]1722 464872

Systematic evaluation of benzoylation for liquid chromatography-mass spectrometry analysis of different analyte classes.

Ashley E Lenhart, Peter-Philip M Booth, Kaley M Simcox, Brianna A Ramos, Robert T Kennedy.

  LC-MS is an indispensable tool for small molecule analysis in many fields; however, many small molecules require chemical derivatization to improve retention on commonly used reversed-phase columns and increase ionization. Benzoyl chloride (BzCl) derivatization is commonly used for derivatization of primary and secondary amines and phenolic alcohols, though evidence exists that with proper reaction conditions (i.e., specific bases), other hydroxyl groups may be derivatized too. Previous studies have examined BzCl concentration, reaction times, and reaction temperatures for derivatization of amines and phenols for LC-MS analysis; however, use of different bases, base concentration, and extending to conditions to hydroxyl groups for LC-MS analysis has not been well-studied. To address this understudied area and identify reaction conditions for both amino and hydroxyl groups, we performed a systematic study of reaction conditions on multiple classes of potential targets. For selected derivatization methods, detection limits and performance in a variety of biological matrices were assessed. Results highlight the importance of tailoring derivatization methods for a given application as they varied by molecule and/or molecule class. Compared to the standard BzCl method commonly used, alternative methods were identified to better derivatize challenging analytes (glucosamine, choline, cortisol, uridine, cytidine) with detection limits reaching 1100, 9, 38, 170, and 67 nM compared to undetectable, 170, 86, 1000, and 86 nM respectively. Sub-nanomolar detection limits were achieved for norepinephrine with alternative derivatization approaches. Improved derivatization methods for several classes and molecules including nucleosides, steroids, and molecules containing hydroxyl groups were also identified.

Keywords:  Benzoyl chloride; Chemical derivatization; Liquid chromatography-mass spectrometry; Small molecules

DOI:  https://doi.org/10.1016/j.chroma.2024.464872