bims-mascan 2023-06-18 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2023–06–18
27 papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz

Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids.
IDSL.CSA: Composite Spectra Analysis for Chemical Annotation of Untargeted Metabolomics Datasets.
Characterization of Oxidized Glycerophosphoethanolamines via Radical-Directed Dissociation Tandem Mass Spectrometry and the Paternò-Büchi Derivatization.
Comparison of Data-Dependent Acquisition, Data-Independent Acquisition, and Parallel Reaction Monitoring in Trapped Ion Mobility Spectrometry-Time-of-Flight Tandem Mass Spectrometry-Based Lipidomics.
Generalized precursor prediction boosts identification rates and accuracy in mass spectrometry based proteomics.
Advantages of using biologically generated 13C-labelled multiple internal standards for stable isotope-assisted LC-MS-based lipidomics.
Comprehensive Insight into Colorectal Cancer Metabolites and Lipids for Human Serum: A Proof-of-Concept Study.
Evaluation of a 'plug and play' nanoflow liquid chromatography system for MS-based proteomic characterization of clinical FFPE specimens.
DiffN Selection of Tandem Mass Spectrometry Precursors.
Mass Spectrometry-Based Vitreous Proteomics: Validated Methods and Analysis Pipeline.
mzapy: An Open-Source Python Library Enabling Efficient Extraction and Processing of Ion Mobility Spectrometry-Mass Spectrometry Data in the MZA File Format.
Host cell protein quantification workflow using optimized standards combined with data-independent acquisition mass spectrometry.
The remodeling roles of lipid metabolism in colorectal cancer cells and immune microenvironment.
Statistical methods and resources for biomarker discovery using metabolomics.
Editorial: Rising stars in cancer metabolism 2022.
Comprehensive serum lipidomic analyses reveal potential biomarkers for malignant breast cancer: A case-control study.
Formation and Tandem Mass Spectrometry of Doubly Charged Lipid-Metal Ion Complexes.
Coenzyme biosynthesis in response to precursor availability reveals incorporation of β-alanine from pantothenate in prototrophic bacteria.
UniProt and mass spectrometry-based proteomics - a 2-way working relationship.
Multidimensional separation methods can improve separation performance for extremely complicated proteomics samples and provide a powerful tool for comprehensive proteome profiling of low-microgram samples.
Optimized Suspension Trapping Method for Phosphoproteomics Sample Preparation.
Phosphoinositide and redox dysregulation by the anticancer methylthioadenosine phosphorylase transition state inhibitor.
Automated Quantitative Analysis of Shape Features in Human Epithelial Monolayers and Spheroids Generated from Colorectal Cancer Cells.
Ketogenic diet promotes tumor ferroptosis but induces relative corticosterone deficiency that accelerates cachexia.
DiLeu Isobaric Labeling Coupled with Limited Proteolysis Mass Spectrometry for High-Throughput Profiling of Protein Structural Changes in Alzheimer's Disease.
One-STAGE Tip Method for TMT-Based Proteomic Analysis of a Minimal Amount of Cells.
Amino acid metabolism in tumor: New shine in the fog?

Nat Commun. 2023 Jun 16. 14(1): 3599

Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids.

Ana Martínez-Val, Kyle Fort, Claire Koenig, Leander Van der Hoeven, Giulia Franciosa, Thomas Moehring, Yasushi Ishihama, Yu-Ju Chen, Alexander Makarov, Yue Xuan, Jesper V Olsen.

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging, especially when analyzing tiny sample amounts. To address this, we present a hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem mass spectrometry (MSx) scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmark hybrid-DIA against state-of-the-art targeted MS methods (i.e., SureQuant) using EGF-stimulated HeLa cells and find the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiles the global phosphoproteome. To demonstrate the robustness, sensitivity, and biomedical potential of hybrid-DIA, we profile chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluate the phospho-signaling difference of cancer cells in 2D vs 3D culture.

DOI: https://doi.org/10.1038/s41467-023-39347-y
Anal Chem. 2023 Jun 13.

IDSL.CSA: Composite Spectra Analysis for Chemical Annotation of Untargeted Metabolomics Datasets.

Sadjad Fakouri Baygi, Yashwant Kumar, Dinesh Kumar Barupal.

Poor chemical annotation of high-resolution mass spectrometry data limits applications of untargeted metabolomics datasets. Our new software, the Integrated Data Science Laboratory for Metabolomics and Exposomics─Composite Spectra Analysis (IDSL.CSA) R package, generates composite mass spectra libraries from MS1-only data, enabling the chemical annotation of high-resolution mass spectrometry coupled with liquid chromatography peaks regardless of the availability of MS2 fragmentation spectra. We demonstrate comparable annotation rates for commonly detected endogenous metabolites in human blood samples using IDSL.CSA libraries versus MS/MS libraries in validation tests. IDSL.CSA can create and search composite spectra libraries from any untargeted metabolomics dataset generated using high-resolution mass spectrometry coupled to liquid or gas chromatography instruments. The cross-applicability of these libraries across independent studies may provide access to new biological insights that may be missed due to the lack of MS2 fragmentation data. The IDSL.CSA package is available in the R-CRAN repository at https://cran.r-project.org/package=IDSL.CSA. Detailed documentation and tutorials are provided at https://github.com/idslme/IDSL.CSA.

DOI: https://doi.org/10.1021/acs.analchem.3c00376
Anal Chem. 2023 Jun 12.

Characterization of Oxidized Glycerophosphoethanolamines via Radical-Directed Dissociation Tandem Mass Spectrometry and the Paternò-Büchi Derivatization.

Qiaohong Lin, Ruijun Jian, Shengzhuo Wang, Yu Xia.

Oxidized glycerophosphoethanolamines (oxPEs) represent a subclass of bioactive lipids that have intricate roles in various physiological and pathological events. Conventional mass spectrometric methods cannot provide unambiguous information to locate the OH group and the sites of unsaturation. Herein, we report a combined strategy for in-depth structural characterization of oxPEs, including radical-directed dissociation tandem mass spectrometry (RDD-MS/MS) for localizing the OH group and the Paternò-Büchi derivatization coupled with tandem mass spectrometry for pinpointing carbon-carbon double-bond locations. The RDD-MS/MS method has been integrated on a reversed-phase liquid chromatography-mass spectrometry workflow. It enables the profiling of 24 distinct oxPE molecules with unequivocal assignment of the OH sites at nM sensitivity in bovine liver lipid extract treated by soybean 15-lipoxygenase. These findings showcase that the developed method has a good potential in analyzing biological systems where oxPEs may play important roles.

DOI: https://doi.org/10.1021/acs.analchem.3c00792
Anal Chem. 2023 Jun 12.

Comparison of Data-Dependent Acquisition, Data-Independent Acquisition, and Parallel Reaction Monitoring in Trapped Ion Mobility Spectrometry-Time-of-Flight Tandem Mass Spectrometry-Based Lipidomics.

E Rudt, M Feldhaus, C G Margraf, S Schlehuber, A Schubert, S Heuckeroth, U Karst, V Jeck, S W Meyer, A Korf, H Hayen.

The parallel accumulation-serial fragmentation (PASEF) approach based on trapped ion mobility spectrometry (TIMS) enables mobility-resolved fragmentation and a higher number of fragments in the same time period compared to conventional MS/MS experiments. Furthermore, the ion mobility dimension offers novel approaches for fragmentation. Using parallel reaction monitoring (prm), the ion mobility dimension allows a more accurate selection of precursor windows, while using data-independent aquisition (dia) spectral quality is improved through ion-mobility filtering. Owing to favorable implementation in proteomics, the transferability of these PASEF modes to lipidomics is of great interest, especially as a result of the high complexity of analytes with similar fragments. However, these novel PASEF modes have not yet been thoroughly evaluated for lipidomics applications. Therefore, data-dependent acquisition (dda)-, dia-, and prm-PASEF were compared using hydrophilic interaction liquid chromatography (HILIC) for phospholipid class separation in human plasma samples. Results show that all three PASEF modes are generally suitable for usage in lipidomics. Although dia-PASEF achieves a high sensitivity in generating MS/MS spectra, the fragment-to-precursor assignment for lipids with both, similar retention time as well as ion mobility, was difficult in HILIC-MS/MS. Therefore, dda-PASEF is the method of choice to investigate unknown samples. However, the best data quality was achieved by prm-PASEF, owing to the focus on fragmentation of specified targets. The high selectivity and sensitivity in generating MS/MS spectra of prm-PASEF could be a potential alternative for targeted lipidomics, e.g., in clinical applications.

DOI: https://doi.org/10.1021/acs.analchem.3c00440
Commun Biol. 2023 Jun 10. 6(1): 628

Generalized precursor prediction boosts identification rates and accuracy in mass spectrometry based proteomics.

Aaron M Scott, Christofer Karlsson, Tirthankar Mohanty, Erik Hartman, Suvi T Vaara, Adam Linder, Johan Malmström, Lars Malmström.

Data independent acquisition mass spectrometry (DIA-MS) has recently emerged as an important method for the identification of blood-based biomarkers. However, the large search space required to identify novel biomarkers from the plasma proteome can introduce a high rate of false positives that compromise the accuracy of false discovery rates (FDR) using existing validation methods. We developed a generalized precursor scoring (GPS) method trained on 2.75 million precursors that can confidently control FDR while increasing the number of identified proteins in DIA-MS independent of the search space. We demonstrate how GPS can generalize to new data, increase protein identification rates, and increase the overall quantitative accuracy. Finally, we apply GPS to the identification of blood-based biomarkers and identify a panel of proteins that are highly accurate in discriminating between subphenotypes of septic acute kidney injury from undepleted plasma to showcase the utility of GPS in discovery DIA-MS proteomics.

DOI: https://doi.org/10.1038/s42003-023-04977-x
Anal Methods. 2023 Jun 13.

Advantages of using biologically generated 13C-labelled multiple internal standards for stable isotope-assisted LC-MS-based lipidomics.

Malak A Jaber, Bruna de Falco, Salah Abdelrazig, Catharine A Ortori, David A Barrett, Dong-Hyun Kim.

In comprehensive lipidomics studies, accurate quantification is essential but biological and/or clinical relevance is often hindered due to unwanted variations such as lipid degradation during sample preparation, matrix effects and non-linear responses of analytical instruments. In addition, the wide chemical diversity of lipids can complicate the accurate identification of individual lipids. These analytical limitations can potentially be corrected efficiently by the use of lipid-specific isotopically labelled internal standards (IS) but currently such IS mixtures have limited coverage of the mammalian lipidome. In this study, an in vivo13C labelling strategy was employed to explore four species (Escherichia coli, Arthrospira platensis, Saccharomyces cerevisiae and Pichia pastoris) as a source of 13C-labelled internal standards (13C-ISs) for more accurate and quantitative liquid chromatography (LC)-mass spectrometry (MS)-based lipidomics. Results showed that extracts from 13C-labelled P. pastoris and S. cerevisiae contain the highest percentage of uniformly labelled lipids (both 83% compared to 67% and 69% in A. platensis and E. coli, respectively) and 13C-labelled P. pastoris extract was identified as the optimum source of 13C-ISs for comprehensive data normalisation to correct unwanted variations during sample preparation and LC-MS analysis. Overall, use of a biologically generated 13C-IS lipid mixture of 357 identified lipid ions resulted in significant reduction in the lipid CV% of normalisation compared with other normalisation methods using total ion counts or a commercially available deuterated internal standard mixture. This improved normalisation using 13C-IS was confirmed in a typical lipidomics analysis using a large number of samples (>100+) and long analysis time (>70 h). This study highlights the benefit of an in vivo labelling strategy for reducing technical and analytical variations introduced during sample preparation and analysis in lipidomics studies.

DOI: https://doi.org/10.1039/d3ay00460k
Int J Mol Sci. 2023 Jun 01. pii: 9614. [Epub ahead of print]24(11):

Comprehensive Insight into Colorectal Cancer Metabolites and Lipids for Human Serum: A Proof-of-Concept Study.

Kinjal Bhatt, Titziana Orlando, Marie-Alice Meuwis, Edouard Louis, Pierre-Hugues Stefanuto, Jean-François Focant.

  Colorectal cancer (CRC) ranks as the third most frequently diagnosed cancer and the second leading cause of cancer-related deaths. The current endoscopic-based or stool-based diagnostic techniques are either highly invasive or lack sufficient sensitivity. Thus, there is a need for less invasive and more sensitive screening approaches. We, therefore, conducted a study on 64 human serum samples representing three different groups (adenocarcinoma, adenoma, and control) using cutting-edge GC×GC-LR/HR-TOFMS (comprehensive two-dimensional gas chromatography coupled with low/high-resolution time-of-flight mass spectrometry). We analyzed samples with two different specifically tailored sample preparation approaches for lipidomics (fatty acids) (25 μL serum) and metabolomics (50 μL serum). In-depth chemometric screening with supervised and unsupervised approaches and metabolic pathway analysis were applied to both datasets. A lipidomics study revealed that specific PUFA (ω-3) molecules are inversely associated with increased odds of CRC, while some PUFA (ω-6) analytes show a positive correlation. The metabolomics approach revealed downregulation of amino acids (alanine, glutamate, methionine, threonine, tyrosine, and valine) and myo-inositol in CRC, while 3-hydroxybutyrate levels were increased. This unique study provides comprehensive insight into molecular-level changes associated with CRC and allows for a comparison of the efficiency of two different analytical approaches for CRC screening using same serum samples and single instrumentation.

Keywords:  GC×GC–TOFMS; blood; chemometrics; colorectal cancer; comprehensive gas chromatography; lipidomics; mass spectrometry; metabolomics; sample preparation; separation science; serum

DOI:  https://doi.org/10.3390/ijms24119614
Expert Rev Proteomics. 2023 Jun 13. 1-6

Evaluation of a 'plug and play' nanoflow liquid chromatography system for MS-based proteomic characterization of clinical FFPE specimens.

Georgia Mitsa, Vincent R Richard, Yasamin Majedi, Josiane Lafleur, Adriana Aguilar-Mahecha, Mark Basik, Christoph H Borchers.

   INTRODUCTION: Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens has gained interest in the last 5 years due to technological advances and improved sample collection, as well as biobanking for clinical trials. The real-world implementation of clinical proteomics to these specimens, however, is hampered by tedious sample preparation steps and long instrument acquisition times.
AREAS COVERED: To advance the translation of quantitative proteomics into the clinic, we are comparing the performance of the leading commercial nanoflow liquid chromatography (nLC) system (based on literature reviews), the Easy-nLC 1200 (Thermo Fisher Scientific, Waltham, MA, U.S.A.), to the Evosep One HPLC (Evosep Biosystems, Odense, Denmark). We measured FFPE-tissue digests from 21 biological replicates with a similar gradient on both of the LC systems while keeping the on-column amount (1 µg total protein) and the single-shot data-dependent acquisition-based MS/MS method constant.
EXPERT OPINION: Overall, the Evosep One facilitates robust and sensitive high-throughput sample acquisition, making it suitable for clinical MS. We found the Evosep One to be a useful platform for positioning mass spectrometry-based proteomics in the clinical setting. The clinical application of nLC/MS will inform clinical decision-making in oncology and other diseases.

Keywords:  Cancer research; clinical mass spectrometry; clinical proteomics; formalin-fixed paraffin-embedded (FFPE); label-free quantitation; nanoflow LC

DOI:  https://doi.org/10.1080/14789450.2023.2219844
Anal Chem. 2023 Jun 13.

DiffN Selection of Tandem Mass Spectrometry Precursors.

Tyler S Larson, Cameron D Worthington, Matthew D Verber, James E Keating, Matthew R Lockett, Gary L Glish.

Current data-dependent acquisition (DDA) approaches select precursor ions for tandem mass spectrometry (MS/MS) characterization based on their absolute intensity, known as a TopN approach. Low-abundance species may not be identified as biomarkers in a TopN approach. Herein, a new DDA approach is proposed, DiffN, which uses the relative differential intensity of ions between two samples to selectively target species undergoing the largest fold changes for MS/MS. Using a dual nano-electrospray (nESI) ionization source which allows samples contained in separate capillaries to be analyzed in parallel, the DiffN approach was developed and validated with well-defined lipid extracts. A dual nESI source and DiffN DDA approach was applied to quantify the differences in lipid abundance between two colorectal cancer cell lines. The SW480 and SW620 lines represent a matched pair from the same patient: the SW480 cells from a primary tumor and the SW620 cells from a metastatic lesion. A comparison of TopN and DiffN DDA approaches on these cancer cell samples highlights the ability of DiffN to increase the likelihood of biomarker discovery and the decreased probability of TopN to efficiently select lipid species that undergo large fold changes. The ability of the DiffN approach to efficiently select precursor ions of interest makes it a strong candidate for lipidomic analyses. This DiffN DDA approach may also apply to other molecule classes (e.g., other metabolites or proteins) that are amenable to shotgun analyses.

DOI: https://doi.org/10.1021/acs.analchem.3c01085
Methods Mol Biol. 2023 ;2678 157-167

Mass Spectrometry-Based Vitreous Proteomics: Validated Methods and Analysis Pipeline.

Sarah Weber, Nick Carruthers, Chris Gates, Yuanjun Zhao, Jeffrey Sundstrom.

  Retinal diseases like diabetic retinopathy and age-related macular degeneration affect millions of individuals worldwide and often lead to vision loss. Vitreous fluid abuts the retina, is accessible for sampling, and contains many proteins related to retinal disease. Therefore, analysis of vitreous is an important tool for studying retinal disease. Because it is rich in proteins and extracellular vesicles, mass spectrometry-based proteomics is an excellent method for vitreous analysis. Here, we discuss important variables to consider when performing vitreous proteomics via mass spectrometry.

Keywords:  Extracellular vesicles; Mass spectrometry; Proteomics; Retinal disease; Vitreous

DOI:  https://doi.org/10.1007/978-1-0716-3255-0_11
Anal Chem. 2023 Jun 12.

mzapy: An Open-Source Python Library Enabling Efficient Extraction and Processing of Ion Mobility Spectrometry-Mass Spectrometry Data in the MZA File Format.

Dylan Ross, Aivett Bilbao, Joon-Yong Lee, Xueyun Zheng.

Analysis of ion mobility spectrometry (IMS) data has been challenging and limited the full utility of these measurements. Unlike liquid chromatography-mass spectrometry, where a plethora of tools with well-established algorithms exist, the incorporation of the additional IMS dimension requires upgrading existing computational pipelines and developing new algorithms to fully exploit the advantages of the technology. We have recently reported MZA, a new and simple mass spectrometry data structure based on the broadly supported HDF5 format and created to facilitate software development. While this format is inherently supportive of application development, the availability of core libraries in popular programming languages with standard mass spectrometry utilities will facilitate fast software development and broader adoption of the format. To this end, we present a Python package, mzapy, for efficient extraction and processing of mass spectrometry data in the MZA format, especially for complex data containing ion mobility spectrometry dimension. In addition to raw data extraction, mzapy contains supporting utilities enabling tasks including calibration, signal processing, peak finding, and generating plots. Being implemented in pure Python and having minimal and largely standardized dependencies makes mzapy uniquely suited to application development in the multiomics domain. The mzapy package is free and open-source, includes comprehensive documentation, and is structured to support future extension to meet the evolving needs of the MS community. The software source code is freely available at https://github.com/PNNL-m-q/mzapy.

DOI: https://doi.org/10.1021/acs.analchem.3c01653
J Pharm Anal. 2023 May;13(5): 494-502

Host cell protein quantification workflow using optimized standards combined with data-independent acquisition mass spectrometry.

Steve Hessmann, Cyrille Chery, Anne-Sophie Sikora, Annick Gervais, Christine Carapito.

  Monitoring of host cell proteins (HCPs) during the manufacturing of monoclonal antibodies (mAb) has become a critical requirement to provide effective and safe drug products. Enzyme-linked immunosorbent assays are still the gold standard methods for the quantification of protein impurities. However, this technique has several limitations and does, among others, not enable the precise identification of proteins. In this context, mass spectrometry (MS) became an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs. However, in order to be routinely implemented in biopharmaceutical companies, liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification. Here, we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard, the HCP Profiler solution, with a spectral library-based data-independent acquisition (DIA) method and strict data validation criteria. The performances of the HCP Profiler solution were compared to more conventional standard protein spikes and the DIA approach was benchmarked against a classical data-dependent acquisition on a series of samples produced at various stages of the manufacturing process. While we also explored spectral library-free DIA interpretation, the spectral library-based approach still showed highest accuracy and reproducibility (coefficients of variation < 10%) with a sensitivity down to the sub-ng/mg mAb level. Thus, this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.

Keywords:  Absolute quantification standards; Data-independent acquisition; Host cell proteins

DOI:  https://doi.org/10.1016/j.jpha.2023.03.009
Oncol Res. 2022 ;30(5): 231-242

The remodeling roles of lipid metabolism in colorectal cancer cells and immune microenvironment.

Jiateng Zhong, Jingyu Guo, Xinyu Zhang, Shuang Feng, Wenyu DI, Yanling Wang, Huifang Zhu.

  Lipid is a key component of plasma membrane, which plays an important role in the regulation of various cell biological behaviors, including cell proliferation, growth, differentiation and intracellular signal transduction. Studies have shown that abnormal lipid metabolism is involved in many malignant processes, including colorectal cancer (CRC). Lipid metabolism in CRC cells can be regulated not only by intracellular signals, but also by various components in the tumor microenvironment, including various cells, cytokines, DNA, RNA, and nutrients including lipids. In contrast, abnormal lipid metabolism provides energy and nutrition support for abnormal malignant growth and distal metastasis of CRC cells. In this review, we highlight the remodeling roles of lipid metabolism crosstalk between the CRC cells and the components of tumor microenvironment.

Keywords:  Colorectal cancer; Lipid metabolism; Tumor microenvironment (TME)

DOI:  https://doi.org/10.32604/or.2022.027900
BMC Bioinformatics. 2023 Jun 15. 24(1): 250

Statistical methods and resources for biomarker discovery using metabolomics.

Najeha R Anwardeen, Ilhame Diboun, Younes Mokrab, Asma A Althani, Mohamed A Elrayess.

  Metabolomics is a dynamic tool for elucidating biochemical changes in human health and disease. Metabolic profiles provide a close insight into physiological states and are highly volatile to genetic and environmental perturbations. Variation in metabolic profiles can inform mechanisms of pathology, providing potential biomarkers for diagnosis and assessment of the risk of contracting a disease. With the advancement of high-throughput technologies, large-scale metabolomics data sources have become abundant. As such, careful statistical analysis of intricate metabolomics data is essential for deriving relevant and robust results that can be deployed in real-life clinical settings. Multiple tools have been developed for both data analysis and interpretations. In this review, we survey statistical approaches and corresponding statistical tools that are available for discovery of biomarkers using metabolomics.

Keywords:  Analytical workflow; Metabolomics; Metabolomics tools; Multivariate; Statistical methods; Univariate

DOI:  https://doi.org/10.1186/s12859-023-05383-0
Front Oncol. 2023 ;13 1223630

Editorial: Rising stars in cancer metabolism 2022.

Jose Luis Izquierdo-Garcia, Domenica Scumaci.



Keywords:  glutamine metabolism; metabolic reprogramming; metabolic symbiosis; metabolism and chemoresistance; metabolism and redox homeostasis; personalized medicine

DOI:  https://doi.org/10.3389/fonc.2023.1223630
Cancer Biomark. 2023 May 24.

Comprehensive serum lipidomic analyses reveal potential biomarkers for malignant breast cancer: A case-control study.

Bin Cao, Siyu Yang, Lailai Yan, Nan Li.

   BACKGROUND: Breast cancer is the most worldwide commonly found malignancy among women. The evidence for lipidomic studies of breast cancer in the Chinese population is relatively limited.
OBJECTIVE: Our current study aimed to identify peripheral lipids capable of distinguishing adults with and without malignant breast cancer in a Chinese population and to explore the potential lipid metabolism pathways implicated in breast cancer.
METHODS: Lipidomics was performed with an Ultimate 3000 UHPLC system coupled with a Q-Exactive HF MS platform by using the serum of 71 female patients with malignant breast cancer and 92 age-matched (± 2 years) healthy women. The data were uploaded to and processed by the specialized online software Metaboanalyst 5.0. Both univariate and multivariate analyses were carried out for potential biomarker screening. Areas under the receiver-operating characteristic (ROC) curves (AUCs) of identified differential lipids were obtained for evaluating their classification capacity.
RESULTS: A total of 47 significantly different lipids were identified by applying the following criteria: false discovery rate-adjusted P < 0.05, variable importance in projection ⩾ 1.0, and fold change ⩾ 2.0 or ⩽ 0.5. Among them, 13 lipids were identified as diagnostic biomarkers with the area under curve (AUC) greater than 0.7. Multivariate ROC curves indicated that AUCs greater than 0.8 could be achieved with 2-47 lipids.
CONCLUSIONS: Using an untargeted LC-MS-based metabolic profiling approach, our study provides preliminary evidence that extensive dysregulations of OxPCs, PCs, SMs and TAGs were involved in the pathological processes of breast cancer. We provided clues for furtherly investigating the role of lipid alterations in the pathoetiology of breast cancer.

Keywords:  LC-MS; Lipidomics; biomarker; breast cancer; metabolism

DOI:  https://doi.org/10.3233/CBM-220462
J Am Soc Mass Spectrom. 2023 Jun 14.

Formation and Tandem Mass Spectrometry of Doubly Charged Lipid-Metal Ion Complexes.

Felix-Levin Hormann, Simon Sommer, Sven Heiles.

  Phospholipids are major components of most eukaryotic cell membranes. Changes in metabolic states are often accompanied by phospholipid structure variations. The structural changes of phospholipids are the hallmark of disease states, or specific lipid structures have been associated with distinct organisms. Prime examples are microorganisms that synthesize phospholipids with, for example, different branched chain fatty acids. Assignment and relative quantitation of structural isomers of phospholipids that arise from attachment of different fatty acids to the glycerophospholipid backbone are difficult with routine tandem mass spectrometry or with liquid chromatography without authentic standards. In this work, we report on the observation that all investigated phospholipid classes form doubly charged lipid-metal ion complexes during electrospray ionization (ESI) and show that these complexes can be used to assign lipid classes and fatty acid moieties, distinguish isomers of branched chain fatty acids, and relatively quantify these isomers in positive-ion mode. Use of water free methanol and addition of divalent metal salts (100 mol %) to ESI spray solutions afford highly abundant doubly charged lipid-metal ion complexes (up to 70 times of protonated compounds). Higher-energy collisional dissociation and collision-induced dissociation of doubly charged complexes yield a diverse set of lipid class-dependent fragment ions. In common for all lipid classes is the liberation of fatty acid-metal adducts that yield fragment ions from the fatty acid hydrocarbon chain upon activation. This ability is used to pinpoint sites of branching in saturated fatty acids and is showcased for free fatty acids as well as glycerophospholipids. The analytical utility of doubly charged phospholipid-metal ion complexes is demonstrated by distinguishing fatty acid branching-site isomers in phospholipid mixtures and relatively quantifying the corresponding isomeric compounds.

Keywords:  Branched chain fatty acids; Electrospray ionization; Hydrocarbons; Ions; Lipids; Tandem mass spectrometry

DOI:  https://doi.org/10.1021/jasms.3c00126
J Biol Chem. 2023 Jun 12. pii: S0021-9258(23)01947-6. [Epub ahead of print] 104919

Coenzyme biosynthesis in response to precursor availability reveals incorporation of β-alanine from pantothenate in prototrophic bacteria.

Birgitta Ryback, Julia A Vorholt.

  Coenzymes are important for all classes of enzymatic reactions and essential for cellular metabolism. Most coenzymes are synthesized from dedicated precursors, also referred to as vitamins, which prototrophic bacteria can either produce themselves from simpler substrates or take up from the environment. The extent to which prototrophs use supplied vitamins and whether externally available vitamins affect the size of intracellular coenzyme pools and control endogenous vitamin synthesis is currently largely unknown. Here, we studied coenzyme pool sizes and vitamin incorporation into coenzymes during growth on different carbon source and vitamin supplementation regimes using metabolomics approaches. We found that the model bacterium Escherichia coli incorporated pyridoxal, niacin, and pantothenate into pyridoxal 5'-phosphate, NAD, and coenzyme A (CoA), respectively. In contrast, riboflavin was not taken up and was produced exclusively endogenously. Coenzyme pools were mostly homeostatic and not affected by externally supplied precursors. Remarkably, we found that pantothenate is not incorporated into CoA as such but is first degraded to pantoate and β-alanine and then rebuilt. This pattern was conserved in various bacterial isolates, suggesting a preference for β-alanine over pantothenate utilization in CoA synthesis. Finally, we found that the endogenous synthesis of coenzyme precursors remains active when vitamins are supplied, which is consistent with described expression data of genes for enzymes involved in coenzyme biosynthesis under these conditions. Continued production of endogenous coenzymes may ensure rapid synthesis of the mature coenzyme under changing environmental conditions, protect against coenzyme limitation, and explain vitamin availability in naturally oligotrophic environments.

Keywords:  Bacterial metabolism; NAD biosynthesis; anaplerosis; biotin; biotin.; coenzyme A; coenzyme metabolism; flavin; flavin adenine dinucleotide (FAD); flavin mononucleotide (FMN); homeostasis; mass spectrometry (MS); metabolic shunt; metabolic tracer; metabolomics; microbiology; microbiome; nicotinamide adenine dinucleotide (NAD); overflow metabolism; pyridoxal phosphate; secretion; systems biology; transport; vitamin; β-alanine

DOI:  https://doi.org/10.1016/j.jbc.2023.104919
Mol Cell Proteomics. 2023 Jun 08. pii: S1535-9476(23)00102-0. [Epub ahead of print] 100591

UniProt and mass spectrometry-based proteomics - a 2-way working relationship.

UniProt Consortium

The human proteome comprises of all of the proteins produced by the sequences translated from the human genome with additional modifications in both sequence and function caused by nonsynonymous variants and post-translational modifications including cleavage of the initial transcript into smaller peptides and polypeptides. The UniProtKB database (www.uniprot.org) is the world's leading high-quality, comprehensive and freely accessible resource of protein sequence and functional information and presents a summary of experimentally verified, or computationally predicted, functional information added by our expert biocuration team for each protein in the proteome. Researchers in the field of mass spectrometry-based proteomics both consume and add to the body of data available in UniProtKB and this review highlights the information we provide to this community and the knowledge we in turn obtain from groups via deposition of large-scale datasets in public domain databases.

DOI: https://doi.org/10.1016/j.mcpro.2023.100591
Bioanalysis. 2023 Jun 13.

Multidimensional separation methods can improve separation performance for extremely complicated proteomics samples and provide a powerful tool for comprehensive proteome profiling of low-microgram samples.

Gang Lu, Guojin Ying, Yu He, Yang Li, Perry G Wang, Zhenbin Zhang.

  Tweetable abstract Multidimensional separation methods with improved sensitivity and peak capacity and throughput allow in-depth proteome profiling of low-microgram samples.

Keywords:  high sensitivity; high throughput; low microgram; multidimensional separation; proteomics

DOI:  https://doi.org/10.4155/bio-2023-0100
Anal Chem. 2023 Jun 15.

Optimized Suspension Trapping Method for Phosphoproteomics Sample Preparation.

Fujia Wang, Tim Veth, Marije Kuipers, Maarten Altelaar, Kelly E Stecker.

A successful mass spectrometry-based phosphoproteomics analysis relies on effective sample preparation strategies. Suspension trapping (S-Trap) is a novel, rapid, and universal method of sample preparation that is increasingly applied in bottom-up proteomics studies. However, the performance of the S-Trap protocol for phosphoproteomics studies is unclear. In the existing S-Trap protocol, the addition of phosphoric acid (PA) and methanol buffer creates a fine protein suspension to capture proteins on a filter and is a critical step for subsequent protein digestion. Herein, we demonstrate that this addition of PA is detrimental to downstream phosphopeptide enrichment, rendering the standard S-Trap protocol suboptimal for phosphoproteomics. In this study, the performance of the S-Trap digestion for proteomics and phosphoproteomics is systematically evaluated in large-scale and small-scale samples. The results of this comparative analysis show that an optimized S-Trap approach, where trifluoroacetic acid is substituted for PA, is a simple and effective method to prepare samples for phosphoproteomics. Our optimized S-Trap protocol is applied to extracellular vesicles to demonstrate superior sample preparation workflow for low-abundance, membrane-rich samples.

DOI: https://doi.org/10.1021/acs.analchem.3c00324
Biochim Biophys Acta Mol Cell Biol Lipids. 2023 Jun 08. pii: S1388-1981(23)00070-7. [Epub ahead of print] 159346

Phosphoinositide and redox dysregulation by the anticancer methylthioadenosine phosphorylase transition state inhibitor.

Timothy Salita, Yepy H Rustam, Vinzenz Hofferek, Michael Jackson, Isaac Tollestrup, Jeffrey P Sheridan, Vern L Schramm, Gary B Evans, Gavin E Reid, Andrew B Munkacsi.

  Methylthio-DADMe-immucillin-A (MTDIA) is an 86 picomolar inhibitor of 5'-methylthioadenosine phosphorylase (MTAP) with potent and specific anti-cancer efficacy. MTAP salvages S-adenosylmethionine (SAM) from 5'-methylthioadenosine (MTA), a toxic metabolite produced during polyamine biosynthesis. Changes in MTAP expression are implicated in cancer growth and development, making MTAP an appealing target for anti-cancer therapeutics. Since SAM is involved in lipid metabolism, we hypothesised that MTDIA alters the lipidomes of MTDIA-treated cells. To identify these effects, we analysed the lipid profiles of MTDIA-treated Saccharomyces cerevisiae using ultra-high resolution accurate mass spectrometry (UHRAMS). MTAP inhibition by MTDIA, and knockout of the Meu1 gene that encodes for MTAP in yeast, caused global lipidomic changes and differential abundance of lipids involved in cell signaling. The phosphoinositide kinase/phosphatase signaling network was specifically impaired upon MTDIA treatment, and was independently validated and further characterised via altered localization of proteins integral to this network. Functional consequences of dysregulated lipid metabolism included a decrease in reactive oxygen species (ROS) levels induced by MTDIA that was contemporaneous with changes in immunological response factors (nitric oxide, tumour necrosis factor-alpha and interleukin-10) in mammalian cells. These results indicate that lipid homeostasis alterations and concomitant downstream effects may be associated with MTDIA mechanistic efficacy.

Keywords:  Cancer therapeutics; Lipid metabolism; Lipidomics; Mass spectrometry; Phosphoinositide; Transition-state analog

DOI:  https://doi.org/10.1016/j.bbalip.2023.159346
Methods Mol Biol. 2023 ;2650 261-271

Automated Quantitative Analysis of Shape Features in Human Epithelial Monolayers and Spheroids Generated from Colorectal Cancer Cells.

Hannah M Brown, M Angeles Juanes.

  Advancements in microscopy techniques permit us to acquire endless datasets of images. A major bottleneck in cell imaging is how to analyze petabytes of data in an effective, reliable, objective, and effortless way. Quantitative imaging is becoming crucial to disentangle the complexity of many biological and pathological processes. For instance, cell shape is a summary readout of a myriad of cellular processes. Changes in cell shape use to reflect changes in growth, migration mode (including speed and persistence), differentiation stage, apoptosis, or gene expression, serving to predict health or disease. However, in certain contexts, e.g., tissues or tumors, cells are tightly packed together, and measurement of individual cellular shapes can be challenging and laborious. Bioinformatics solutions like automated computational image methods provide a blind and efficient analysis of large image datasets. Here we describe a detailed and friendly step-by-step protocol to extract various cellular shape parameters quickly and accurately from colorectal cancer cells forming either monolayers or spheroids. We envision those similar settings could be extended to other cell lines, colorectal and beyond, either label/unlabeled or in 2D/3D environments.

Keywords:  Cell size; Colon cancer cells; HCT116; Machine learning; Monolayers; Quantification; Segmentation; Spheroids

DOI:  https://doi.org/10.1007/978-1-0716-3076-1_20
Cell Metab. 2023 Jun 07. pii: S1550-4131(23)00185-7. [Epub ahead of print]

Ketogenic diet promotes tumor ferroptosis but induces relative corticosterone deficiency that accelerates cachexia.

Miriam Ferrer, Nicholas Mourikis, Emma E Davidson, Sam O Kleeman, Marta Zaccaria, Jill Habel, Rachel Rubino, Qing Gao, Thomas R Flint, Lisa Young, Claire M Connell, Michael J Lukey, Marcus D Goncalves, Eileen P White, Ashok R Venkitaraman, Tobias Janowitz.

  Glucose dependency of cancer cells can be targeted with a high-fat, low-carbohydrate ketogenic diet (KD). However, in IL-6-producing cancers, suppression of the hepatic ketogenic potential hinders the utilization of KD as energy for the organism. In IL-6-associated murine models of cancer cachexia, we describe delayed tumor growth but accelerated cachexia onset and shortened survival in mice fed KD. Mechanistically, this uncoupling is a consequence of the biochemical interaction of two NADPH-dependent pathways. Within the tumor, increased lipid peroxidation and, consequently, saturation of the glutathione (GSH) system lead to the ferroptotic death of cancer cells. Systemically, redox imbalance and NADPH depletion impair corticosterone biosynthesis. Administration of dexamethasone, a potent glucocorticoid, increases food intake, normalizes glucose levels and utilization of nutritional substrates, delays cachexia onset, and extends the survival of tumor-bearing mice fed KD while preserving reduced tumor growth. Our study emphasizes the need to investigate the effects of systemic interventions on both the tumor and the host to accurately assess therapeutic potential. These findings may be relevant to clinical research efforts that investigate nutritional interventions such as KD in patients with cancer.

Keywords:  GDF-15; IL-6; NADPH; cachexia; cancer; corticosterone; ferroptosis; ketogenic diet; lipid peroxidation; steroid

DOI:  https://doi.org/10.1016/j.cmet.2023.05.008
Anal Chem. 2023 Jun 12.

DiLeu Isobaric Labeling Coupled with Limited Proteolysis Mass Spectrometry for High-Throughput Profiling of Protein Structural Changes in Alzheimer's Disease.

Haiyan Lu, Bin Wang, Yuan Liu, Danqing Wang, Lauren Fields, Hua Zhang, Miyang Li, Xudong Shi, Henrik Zetterberg, Lingjun Li.

High-throughput quantitative analysis of protein conformational changes has a profound impact on our understanding of the pathological mechanisms of Alzheimer's disease (AD). To establish an effective workflow enabling quantitative analysis of changes in protein conformation within multiple samples simultaneously, here we report the combination of N,N-dimethyl leucine (DiLeu) isobaric tag labeling with limited proteolysis mass spectrometry (DiLeu-LiP-MS) for high-throughput structural protein quantitation in serum samples collected from AD patients and control donors. Twenty-three proteins were discovered to undergo structural changes, mapping to 35 unique conformotypic peptides with significant changes between the AD group and the control group. Seven out of 23 proteins, including CO3, CO9, C4BPA, APOA1, APOA4, C1R, and APOA, exhibited a potential correlation with AD. Moreover, we found that complement proteins (e.g., CO3, CO9, and C4BPA) related to AD exhibited elevated levels in the AD group compared to those in the control group. These results provide evidence that the established DiLeu-LiP-MS method can be used for high-throughput structural protein quantitation, which also showed great potential in achieving large-scale and in-depth quantitative analysis of protein conformational changes in other biological systems.

DOI: https://doi.org/10.1021/acs.analchem.2c05731
ACS Omega. 2023 Jun 06. 8(22): 19741-19751

One-STAGE Tip Method for TMT-Based Proteomic Analysis of a Minimal Amount of Cells.

Narae Park, Hankyul Lee, Yumi Kwon, Shinyeong Ju, Seonjeong Lee, Seongjin Yoo, Kang-Sik Park, Cheolju Lee.

Liquid chromatography-tandem mass spectrometry (LC-MS)-based profiling of proteomes with isobaric tag labeling from low-quantity biological and clinical samples, including needle-core biopsies and laser capture microdissection, has been challenging due to the limited amount and sample loss during preparation. To address this problem, we developed OnM (On-Column from Myers et al. and mPOP)-modified on-column method combining freeze-thaw lysis of mPOP with isobaric tag labeling of On-Column method to minimize sample loss. OnM is a method that processes the sample in one-STAGE tip from cell lysis to tandem mass tag (TMT) labeling without any transfer of the sample. In terms of protein coverage, cellular components, and TMT labeling efficiency, the modified On-Column (or OnM) displayed similar performance to the results from Myers et al. To evaluate the lower-limit processing capability of OnM, we utilized OnM for multiplexing and were able to quantify 301 proteins in a TMT 9-plex with 50 cells per channel. We optimized the method as low as 5 cells per channel in which we identified 51 quantifiable proteins. OnM method is a low-input proteomics method widely applicable and capable of identifying and quantifying proteomes from limited samples, with tools that are readily available in a majority of proteomic laboratories.

DOI: https://doi.org/10.1021/acsomega.3c01392
Clin Nutr. 2023 Jun 07. pii: S0261-5614(23)00184-X. [Epub ahead of print]

Amino acid metabolism in tumor: New shine in the fog?

Hui Qiu, Nan Shao, Jing Liu, Juanjuan Zhao, Chao Chen, Qihong Li, Zhixu He, Xu Zhao, Lin Xu.

  Alterations in amino acid metabolism is closely related to the occurrence of clinical diseases. The mechanism of tumorigenesis is complex, involving the complicated relationship between tumor cells and immune cells in local tumor microenvironment. A series of recent studies have shown that metabolic remodeling is intimately related to tumorigenesis. And amino acid metabolic reprogramming is one of the important characteristics of tumor metabolic remodeling, which participates in tumor cells growth, survival as well as the immune cell activation and function in the local tumor microenvironment, thereby affecting tumor immune escape. Recent studies have further shown that controlling the intake of specific amino acids can significantly improve the effect of clinical intervention in tumors, suggesting that amino acid metabolism is gradually becoming one of the new promising targets of clinical intervention in tumors. Therefore, developing new intervention strategies based on amino acid metabolism has broad prospects. In this article, we review the abnormal changes in the metabolism of some typical amino acids, including glutamine, serine, glycine, asparagine and so on in tumor cells and summarize the relationship among amino acid metabolism, tumor microenvironment and the function of T cells. In particular, we discuss the current issues that need to be addressed in the related fields of tumor amino acid metabolism, aiming to provide a theoretical basis for the development of new strategies for clinical interventions in tumors based on amino acid metabolism reprogramming.

Keywords:  Amino acid metabolism; Immune cells; Immune escape; Tumor

DOI:  https://doi.org/10.1016/j.clnu.2023.06.011