bims-mascan 2024-07-28 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2024‒07‒28
twelve papers selected by
Giovanny Rodriguez Blanco, University of Edinburgh

Targeted Mass Spectrometry for Quantification of Receptor Tyrosine Kinase Signaling.
13C Tracer Analysis and Metabolomics in Dormant Cancer Cells.
12-Plex Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tag-Enabled High-Throughput Quantitative Glycomics.
Automated Liquid Handling Extraction and Rapid Quantification of Underivatized Amino Acids and Tryptophan Metabolites from Human Serum and Plasma Using Dual-Column U(H)PLC-MRM-MS and Its Application to Prostate Cancer Study.
Using Photoreactive Probes to Identify Viable Drug Targets in Non-small Cell Lung Cancer.
Spatially and temporally probing distinctive glycerophospholipid alterations in Alzheimer's disease mouse brain via high-resolution ion mobility-enabled sn-position resolved lipidomics.
Oxidative Metabolism as a Cause of Lipid Peroxidation in the Execution of Ferroptosis.
Multiplexed quantitative proteomics in prostate cancer biomarker development.
Monitoring Epoxidized Soybean Oil Degradation Using Liquid Chromatography-Mass Spectrometry and In Silico Spectral Libraries.
A Fast-Tracking Sample Preparation Protocol for Proteomics of Formalin-Fixed Paraffin-Embedded Tumor Tissues.
Automated Immunoprecipitation Workflow for Comprehensive Acetylome Analysis.
The Utility of Lipidomic Analysis in Colorectal Cancer Diagnosis and Prognosis-A Systematic Review of Recent Literature.

Methods Mol Biol. 2024 ;2823 253-267

Targeted Mass Spectrometry for Quantification of Receptor Tyrosine Kinase Signaling.

Jeffrey R Whiteaker, Lei Zhao, Jacob J Kennedy, Richard G Ivey, Amanda G Paulovich.

  Targeted proteomics enables sensitive and specific quantification of proteins and post-translational modifications. By coupling peptide immunoaffinity enrichment with targeted mass spectrometry, we have developed the methodology for multiplexed quantification of proteins and phosphosites involved in the RAS/MAPK signaling network. The method uses anti-peptide antibodies to enrich analytes and heavy stable isotope-labeled internal standards, spiked in at known concentrations. The enriched peptides are directly measured by multiple-reaction monitoring (MRM), a well-characterized quantitative mass spectrometry-based method. The analyte (light) peptide response is measured relative to the heavy standard. The method described provides quantitative measurements of phospho-signaling and is generally applicable to other phosphopeptides and sample types.

Keywords:   LC–MS; Multiplex; Phospho-signaling; Quantitation; RAS/MAPK; Targeted proteomics

DOI:  https://doi.org/10.1007/978-1-0716-3922-1_16
Methods Mol Biol. 2024 ;2811 195-206

13C Tracer Analysis and Metabolomics in Dormant Cancer Cells.

Patricia Altea-Manzano, Sarah-Maria Fendt, Laura Vera-Ramirez.

  Over the last two decades, major advances in the field of tumor dormancy have been made. Yet, it is not completely understood how dormant disseminated tumor cells survive and transition to a proliferative state to generate a metastatic lesion. On the other hand, metabolic rewiring has been shown to influence metastasis development through the modulation of both intracellular signaling and the crosstalk between metastatic cells and their microenvironment. Thus, studying the metabolic features of dormant disseminated tumor cells has gained importance in understanding the dormancy process. Here, we describe a method to perform metabolomics and 13C tracer analysis in 3D cultures of dormant breast cancer cells.

Keywords:  13C tracing; 3D cell culture; Cancer; Disseminated tumor cells; Mass spectrometry; Metabolomics; Metastasis; Tumor cell dormancy

DOI:  https://doi.org/10.1007/978-1-0716-3882-8_15
Methods Mol Biol. 2024 ;2823 155-172

12-Plex Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tag-Enabled High-Throughput Quantitative Glycomics.

Miyang Li, Yu Feng, Min Ma, Lingjun Li.

  Glycans, which are ubiquitously distributed on most proteins and cell surfaces, are a class of important biomolecules playing crucial roles in various biological processes such as molecular recognition and cellular communication. Modern mass spectrometry (MS) coupled with novel chemical probe labeling strategies has greatly advanced analysis of glycans. However, the requirement of high-throughput and robust quantitative analysis still calls for the development of more advanced tools. Recently, we devised isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags for 4-plex N-glycan analysis. To further improve the throughput, we utilized the mass-defect strategy and expanded the multiplexing capacity to 12 channels without changing the chemical structure of the SUGAR tag, achieving a threefold enhancement in throughput compared with the original design and managing to perform high-throughput N-glycan analysis in a single LC - MS/MS injection. Herein, we present detailed methods for the synthesis of 12-plex SUGAR isobaric tags, the procedure to release and label the N-glycans from proteins, and the analysis by high-resolution LC-MS/MS, as well as data processing to achieve multiplexed quantitative glycomics.

Keywords:  Isobaric labeling; Mass defect; Mass spectrometry; Multiplexed quantification; Quantitative glycomics; SUGAR; Tag synthesis

DOI:  https://doi.org/10.1007/978-1-0716-3922-1_11
Metabolites. 2024 Jun 30. pii: 370. [Epub ahead of print]14(7):

Automated Liquid Handling Extraction and Rapid Quantification of Underivatized Amino Acids and Tryptophan Metabolites from Human Serum and Plasma Using Dual-Column U(H)PLC-MRM-MS and Its Application to Prostate Cancer Study.

Tobias Kipura, Madlen Hotze, Alexa Hofer, Anna-Sophia Egger, Lea E Timpen, Christiane A Opitz, Paul A Townsend, Lee A Gethings, Kathrin Thedieck, Marcel Kwiatkowski.

  Amino acids (AAs) and their metabolites are important building blocks, energy sources, and signaling molecules associated with various pathological phenotypes. The quantification of AA and tryptophan (TRP) metabolites in human serum and plasma is therefore of great diagnostic interest. Therefore, robust, reproducible sample extraction and processing workflows as well as rapid, sensitive absolute quantification are required to identify candidate biomarkers and to improve screening methods. We developed a validated semi-automated robotic liquid extraction and processing workflow and a rapid method for absolute quantification of 20 free, underivatized AAs and six TRP metabolites using dual-column U(H)PLC-MRM-MS. The extraction and sample preparation workflow in a 96-well plate was optimized for robust, reproducible high sample throughput allowing for transfer of samples to the U(H)PLC autosampler directly without additional cleanup steps. The U(H)PLC-MRM-MS method, using a mixed-mode reversed-phase anion exchange column with formic acid and a high-strength silica reversed-phase column with difluoro-acetic acid as mobile phase additive, provided absolute quantification with nanomolar lower limits of quantification within 7.9 min. The semi-automated extraction workflow and dual-column U(H)PLC-MRM-MS method was applied to a human prostate cancer study and was shown to discriminate between treatment regimens and to identify metabolites responsible for discriminating between healthy controls and patients on active surveillance.

Keywords:  LC-MS; amino acids; automation; mixed-mode chromatography; prostate cancer; tryptophan metabolites analysis

DOI:  https://doi.org/10.3390/metabo14070370
Methods Mol Biol. 2024 ;2823 47-53

Using Photoreactive Probes to Identify Viable Drug Targets in Non-small Cell Lung Cancer.

Yi Liao.

  Recent advancements in chemoproteomics have accelerated new chemical tools for exploring protein ligandability in native biological systems. However, a large fraction of ligandable proteome in cancer cells remains poorly studied. Here, we present a practical and efficient sample processing method for liquid chromatography high-resolution-tandem mass spectrometry (HPLC-MS/MS) analysis. This method uses fully functionalized photoreactive fragment-like probes for profiling protein-ligand interactions in live cancer cells. This method adopts "on-bead" digestion in conjunction with ZipTip desalting prior sample injection to MS. By using this protocol, fragment protein interactions can be visualized using fluorescent imaging, and fragment-associated proteins can be identified via HPLC-MS/MS analysis. Approximately 16 samples would generally expect to be processed within 3 days by following this protocol.

Keywords:  Chemical proteomics; Fluorescent imaging; HPLC-MS/MS; On-bead digestion; Photoactivable fragment probes; ZipTip

DOI:  https://doi.org/10.1007/978-1-0716-3922-1_4
Nat Commun. 2024 Jul 24. 15(1): 6252

Spatially and temporally probing distinctive glycerophospholipid alterations in Alzheimer's disease mouse brain via high-resolution ion mobility-enabled sn-position resolved lipidomics.

Shuling Xu, Zhijun Zhu, Daniel G Delafield, Michael J Rigby, Gaoyuan Lu, Megan Braun, Luigi Puglielli, Lingjun Li.

Dysregulated glycerophospholipid (GP) metabolism in the brain is associated with the progression of neurodegenerative diseases including Alzheimer's disease (AD). Routine liquid chromatography-mass spectrometry (LC-MS)-based large-scale lipidomic methods often fail to elucidate subtle yet important structural features such as sn-position, hindering the precise interrogation of GP molecules. Leveraging high-resolution demultiplexing (HRdm) ion mobility spectrometry (IMS), we develop a four-dimensional (4D) lipidomic strategy to resolve GP sn-position isomers. We further construct a comprehensive experimental 4D GP database of 498 GPs identified from the mouse brain and an in-depth extended 4D library of 2500 GPs predicted by machine learning, enabling automated profiling of GPs with detailed acyl chain sn-position assignment. Analyzing three mouse brain regions (hippocampus, cerebellum, and cortex), we successfully identify a total of 592 GPs including 130 pairs of sn-position isomers. Further temporal GPs analysis in the three functional brain regions illustrates their metabolic alterations in AD progression.

DOI: https://doi.org/10.1038/s41467-024-50299-9
Int J Mol Sci. 2024 Jul 09. pii: 7544. [Epub ahead of print]25(14):

Oxidative Metabolism as a Cause of Lipid Peroxidation in the Execution of Ferroptosis.

Junichi Fujii, Hirotaka Imai.

  Ferroptosis is a type of nonapoptotic cell death that is characteristically caused by phospholipid peroxidation promoted by radical reactions involving iron. Researchers have identified many of the protein factors that are encoded by genes that promote ferroptosis. Glutathione peroxidase 4 (GPX4) is a key enzyme that protects phospholipids from peroxidation and suppresses ferroptosis in a glutathione-dependent manner. Thus, the dysregulation of genes involved in cysteine and/or glutathione metabolism is closely associated with ferroptosis. From the perspective of cell dynamics, actively proliferating cells are more prone to ferroptosis than quiescent cells, which suggests that radical species generated during oxygen-involved metabolism are responsible for lipid peroxidation. Herein, we discuss the initial events involved in ferroptosis that dominantly occur in the process of energy metabolism, in association with cysteine deficiency. Accordingly, dysregulation of the tricarboxylic acid cycle coupled with the respiratory chain in mitochondria are the main subjects here, and this suggests that mitochondria are the likely source of both radical electrons and free iron. Since not only carbohydrates, but also amino acids, especially glutamate, are major substrates for central metabolism, dealing with nitrogen derived from amino groups also contributes to lipid peroxidation and is a subject of this discussion.

Keywords:  glycolysis; metabolic remodeling; tricarboxylic acid cycle; urea cycle

DOI:  https://doi.org/10.3390/ijms25147544
Adv Cancer Res. 2024 ;pii: S0065-230X(24)00003-4. [Epub ahead of print]161 31-69

Multiplexed quantitative proteomics in prostate cancer biomarker development.

Yuqian Gao, Hyeyoon Kim, Reta Birhanu Kitata, Tai-Tu Lin, Adam C Swensen, Tujin Shi, Tao Liu.

  Prostate cancer (PCa) is the most common non-skin cancer among men in the United States. However, the widely used protein biomarker in PCa, prostate-specific antigen (PSA), while useful for initial detection, its use alone cannot detect aggressive PCa and can lead to overtreatment. This chapter provides an overview of PCa protein biomarker development. It reviews the state-of-the-art liquid chromatography-mass spectrometry-based proteomics technologies for PCa biomarker development, such as enhancing the detection sensitivity of low-abundance proteins through antibody-based or antibody-independent protein/peptide enrichment, enriching post-translational modifications such as glycosylation as well as information-rich extracellular vesicles, and increasing accuracy and throughput using advanced data acquisition methodologies. This chapter also summarizes recent PCa biomarker validation studies that applied those techniques in diverse specimen types, including cell lines, tissues, proximal fluids, urine, and blood, developing novel protein biomarkers for various clinical applications, including early detection and diagnosis, prognosis, and therapeutic intervention of PCa.

Keywords:  DIA; Early detection; PRM; Prostate cancer; Protein biomarker; Risk stratification; SRM

DOI:  https://doi.org/10.1016/bs.acr.2024.04.003
J Am Soc Mass Spectrom. 2024 Jul 23.

Monitoring Epoxidized Soybean Oil Degradation Using Liquid Chromatography-Mass Spectrometry and In Silico Spectral Libraries.

Paul D Hutchins, Camila A Saez Cabezas, Joshua S Enokida, Yushan Hu, Yuming Lai, Victoria Mazure, Marie Martin, Kelly Setula, John R Stutzman, James H Wade.

Epoxidized soybean oil (ESO) is routinely used as a bioderived plasticizer and stabilizer in polyvinyl chloride (PVC), as it prolongs material integrity during dehydrochlorination. During this process, the epoxide moieties of ESO are progressively converted to chlorohydrins, which amplify ESO's inherent structural complexity. Past characterization efforts utilized separation-mass spectrometry (MS) analysis of the hydrolyzed acyl chains to simplify the complexity. However, this approach significantly increases the complexity of sample preparation and cannot directly monitor the chlorination of individual ESO species during aging. Here, we present a comprehensive LC-MS/MS data acquisition and in silico spectral library identification workflow optimized for intact ESO byproduct analysis. Detailed MS/MS fragmentation rules derived from synthesized standards were coupled with improved fragment ion intensity modeling capabilities to generate a high-fidelity spectral library for rapid ESO byproduct identification. Identification confidence was further bolstered by using retention time modeling to filter spurious MS/MS matches. Finally, we paired this informatic approach with an optimized extraction procedure and reversed-phase separation to generate a detailed timeline of more than 400 ESO species and byproducts during PVC thermal aging. These developments significantly improve our ability to detect, characterize, and understand ESO degradation in complex PVC formulations with new levels of molecular resolution.

DOI: https://doi.org/10.1021/jasms.4c00212
Methods Mol Biol. 2024 ;2823 193-223

A Fast-Tracking Sample Preparation Protocol for Proteomics of Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Lancia N F Darville, John H Lockhart, Sudhir Putty Reddy, Bin Fang, Victoria Izumi, Theresa A Boyle, Eric B Haura, Elsa R Flores, John M Koomen.

  Archived tumor specimens are routinely preserved by formalin fixation and paraffin embedding. Despite the conventional wisdom that proteomics might be ineffective due to the cross-linking and pre-analytical variables, these samples have utility for both discovery and targeted proteomics. Building on this capability, proteomics approaches can be used to maximize our understanding of cancer biology and clinical relevance by studying preserved tumor tissues annotated with the patients' medical histories. Proteomics of formalin-fixed paraffin-embedded (FFPE) tissues also integrates with histological evaluation and molecular pathology strategies, so that additional collection of research biopsies or resected tumor aliquots is not needed. The acquisition of data from the same tumor sample also overcomes concerns about biological variation between samples due to intratumoral heterogeneity. However, the protein extraction and proteomics sample preparation from FFPE samples can be onerous, particularly for small (i.e., limited or precious) samples. Therefore, we provide a protocol for a recently introduced kit-based EasyPep method with benchmarking against a modified version of the well-established filter-aided sample preparation strategy using laser-capture microdissected lung adenocarcinoma tissues from a genetically engineered mouse model. This model system allows control over the tumor preparation and pre-analytical variables while also supporting the development of methods for spatial proteomics to examine intratumoral heterogeneity. Data are posted in ProteomeXchange (PXD045879).

Keywords:   EasyPep; Filter-aided sample preparation; Lung cancer; Proteomics; Formalin-fixed paraffin-embedded tissue

DOI:  https://doi.org/10.1007/978-1-0716-3922-1_13
Methods Mol Biol. 2024 ;2823 173-191

Automated Immunoprecipitation Workflow for Comprehensive Acetylome Analysis.

Marina A Gritsenko, Chia-Feng Tsai, Hyeyoon Kim, Tao Liu.

  Immunoprecipitation is one of the most effective methods for enrichment of lysine-acetylated peptides for comprehensive acetylome analysis using mass spectrometry. Manual acetyl peptide enrichment method using non-conjugated antibodies and agarose beads has been developed and applied in various studies. However, it is time-consuming and can introduce contaminants and variability that leads to potential sample loss and decreased sensitivity and robustness of the analysis. Here we describe a fast, automated enrichment protocol that enables reproducible and comprehensive acetylome analysis using a magnetic bead-based immunoprecipitation reagent.

Keywords:   Acetyl peptide enrichment; Acetylome; Automation; Immunoprecipitation; Magnetic beads; Mass spectrometry; Post-translational modification; Proteomics; Lysine acetylation

DOI:  https://doi.org/10.1007/978-1-0716-3922-1_12
Int J Mol Sci. 2024 Jul 14. pii: 7722. [Epub ahead of print]25(14):

The Utility of Lipidomic Analysis in Colorectal Cancer Diagnosis and Prognosis-A Systematic Review of Recent Literature.

Jakub Klekowski, Mariusz Chabowski, Małgorzata Krzystek-Korpacka, Mariusz Fleszar.

  Colorectal cancer (CRC) is among the most prevalent and lethal malignancies. Lipidomic investigations have revealed numerous disruptions in lipid profiles across various cancers. Studies on CRC exhibit potential for identifying novel diagnostic or prognostic indicators through lipidomic signatures. This review examines recent literature regarding lipidomic markers for CRC. PubMed database was searched for eligible articles concerning lipidomic biomarkers of CRC. After selection, 36 articles were included in the review. Several studies endeavor to establish sets of lipid biomarkers that demonstrate promising potential to diagnose CRC based on blood samples. Phosphatidylcholine, phosphatidylethanolamine, ceramides, and triacylglycerols (TAGs) appear to offer the highest diagnostic accuracy. In tissues, lysophospholipids, ceramides, and TAGs were among the most altered lipids, while unsaturated fatty acids also emerged as potential biomarkers. In-depth analysis requires both cell culture and animal studies. CRC involves multiple lipid metabolism alterations. Although numerous lipid species have been suggested as potential diagnostic markers, the establishment of standardized methods and the conduct of large-scale studies are necessary to facilitate their clinical application.

Keywords:  biomarkers; colorectal cancer; diagnosis; lipidomics; mass spectrometry; prognosis

DOI:  https://doi.org/10.3390/ijms25147722