bims-mascan 2023-12-24 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2023–12–24
29 papers selected by
Giovanny Rodriguez Blanco, University of Edinburgh

High-Throughput Microbore LC-MS Lipidomics to Investigate APOE Phenotypes.
Liquid chromatography-mass spectrometry-based metabolomics and fluxomics reveals the metabolic alterations in glioma U87MG multicellular tumor spheroids versus two-dimensional cell cultures.
Targeting lipid metabolism in cancer metastasis.
Recent progress in quantitative phosphoproteomics.
Open access repository-scale propagated nearest neighbor suspect spectral library for untargeted metabolomics.
Oleic Acid Metabolism in Response to Glucose in C. elegans.
Membranes that make fat: roles of membrane lipids as acyl donors for triglyceride synthesis and organelle function.
Mapping protein states and interactions across the tree of life with co-fractionation mass spectrometry.
Wide-scope targeted analysis of bioactive lipids in human plasma by LC/MS/MS.
Immunopeptidomics in the Era of Single-Cell Proteomics.
Effect of statin treatment on metabolites, lipids and prostanoids in patients with Statin Associated Muscle Symptoms (SAMS).
FLiPPR: A Processor for Limited Proteolysis (LiP) Mass Spectrometry Datasets Built on FragPipe.
Untargeted serum metabolomics reveals novel metabolite associations and disruptions in amino acid and lipid metabolism in Parkinson's disease.
RNA Modifications in Cancer Metabolism and Tumor Microenvironment.
Comparison of 3-nitrophenylhydrazine, O-benzyl hydroxylamine, and 2-picolylamine derivatizations for analysis of short-chain fatty acids through liquid chromatography coupled with tandem mass spectrometry.
Mitochondrial fatty acid synthesis is an emergent central regulator of mammalian oxidative metabolism.
Exploring protein lipidation by mass spectrometry-based proteomics.
Integration of lipidomics and metabolomics reveals plasma and urinary profiles associated with pediatric Mycoplasma pneumoniae infections and its severity.
Role of Glycolytic and Glutamine Metabolism Reprogramming on the Proliferation, Invasion, and Apoptosis Resistance through Modulation of Signaling Pathways in Glioblastoma.
Protein kinase D (PKD) on the crossroad of lipid absorption, synthesis and utilization.
Application of Normal-Phase Silica Column in Hydrophilic Interaction Liquid Chromatography Mode for Simultaneous Determination of Underivatized Amino Acids from Human Serum Samples via Liquid Chromatography-Tandem Mass Spectrometry.
Effect of metabolic reprogramming on the immune microenvironment in gastric cancer.
An Overview on Lipid Droplets Accumulation as Novel Target for Acute Myeloid Leukemia Therapy.
Development of a single mobile phase for LC-IM-MS-based discovery lipidomics and metabolic phenotyping: Application to methapyrilene hepatotoxicity in the rat.
Efficient lipidomic approach for the discovery of lipid ligands for immune receptors by combining LC-HRMS/MS analysis with fractionation and reporter cell assay.
The Role of Tumor Metabolic Reprogramming in Tumor Immunity.
Sulfoproteomics Workflow with Precursor Ion Accurate Mass Shift Analysis Reveals Novel Tyrosine Sulfoproteins in the Golgi.
Proteomics-driven noninvasive screening of circulating serum protein panels for the early diagnosis of hepatocellular carcinoma.
A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry.

Anal Chem. 2023 Dec 19.

High-Throughput Microbore LC-MS Lipidomics to Investigate APOE Phenotypes.

Darshak Gadara, Vratislav Berka, Zdenek Spacil.

Microflow liquid chromatography interfaced with mass spectrometry (μLC-MS/MS) is increasingly applied for high-throughput profiling of biological samples and has been proven to have an acceptable trade-off between sensitivity and reproducibility. However, lipidomics applications are scarce. We optimized a μLC-MS/MS system utilizing a 1 mm inner diameter × 100 mm column coupled to a triple quadrupole mass spectrometer to establish a sensitive, high-throughput, and robust single-shot lipidomics workflow. Compared to conventional lipidomics methods, we achieve a ∼4-fold increase in response, facilitating quantification of 351 lipid species from a single iPSC-derived cerebral organoid during a 15 min LC-MS analysis. Consecutively, we injected 303 samples over ∼75 h to prove the robustness and reproducibility of the microflow separation. As a proof of concept, μLC-MS/MS analysis of Alzheimer's disease patient-derived iPSC cerebral organoid reveals differential lipid metabolism depending on APOE phenotype (E3/3 vs E4/4). Microflow separation proves to be an environmentally friendly and cost-effective method as it reduces the consumption of harmful solvents. Also, the data demonstrate robust, in-depth, high-throughput performance to enable routine clinical or biomedical applications.

DOI: https://doi.org/10.1021/acs.analchem.3c02652
Rapid Commun Mass Spectrom. 2024 Jan 30. 38(2): e9670

Liquid chromatography-mass spectrometry-based metabolomics and fluxomics reveals the metabolic alterations in glioma U87MG multicellular tumor spheroids versus two-dimensional cell cultures.

Shanjing Wen, Xinyi Tu, Qingce Zang, Ying Zhu, Limei Li, Ruiping Zhang, Zeper Abliz.

RATIONALE: Multicellular tumor spheroids (MCTSs) that reconstitute the metabolic characteristics of in vivo tumor tissue may facilitate the discovery of molecular biomarkers and effective anticancer therapies. However, little is known about how cancer cells adapt their metabolic changes in complex three-dimensional (3D) microenvironments. Here, using the two-dimensional (2D) cell model as control, the metabolic phenotypes of glioma U87MG multicellular tumor spheroids were systematically investigated based on static metabolomics and dynamic fluxomics analysis.
METHODS: A liquid chromatography-mass spectrometry-based global metabolomics and lipidomics approach was adopted to survey the cellular samples from 2D and 3D culture systems, revealing marked molecular differences between them. Then, by means of metabolomic pathway analysis, the metabolic pathways altered in glioma MCTSs were found using 13 C6 -glucose as a tracer to map the metabolic flux of glycolysis, the tricarboxylic acid (TCA) cycle, de novo nucleotide synthesis, and de novo lipid biosynthesis in the MCTS model.
RESULTS: We found nine metabolic pathways as well as glycerolipid, glycerophospholipid and sphingolipid metabolism to be predominantly altered in glioma MCTSs. The reduced nucleotide metabolism, amino acid metabolism and glutathione metabolism indicated an overall lower cellular activity in MCTSs. Through dynamic fluxomics analysis in the MCTS model, we found that cells cultured in MCTSs exhibited increased glycolysis activity and de novo lipid biosynthesis activity, and decreased the TCA cycle and de novo purine nucleotide biosynthesis activity.
CONCLUSIONS: Our study highlights specific, altered biochemical pathways in MCTSs, emphasizing dysregulation of energy metabolism and lipid metabolism, and offering novel insight into metabolic events in glioma MCTSs.

DOI: https://doi.org/10.1002/rcm.9670
Biochim Biophys Acta Rev Cancer. 2023 Dec 13. pii: S0304-419X(23)00200-7. [Epub ahead of print] 189051

Targeting lipid metabolism in cancer metastasis.

Gloria Pascual, Blanca Majem, Salvador Aznar-Benitah.

  This review delves into the most recent research on the metabolic adaptability of cancer cells and examines how their metabolic functions can impact their progression into metastatic forms. We emphasize the growing significance of lipid metabolism and dietary lipids within the tumor microenvironment, underscoring their influence on tumor progression. Additionally, we present an outline of the interplay between metabolic processes and the epigenome of cancer cells, underscoring the importance regarding the metastatic process. Lastly, we examine the potential of targeting metabolism as a therapeutic approach in combating cancer progression, shedding light on innovative drugs/targets currently undergoing preclinical evaluation.

Keywords:  Epigenetics; Lipid metabolism; Metastasis; Microenvironment; Therapies

DOI:  https://doi.org/10.1016/j.bbcan.2023.189051
Expert Rev Proteomics. 2023 Dec 20.

Recent progress in quantitative phosphoproteomics.

Katharina Zittlau, Payal Nashier, Claudia Cavarischia-Rega, Boris Macek, Philipp Spät, Nicolas Nalpas.

   INTRODUCTION: Protein phosphorylation is a critical post-translational modification involved in the regulation of numerous cellular processes from signal transduction to modulation of enzyme activities. Knowledge on dynamic changes of phosphorylation levels during biological processes, under various treatments or between healthy and disease models is fundamental for understanding the role of each phosphorylation event. Thereby, LC-MS/MS based technologies in combination with quantitative proteomics strategies evolved as a powerful strategy to investigate the function of individual protein phosphorylation events.
AREAS COVERED: Stateoftheart labeling techniques including stable isotope and isobaric labeling provide precise and accurate quantification of phosphorylation events. Here, we review the strengths and limitations of recent quantification methods and provide examples based on current studies, how quantitative phosphoproteomics can be further optimized for enhanced analytic depth, dynamic range, site localization and data integrity. Specifically, reducing the input material demands is key to a broader implementation of quantitative phosphoproteomics, not least for clinical samples.
EXPERT OPINION: Despite quantitative phosphoproteomics is one of the most thriving fields in the proteomics world, many challenges still have to be overcome to facilitate even deeper and more comprehensive analyses as required in current research, especially at single cell levels and in clinical diagnostics.

Keywords:  DIA; Hotspot thermal profiling; Isobaric labeling; Isobaric tags; LC-MS; PTM; Phosphopeptide enrichment; protein phosphorylation; quantitative phosphoproteomics; stable isotope labeling

DOI:  https://doi.org/10.1080/14789450.2023.2295872
Nat Commun. 2023 Dec 20. 14(1): 8488

Open access repository-scale propagated nearest neighbor suspect spectral library for untargeted metabolomics.

Wout Bittremieux, Nicole E Avalon, Sydney P Thomas, Sarvar A Kakhkhorov, Alexander A Aksenov, Paulo Wender P Gomes, Christine M Aceves, Andrés Mauricio Caraballo-Rodríguez, Julia M Gauglitz, William H Gerwick, Tao Huan, Alan K Jarmusch, Rima F Kaddurah-Daouk, Kyo Bin Kang, Hyun Woo Kim, Todor Kondić, Helena Mannochio-Russo, Michael J Meehan, Alexey V Melnik, Louis-Felix Nothias, Claire O'Donovan, Morgan Panitchpakdi, Daniel Petras, Robin Schmid, Emma L Schymanski, Justin J J van der Hooft, Kelly C Weldon, Heejung Yang, Shipei Xing, Jasmine Zemlin, Mingxun Wang, Pieter C Dorrestein.

Despite the increasing availability of tandem mass spectrometry (MS/MS) community spectral libraries for untargeted metabolomics over the past decade, the majority of acquired MS/MS spectra remain uninterpreted. To further aid in interpreting unannotated spectra, we created a nearest neighbor suspect spectral library, consisting of 87,916 annotated MS/MS spectra derived from hundreds of millions of MS/MS spectra originating from published untargeted metabolomics experiments. Entries in this library, or "suspects," were derived from unannotated spectra that could be linked in a molecular network to an annotated spectrum. Annotations were propagated to unknowns based on structural relationships to reference molecules using MS/MS-based spectrum alignment. We demonstrate the broad relevance of the nearest neighbor suspect spectral library through representative examples of propagation-based annotation of acylcarnitines, bacterial and plant natural products, and drug metabolism. Our results also highlight how the library can help to better understand an Alzheimer's brain phenotype. The nearest neighbor suspect spectral library is openly available for download or for data analysis through the GNPS platform to help investigators hypothesize candidate structures for unknown MS/MS spectra in untargeted metabolomics data.

DOI: https://doi.org/10.1038/s41467-023-44035-y
Metabolites. 2023 Dec 06. pii: 1185. [Epub ahead of print]13(12):

Oleic Acid Metabolism in Response to Glucose in C. elegans.

Andre F C Vieira, Mark A Xatse, Sofi Y Murray, Carissa Perez Olsen.

  A key response to glucose stress is an increased production of unsaturated fatty acids to balance the increase in saturated fatty acids in the membrane. The C. elegans homolog of stearoyl-CoA desaturase, FAT-7, introduces the first double bond into saturated C18 fatty acids yielding oleic acid, and is a critical regulatory point for surviving cold and glucose stress. Here, we incorporated 13C stable isotopes into the diet of nematodes and quantified the 13C-labelled fatty acid using GC-MS and HPLC/MS-MS to track its metabolic response to various concentrations of glucose. Previous work has analyzed the membrane composition of C. elegans when responding to mild glucose stress and showed few alterations in the overall fatty acid composition in the membrane. Here, in nematodes exposed to higher concentrations of glucose, a specific reduction in oleic acid and linoleic acid was observed. Using time courses and stable isotope tracing, the response of fatty acid metabolism to increasing levels of glucose stress is characterized, revealing the funneling of monounsaturated fatty acids to preserve the abundance of polyunsaturated fatty acids. Taken together, higher levels of glucose unveil a specific reduction in oleic and linolenic acid in the metabolic rewiring required to survive glucose stress.

Keywords:  fatty acid metabolism; glucose; lipidomics; mass spectrometry; oleic acid; phospholipids; stable isotopes

DOI:  https://doi.org/10.3390/metabo13121185
FEBS Lett. 2023 Dec 23.

Membranes that make fat: roles of membrane lipids as acyl donors for triglyceride synthesis and organelle function.

Antonio D Barbosa, Symeon Siniossoglou.

  Triglycerides constitute an inert storage form for fatty acids deposited in lipid droplets, and are mobilized to provide metabolic energy or membrane building blocks. The biosynthesis of triglycerides is highly conserved within eukaryotes and normally involves the sequential esterification of activated fatty acids with a glycerol backbone. Some eukaryotes, however, can also use cellular membrane lipids as direct fatty acid donors for triglyceride synthesis. The biological significance of a pathway that generates triglycerides at the expense of organelle membranes has remained elusive. Here we review current knowledge on how cells use membrane lipids as fatty acid donors for triglyceride synthesis, and discuss the hypothesis that a primary function of this pathway is to regulate membrane lipid remodelling and organelle function.

Keywords:  Lipid droplet; endoplasmic reticulum; fat; membrane; nuclear membrane; phospholipid; triglyceride

DOI:  https://doi.org/10.1002/1873-3468.14793
Nat Commun. 2023 Dec 15. 14(1): 8365

Mapping protein states and interactions across the tree of life with co-fractionation mass spectrometry.

Michael A Skinnider, Mopelola O Akinlaja, Leonard J Foster.

We present CFdb, a harmonized resource of interaction proteomics data from 411 co-fractionation mass spectrometry (CF-MS) datasets spanning 21,703 fractions. Meta-analysis of this resource charts protein abundance, phosphorylation, and interactions throughout the tree of life, including a reference map of the human interactome. We show how large-scale CF-MS data can enhance analyses of individual CF-MS datasets, and exemplify this strategy by mapping the honey bee interactome.

DOI: https://doi.org/10.1038/s41467-023-44139-5
J Lipid Res. 2023 Dec 20. pii: S0022-2275(23)00165-7. [Epub ahead of print] 100492

Wide-scope targeted analysis of bioactive lipids in human plasma by LC/MS/MS.

Kohta Nakatani, Yoshihiro Izumi, Hironobu Umakoshi, Maki Yokomoto-Umakoshi, Tomoko Nakaji, Hiroki Kaneko, Hiroshi Nakao, Yoshihiro Ogawa, Kazutaka Ikeda, Takeshi Bamba.

  Quantitative information on blood metabolites can be used in developing advanced medical strategies such as early detection and prevention of disease. Monitoring bioactive lipids such as steroids, bile acids, and polyunsaturated fatty acid (PUFA) metabolites could be a valuable indicator of health status. However, a method for simultaneously measuring these bioactive lipids has not yet been developed. Here, we report a liquid chromatography tandem mass spectrometry (LC/MS/MS) method that can simultaneously measure 144 bioactive lipids, including steroids, bile acids, and PUFA metabolites, from human plasma, and a sample preparation method for these targets. Protein removal by methanol precipitation and purification of bioactive lipids by solid-phase extraction improved the recovery of the targeted compounds in human plasma samples, demonstrating the importance of sample preparation methods for a wide range of bioactive lipid analyses. Using the developed method, we studied the plasma from healthy human volunteers and confirmed the presence of bioactive lipid molecules associated with sex differences and circadian rhythms. The developed method of bioactive lipid analysis can be applied to health monitoring and disease biomarker discovery in precision medicine.

Keywords:  bile acid; comprehensive analysis; liquid chromatography; polyunsaturated fatty acid; solid-phase extraction; steroid; tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.jlr.2023.100492
Biology (Basel). 2023 Dec 12. pii: 1514. [Epub ahead of print]12(12):

Immunopeptidomics in the Era of Single-Cell Proteomics.

Rupert L Mayer, Karl Mechtler.

  Immunopeptidomics, as the analysis of antigen peptides being presented to the immune system via major histocompatibility complexes (MHC), is being seen as an imperative tool for identifying epitopes for vaccine development to treat cancer and viral and bacterial infections as well as parasites. The field has made tremendous strides over the last 25 years but currently still faces challenges in sensitivity and throughput for widespread applications in personalized medicine and large vaccine development studies. Cutting-edge technological advancements in sample preparation, liquid chromatography as well as mass spectrometry, and data analysis, however, are currently transforming the field. This perspective showcases how the advent of single-cell proteomics has accelerated this transformation of immunopeptidomics in recent years and will pave the way for even more sensitive and higher-throughput immunopeptidomics analyses.

Keywords:  HLA; MHC; antigen discovery; data-independent acquisition; immunopeptidomics; liquid chromatography; mass spectrometry; single-cell proteomics; vaccine development

DOI:  https://doi.org/10.3390/biology12121514
PLoS One. 2023 ;18(12): e0294498

Effect of statin treatment on metabolites, lipids and prostanoids in patients with Statin Associated Muscle Symptoms (SAMS).

Timothy J Garrett, Michelle A Puchowicz, Edwards A Park, Qingming Dong, Gregory Farage, Richard Childress, Joy Guingab, Claire L Simpson, Saunak Sen, Elizabeth C Brogdon, Logan M Buchanan, Rajendra Raghow, Marshall B Elam.

BACKGROUND: Between 5-10% of patients discontinue statin therapy due to statin-associated adverse reactions, primarily statin associated muscle symptoms (SAMS). The absence of a clear clinical phenotype or of biomarkers poses a challenge for diagnosis and management of SAMS. Similarly, our incomplete understanding of the pathogenesis of SAMS hinders the identification of treatments for SAMS. Metabolomics, the profiling of metabolites in biofluids, cells and tissues is an important tool for biomarker discovery and provides important insight into the origins of symptomatology. In order to better understand the pathophysiology of this common disorder and to identify biomarkers, we undertook comprehensive metabolomic and lipidomic profiling of plasma samples from patients with SAMS who were undergoing statin rechallenge as part of their clinical care.
METHODS AND FINDINGS: We report our findings in 67 patients, 28 with SAMS (cases) and 39 statin-tolerant controls. SAMS patients were studied during statin rechallenge and statin tolerant controls were studied while on statin. Plasma samples were analyzed using untargeted LC-MS metabolomics and lipidomics to detect differences between cases and controls. Differences in lipid species in plasma were observed between cases and controls. These included higher levels of linoleic acid containing phospholipids and lower ether lipids and sphingolipids. Reduced levels of acylcarnitines and altered amino acid profile (tryptophan, tyrosine, proline, arginine, and taurine) were observed in cases relative to controls. Pathway analysis identified significant increase of urea cycle metabolites and arginine and proline metabolites among cases along with downregulation of pathways mediating oxidation of branched chain fatty acids, carnitine synthesis, and transfer of acetyl groups into mitochondria.
CONCLUSIONS: The plasma metabolome of patients with SAMS exhibited reduced content of long chain fatty acids and increased levels of linoleic acid (18:2) in phospholipids, altered energy production pathways (β-oxidation, citric acid cycle and urea cycles) as well as reduced levels of carnitine, an essential mediator of mitochondrial energy production. Our findings support the hypothesis that alterations in pro-inflammatory lipids (arachidonic acid pathway) and impaired mitochondrial energy metabolism underlie the muscle symptoms of patients with statin associated muscle symptoms (SAMS).

DOI: https://doi.org/10.1371/journal.pone.0294498
bioRxiv. 2023 Dec 05. pii: 2023.12.04.569947. [Epub ahead of print]

FLiPPR: A Processor for Limited Proteolysis (LiP) Mass Spectrometry Datasets Built on FragPipe.

Edgar Manriquez-Sandoval, Joy Brewer, Gabriela Lule, Samanta Lopez, Stephen D Fried.

Here, we present FLiPPR, or FragPipe LiP (limited proteolysis) Processor, a tool that facilitates the analysis of data from limited proteolysis mass spectrometry (LiP-MS) experiments following primary search and quantification in FragPipe. LiP-MS has emerged as a method that can provide proteome-wide information on protein structure and has been applied to a range of biological and biophysical questions. Although LiP- MS can be carried out with standard laboratory reagents and mass spectrometers, analyzing the data can be slow and poses unique challenges compared to typical quantitative proteomics workflows. To address this, we leverage the fast, sensitive, and accurate search and label-free quantification algorithms in FragPipe and then process its output in FLiPPR. FLiPPR formalizes a specific data imputation heuristic that carefully uses missing data in LiP-MS experiments to report on the most significant structural changes. Moreover, FLiPPR introduces a new data merging scheme (from ions to cut-sites) and a protein-centric multiple hypothesis correction scheme, collectively enabling processed LiP-MS datasets to be more robust and less redundant. These improvements substantially strengthen statistical trends when previously published data are reanalyzed with the FragPipe/FLiPPR workflow. As a final feature, FLiPPR facilitates the collection of structural metadata to identify correlations between experiments and structural features. We hope that FLiPPR will lower the barrier for more users to adopt LiP-MS, standardize statistical procedures for LiP-MS data analysis, and systematize output to facilitate eventual larger-scale integration of LiP-MS data.

DOI: https://doi.org/10.1101/2023.12.04.569947
Mol Neurodegener. 2023 Dec 19. 18(1): 100

Untargeted serum metabolomics reveals novel metabolite associations and disruptions in amino acid and lipid metabolism in Parkinson's disease.

Kimberly C Paul, Keren Zhang, Douglas I Walker, Janet Sinsheimer, Yu Yu, Cynthia Kusters, Irish Del Rosario, Aline Duarte Folle, Adrienne M Keener, Jeff Bronstein, Dean P Jones, Beate Ritz.

BACKGROUND: Untargeted high-resolution metabolomic profiling provides simultaneous measurement of thousands of metabolites. Metabolic networks based on these data can help uncover disease-related perturbations across interconnected pathways.
OBJECTIVE: Identify metabolic disturbances associated with Parkinson's disease (PD) in two population-based studies using untargeted metabolomics.
METHODS: We performed a metabolome-wide association study (MWAS) of PD using serum-based untargeted metabolomics data derived from liquid chromatography with high-resolution mass spectrometry (LC-HRMS) using two distinct population-based case-control populations. We also combined our results with a previous publication of 34 metabolites linked to PD in a large-scale, untargeted MWAS to assess external validation.
RESULTS: LC-HRMS detected 4,762 metabolites for analysis (HILIC: 2716 metabolites; C18: 2046 metabolites). We identified 296 features associated with PD at FDR<0.05, 134 having a log2 fold change (FC) beyond ±0.5 (228 beyond ±0.25). Of these, 104 were independently associated with PD in both discovery and replication studies at p<0.05 (170 at p<0.10), while 27 were associated with levodopa-equivalent dose among the PD patients. Intriguingly, among the externally validated features were the microbial-related metabolites, p-cresol glucuronide (FC=2.52, 95% CI=1.67, 3.81, FDR=7.8e-04) and p-cresol sulfate. P-cresol glucuronide was also associated with motor symptoms among patients. Additional externally validated metabolites associated with PD include phenylacetyl-L-glutamine, trigonelline, kynurenine, biliverdin, and pantothenic acid. Novel associations include the anti-inflammatory metabolite itaconate (FC=0.79, 95% CI=0.73, 0.86; FDR=2.17E-06) and cysteine-S-sulfate (FC=1.56, 95% CI=1.39, 1.75; FDR=3.43E-11). Seventeen pathways were enriched, including several related to amino acid and lipid metabolism.
CONCLUSIONS: Our results revealed PD-associated metabolites, confirming several previous observations, including for p-cresol glucuronide, and newly implicating interesting metabolites, such as itaconate. Our data also suggests metabolic disturbances in amino acid and lipid metabolism and inflammatory processes in PD.

DOI: https://doi.org/10.1186/s13024-023-00694-5
Cancer Treat Res. 2023 ;190 3-24

RNA Modifications in Cancer Metabolism and Tumor Microenvironment.

Ying Qing, Dong Wu, Xiaolan Deng, Jianjun Chen, Rui Su.

  RNA modifications have recently been recognized as essential posttranscriptional regulators of gene expression in eukaryotes. Investigations over the past decade have revealed that RNA chemical modifications have profound effects on tumor initiation, progression, refractory, and recurrence. Tumor cells are notorious for their robust plasticity in response to the stressful microenvironment and undergo metabolic adaptations to sustain rapid cell proliferation, which is termed as metabolic reprogramming. Meanwhile, cancer-associated metabolic reprogramming leads to substantial alterations of intracellular and extracellular metabolites, which further reshapes the tumor microenvironment (TME). Moreover, cancer cells compete with tumor-infiltrating immune cells for the limited nutrients to maintain their proliferation and function in the TME. In this chapter, we review recent interesting findings on the engagement of epitranscriptomic pathways, especially the ones associated with N6-methyladenosine (m6A), in the regulation of cancer metabolism and the surrounding microenvironment. We also discuss the promising therapeutic approaches targeting RNA modifications for anti-tumor therapy.

Keywords:  Aerobic glycolysis; Amino acid metabolism; Cancer metabolism; Immune cells; Immunotherapy; Lipid metabolism; RNA modification; Tumor microenvironment; m6A

DOI:  https://doi.org/10.1007/978-3-031-45654-1_1
Anal Sci. 2023 Dec 19.

Comparison of 3-nitrophenylhydrazine, O-benzyl hydroxylamine, and 2-picolylamine derivatizations for analysis of short-chain fatty acids through liquid chromatography coupled with tandem mass spectrometry.

Ryosuke Nagatomo, Aoi Ichikawa, Haruki Kaneko, Koichi Inoue.

  Short-chain fatty acids (SCFAs) are metabolites derived from gut microbiota and implicated in host homeostasis. Hence, the profiling SCFAs from biological samples plays an important role in revealing the interaction between gut microbiota and pathogens. Previous studies, liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with various derivatization strategies have been performed to obtain the SCFA profiles from biological samples. However, it is poor evidence to compare these derivatization regents and conditions. Thus, we present the evaluation of three major derivatization reagents, namely 3-nitrophenylhydrazine (3-NPH), O-benzylhydroxylamine (O-BHA), and 2-picolylamine (2-PA), for the analysis of eight SCFAs classified as C2-C5 isomers using LC-MS/MS. First, in a reversed-phase LC separation, 3-NPH showed good retention capacity. Although O-BHA derivatization showed higher sensitivity and good retention capacity than 2-PA, only 2-PA derivatization could successfully separate eight SCFAs. The matrix effects in human serum ranged 77.1-99.0% (RSD ≤ 3.4%, n = 6) for 3-NPH derivatives, 91.0-94.6% (RSD ≤ 5.4%, n = 6) for O-BHA derivatives, 81.6-99.5% (RSD ≤ 8.0%, n = 6) for 2-PA derivatives. These compared results showed each characteristic of 3-NPH, O-BHA, and 2-PA for SCFA derivatization based on LC-MS/MS approaches.

Keywords:  2-Picolylamine; 3-Nitrophenylhydrazine; O-benzylhydroxylamine; Short-chain fatty acids

DOI:  https://doi.org/10.1007/s44211-023-00474-7
Cell Metab. 2023 Dec 14. pii: S1550-4131(23)00449-7. [Epub ahead of print]

Mitochondrial fatty acid synthesis is an emergent central regulator of mammalian oxidative metabolism.

Riley J Wedan, Jacob Z Longenecker, Sara M Nowinski.

  Contrary to their well-known functions in nutrient breakdown, mitochondria are also important biosynthetic hubs and express an evolutionarily conserved mitochondrial fatty acid synthesis (mtFAS) pathway. mtFAS builds lipoic acid and longer saturated fatty acids, but its exact products, their ultimate destination in cells, and the cellular significance of the pathway are all active research questions. Moreover, why mitochondria need mtFAS despite their well-defined ability to import fatty acids is still unclear. The identification of patients with inborn errors of metabolism in mtFAS genes has sparked fresh research interest in the pathway. New mammalian models have provided insights into how mtFAS coordinates many aspects of oxidative mitochondrial metabolism and raise questions about its role in diseases such as obesity, diabetes, and heart failure. In this review, we discuss the products of mtFAS, their function, and the consequences of mtFAS impairment across models and in metabolic disease.

Keywords:  fatty acids; inborn errors of metabolism; lipid metabolism; lipids; mitochondria; mitochondrial fatty acid synthesis; mouse models; mtFAS

DOI:  https://doi.org/10.1016/j.cmet.2023.11.017
J Biochem. 2023 Dec 15. pii: mvad109. [Epub ahead of print]

Exploring protein lipidation by mass spectrometry-based proteomics.

Kazuya Tsumagari, Yosuke Isobe, Koshi Imami, Makoto Arita.

  Protein lipidation is a common co- or post-translational modification that plays a crucial role in regulating the localization, interaction, and function of cellular proteins. Dysregulation of lipid modifications can lead to various diseases, including cancer, neurodegenerative diseases, and infectious diseases. Therefore, the identification of proteins undergoing lipidation and their lipidation sites should provide insights into many aspects of lipid biology, as well as providing potential targets for therapeutic strategies. Bottom-up proteomics using liquid chromatography/tandem mass spectrometry is a powerful technique for the global analysis of protein lipidation. Here, we review proteomic methods for profiling protein lipidation, focusing on the two major approaches: the use of chemical probes, such as lipid alkyne probes, and the use of enrichment techniques for endogenous lipid-modified peptides. The challenges facing these methods and the prospects for developing them further to achieve a comprehensive analysis of lipid modifications are discussed.

Keywords:  Chemical probe; Lipidation; Mass spectrometry; Post-translational modification; Proteomics

DOI:  https://doi.org/10.1093/jb/mvad109
Biomed Chromatogr. 2023 Dec 22. e5817

Integration of lipidomics and metabolomics reveals plasma and urinary profiles associated with pediatric Mycoplasma pneumoniae infections and its severity.

Hui Chen, Jia-Bin Chen, Li-Na Du, Hai-Xia Yuan, Jin-Jun Shan, Shou-Chuan Wang, Jin Ye, Li-Li Lin.

  Mycoplasma pneumoniae is a significant contributor to lower respiratory infections in children. However, the lipidomics and metabolics bases of childhood M. pneumoniae infections remain unclear. In this study, lipidomics and metabolomics analyses were conducted using UHPLC-LTQ-Orbitrap XL mass spectrometry and gas chromatography-triple quadrupole mass spectrometry on plasma (n = 65) and urine (n = 65) samples. MS-DIAL software, in combination with LipidBlast and Fiehn BinBase DB, identified 163 lipids and 104 metabolites in plasma samples, as well as 208 metabolites in urine samples. Perturbed lipid species (adjusted p < 0.05) were observed, including lysophosphatidylethanolamines, phosphatidylinositols, phosphatidylcholines, phosphatidylethanol amines, and triglycerides. Additionally, differential metabolites (adjusted p < 0.05) exhibited associations with amino acid metabolism, nucleotide metabolism, and energy metabolism. Thirteen plasma metabolites, namely l-hydroxyproline, 3-phosphoglycerate, citric acid, creatine, inosine, ribitol, α tocopherol, cholesterol, cystine, serine, uric acid, tagatose, and glycine, showed significant associations with disease severity (p < 0.05) and exhibited distinct separation patterns in M. pneumoniae-infected bronchitis and pneumonia, with an area under the curve of 0.927. Nine of them exhibited either positive or negative correlations with neutrophil or lymphocyte percentages. These findings indicated significant systemic metabolic shifts in childhood M. pneumoniae infections, offering valuable insights into the associated metabolic alterations and their relationship with disease severity.

Keywords:  Mycoplasma pneumoniae infections; disease severity; lipidomics; metabolomics; plasma; urine

DOI:  https://doi.org/10.1002/bmc.5817
Int J Mol Sci. 2023 Dec 18. pii: 17633. [Epub ahead of print]24(24):

Role of Glycolytic and Glutamine Metabolism Reprogramming on the Proliferation, Invasion, and Apoptosis Resistance through Modulation of Signaling Pathways in Glioblastoma.

Cristina Trejo-Solis, Daniela Silva-Adaya, Norma Serrano-García, Roxana Magaña-Maldonado, Dolores Jimenez-Farfan, Elizabeth Ferreira-Guerrero, Arturo Cruz-Salgado, Rosa Angelica Castillo-Rodriguez.

  Glioma cells exhibit genetic and metabolic alterations that affect the deregulation of several cellular signal transduction pathways, including those related to glucose metabolism. Moreover, oncogenic signaling pathways induce the expression of metabolic genes, increasing the metabolic enzyme activities and thus the critical biosynthetic pathways to generate nucleotides, amino acids, and fatty acids, which provide energy and metabolic intermediates that are essential to accomplish the biosynthetic needs of glioma cells. In this review, we aim to explore how dysregulated metabolic enzymes and their metabolites from primary metabolism pathways in glioblastoma (GBM) such as glycolysis and glutaminolysis modulate anabolic and catabolic metabolic pathways as well as pro-oncogenic signaling and contribute to the formation, survival, growth, and malignancy of glioma cells. Also, we discuss promising therapeutic strategies by targeting the key players in metabolic regulation. Therefore, the knowledge of metabolic reprogramming is necessary to fully understand the biology of malignant gliomas to improve patient survival significantly.

Keywords:  glioma; glucose; glutamine; metabolism; oncogenic pathways

DOI:  https://doi.org/10.3390/ijms242417633
Biochim Biophys Acta Mol Cell Res. 2023 Dec 15. pii: S0167-4889(23)00226-4. [Epub ahead of print]1871(2): 119653

Protein kinase D (PKD) on the crossroad of lipid absorption, synthesis and utilization.

Magdalena Wit, Andrei Belykh, Grzegorz Sumara.

  Inappropriate lipid levels in the blood, as well as its content and composition in different organs, underlie multiple metabolic disorders including obesity, non-alcoholic fatty liver disease, type 2 diabetes, and atherosclerosis. Multiple processes contribute to the complex metabolism of triglycerides (TGs), fatty acids (FAs), and other lipid species. These consist of digestion and absorption of dietary lipids, de novo FAs synthesis (lipogenesis), uptake of TGs and FAs by peripheral tissues, TGs storage in the intracellular depots as well as lipid utilization for β-oxidation and their conversion to lipid-derivatives. A majority of the enzymatic reactions linked to lipogenesis, TGs synthesis, lipid absorption, and transport are happening at the endoplasmic reticulum, while β-oxidation takes place in mitochondria and peroxisomes. The Golgi apparatus is a central sorting, protein- and lipid-modifying organelle and hence is involved in lipid metabolism as well. However, the impact of the processes taking part in the Golgi apparatus are often overseen. The protein kinase D (PKD) family (composed of three members, PKD1, 2, and 3) is the master regulator of Golgi dynamics. PKDs are also a sensor of different lipid species in distinct cellular compartments. In this review, we discuss the roles of PKD family members in the regulation of lipid metabolism including the processes executed by PKDs at the Golgi apparatus. We also discuss the role of PKDs-dependent signaling in different cellular compartments and organs in the context of the development of metabolic disorders.

Keywords:  Golgi apparatus; Lipid metabolism; Metabolic disease; PKD; Protein kinase D

DOI:  https://doi.org/10.1016/j.bbamcr.2023.119653
Curr Issues Mol Biol. 2023 Nov 22. 45(12): 9354-9367

Application of Normal-Phase Silica Column in Hydrophilic Interaction Liquid Chromatography Mode for Simultaneous Determination of Underivatized Amino Acids from Human Serum Samples via Liquid Chromatography-Tandem Mass Spectrometry.

Krisztina Németh, Ildikó Szatmári, Viktória Tőkési, Pál Tamás Szabó.

  In neonatal screening, amino acids have a significant diagnostic role. Determination of their values may identify abnormal conditions. Early diagnosis and continuous monitoring of amino acid disorders results in a better disease outcome. An easy and simple LC-MS/MS method was developed for the quantitation of underivatized amino acids. Amino acids were separated using a normal-phase HPLC column having a totally porous silica stationary phase and using classical reversed-phase eluents. Mass spectrometry in multiple reaction monitoring mode was used for the analysis, providing high selectivity and sensitivity. A standard addition calibration model was applied for quantitation using only one isotope-labeled internal standard for all amino acids. Five calibration points were used for quantitation, and the method was successfully validated. The slopes of the calibration curves of the individual amino acids in parallel measurements were found to be similar. Since the measured slopes were reproducible, one serum sample could represent every series of serum samples of a given day. The method was tested on human serum samples and adequate results were obtained. This new method can be easily applied in clinical laboratories.

Keywords:  LC-MS/MS; amino acids; normal-phase chromatography; standard addition calibration

DOI:  https://doi.org/10.3390/cimb45120586
Biomed Pharmacother. 2023 Dec 20. pii: S0753-3322(23)01828-0. [Epub ahead of print]170 116030

Effect of metabolic reprogramming on the immune microenvironment in gastric cancer.

Zhengye Shang, Zhiyuan Ma, Enqin Wu, Xingzhao Chen, Biguang Tuo, Taolang Li, Xuemei Liu.

  Gastric cancer (GC) is a malignant tumor of the gastrointestinal tract with a high mortality rate worldwide, a low early detection rate and a poor prognosis. The rise of metabolomics has facilitated the early detection and treatment of GC. Metabolism in the GC tumor microenvironment (TME) mainly includes glucose metabolism, lipid metabolism and amino acid metabolism, which provide energy and nutrients for GC cell proliferation and migration. Abnormal tumor metabolism can influence tumor progression by regulating the functions of immune cells and immune molecules in the TME, thereby contributing to tumor immune escape. Thus, in this review, we summarize the impact of metabolism on the TME during GC progression. We also propose novel strategies to modulate antitumor immune responses by targeting metabolism.

Keywords:  Cancer immunity; Gastric cancer; Immune microenvironment; Metabolism; TIME

DOI:  https://doi.org/10.1016/j.biopha.2023.116030
Biomedicines. 2023 Nov 30. pii: 3186. [Epub ahead of print]11(12):

An Overview on Lipid Droplets Accumulation as Novel Target for Acute Myeloid Leukemia Therapy.

Clelia Nisticò, Emanuela Chiarella.

  Metabolic reprogramming is a key alteration in tumorigenesis. In cancer cells, changes in metabolic fluxes are required to cope with large demands on ATP, NADPH, and NADH, as well as carbon skeletons. In particular, dysregulation in lipid metabolism ensures a great energy source for the cells and sustains cell membrane biogenesis and signaling molecules, which are necessary for tumor progression. Increased lipid uptake and synthesis results in intracellular lipid accumulation as lipid droplets (LDs), which in recent years have been considered hallmarks of malignancies. Here, we review current evidence implicating the biogenesis, composition, and functions of lipid droplets in acute myeloid leukemia (AML). This is an aggressive hematological neoplasm originating from the abnormal expansion of myeloid progenitor cells in bone marrow and blood and can be fatal within a few months without treatment. LD accumulation positively correlates with a poor prognosis in AML since it involves the activation of oncogenic signaling pathways and cross-talk between the tumor microenvironment and leukemic cells. Targeting altered LD production could represent a potential therapeutic strategy in AML. From this perspective, we discuss the main inhibitors tested in in vitro AML cell models to block LD formation, which is often associated with leukemia aggressiveness and which may find clinical application in the future.

Keywords:  PPARγ; acute myeloid leukemia (AML); chemotherapy resistance; lipid droplets (LDs); lipid metabolism

DOI:  https://doi.org/10.3390/biomedicines11123186
J Chromatogr A. 2023 Dec 10. pii: S0021-9673(23)00777-X. [Epub ahead of print]1714 464552

Development of a single mobile phase for LC-IM-MS-based discovery lipidomics and metabolic phenotyping: Application to methapyrilene hepatotoxicity in the rat.

Ian D Wilson, Corey Broeckling, Lee A Gethings, Nyasha C Munjoma, Robert Trengove, Paul D Rainville, Steven K Lai, Giorgis Isaac, Robert S Plumb.

  The untargeted global profiling of endogenous metabolites and lipids has the potential to increase knowledge and understanding in many areas of biology. LC-MS/MS is a key technology for such analyses however, several different LC methodologies, using different mobile phase compositions, are required to cover the diversity in polarity and analyte structure encountered in biological samples. Most notably many lipid screening methods make use of isopropanol (IPA) as a major component of mobile phases employed for comprehensive lipidomic profiling. In order to increase laboratory efficiency, and minimize opportunities for errors, a suite of methods, based on a single acetonitrile (ACN)-aqueous buffer mobile phase combination, has been developed. This mobile phase can be used for hydrophobic interaction liquid chromatography on an amide stationary phase (for polar analytes), reversed-phase (RP) LC analysis on a C8 stationary phase (for moderately polar-non-polar compounds) and RPLC using a CSH phenyl-hexyl bonded column (for lipids). All of these sub 10 minute separations had good throughput and reproducibility with CV's of analyte response <25 % whilst eliminating the need for complex mobile phase preparation and the use of IPA as an organic modifier for lipidomics. Advantages of removing IPA and replacing it with the ACN-based method were a 58 % increase in peak capacity for lipids, with improved resolution for the di- and triglycerides and cholesterol esters compared to current methods. Compared to the IPA-containing solvent system the ACN-based mobile phase also resulted in a 61 % increase in lipid feature detection. The utility of this "universal" mobile phase approach was demonstrated by its application to a rat toxicology study investigating the consequences of methapyrilene administration through on the endogenous metabolite profiles of plasma and urine. Methapyrilene and its metabolites were also profiled in these samples.

Keywords:  Chromatography; Efficiency; LC-MS/MS; Lipidomics; Metabolomics; Mobile phase

DOI:  https://doi.org/10.1016/j.chroma.2023.464552
Anal Bioanal Chem. 2023 Dec 23.

Efficient lipidomic approach for the discovery of lipid ligands for immune receptors by combining LC-HRMS/MS analysis with fractionation and reporter cell assay.

Noriyuki Tomiyasu, Masatomo Takahashi, Kenji Toyonaga, Sho Yamasaki, Takeshi Bamba, Yoshihiro Izumi.

  C-type lectin receptors (CLRs), which are pattern recognition receptors responsible for triggering innate immune responses, recognize damaged self-components and immunostimulatory lipids from pathogenic bacteria; however, several of their ligands remain unknown. Here, we propose a new analytical platform combining liquid chromatography-high-resolution tandem mass spectrometry with microfractionation capability (LC-FRC-HRMS/MS) and a reporter cell assay for sensitive activity measurements to develop an efficient methodology for searching for lipid ligands of CLR from microbial trace samples (crude cell extracts of approximately 5 mg dry cell/mL). We also developed an in-house lipidomic library containing accurate mass and fragmentation patterns of more than 10,000 lipid molecules predicted in silico for 90 lipid subclasses and 35 acyl side chain fatty acids. Using the developed LC-FRC-HRMS/MS system, the lipid extracts of Helicobacter pylori were separated and fractionated, and HRMS and HRMS/MS spectra were obtained simultaneously. The fractionated lipid extract samples in 96-well plates were thereafter subjected to reporter cell assays using nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing mouse or human macrophage-inducible C-type lectin (Mincle). A total of 102 lipid molecules from all fractions were annotated using an in-house lipidomic library. Furthermore, a fraction that exhibited significant activity in the NFAT-GFP reporter cell assay contained α-cholesteryl glucoside, a type of glycolipid, which was successfully identified as a lipid ligand molecule for Mincle. Our analytical platform has the potential to be a useful tool for efficient discovery of lipid ligands for immunoreceptors.

Keywords:  Immune receptor; LC-HRMS/MS; Lipid ligand; Lipidomics; Microfractionation; Reporter cell assay

DOI:  https://doi.org/10.1007/s00216-023-05111-w
Int J Mol Sci. 2023 Dec 13. pii: 17422. [Epub ahead of print]24(24):

The Role of Tumor Metabolic Reprogramming in Tumor Immunity.

Xianhong Zhang, Weiguo Song, Yue Gao, Yu Zhang, Yuqi Zhao, Shuailin Hao, Ting Ni.

  The occurrence and development of tumors require the metabolic reprogramming of cancer cells, namely the alteration of flux in an autonomous manner via various metabolic pathways to meet increased bioenergetic and biosynthetic demands. Tumor cells consume large quantities of nutrients and produce related metabolites via their metabolism; this leads to the remodeling of the tumor microenvironment (TME) to better support tumor growth. During TME remodeling, the immune cell metabolism and antitumor immune activity are affected. This further leads to the escape of tumor cells from immune surveillance and therefore to abnormal proliferation. This review summarizes the regulatory functions associated with the abnormal biosynthesis and activity of metabolic signaling molecules during the process of tumor metabolic reprogramming. In addition, we provide a comprehensive description of the competition between immune cells and tumor cells for nutrients in the TME, as well as the metabolites required for tumor metabolism, the metabolic signaling pathways involved, and the functionality of the immune cells. Finally, we summarize current research targeted at the development of tumor immunotherapy. We aim to provide new concepts for future investigations of the mechanisms underlying the metabolic reprogramming of tumors and explore the association of these mechanisms with tumor immunity.

Keywords:  immune cells; tumor immunity; tumor metabolism; tumor microenvironment; tumor therapy

DOI:  https://doi.org/10.3390/ijms242417422
J Proteome Res. 2023 Dec 19.

Sulfoproteomics Workflow with Precursor Ion Accurate Mass Shift Analysis Reveals Novel Tyrosine Sulfoproteins in the Golgi.

Hye Kyong Kweon, Andy T Kong, Katherine E Hersberger, Shijiao Huang, Alexey I Nesvizhskii, Yanzhuang Wang, Kristina Hakansson, Philip C Andrews.

  Tyrosine sulfation in the Golgi of secreted and membrane proteins is an important post-translational modification (PTM). However, its labile nature has limited analysis by mass spectrometry (MS), a major reason why no sulfoproteome studies have been previously reported. Here, we show that a phosphoproteomics experimental workflow, which includes serial enrichment followed by high resolution, high mass accuracy MS, and tandem MS (MS/MS) analysis, enables sulfopeptide coenrichment and identification via accurate precursor ion mass shift open MSFragger database search. This approach, supported by manual validation, allows the confident identification of sulfotyrosine-containing peptides in the presence of high levels of phosphorylated peptides, thus enabling these two sterically and ionically similar isobaric PTMs to be distinguished and annotated in a single proteomic analysis. We applied this approach to isolated interphase and mitotic rat liver Golgi membranes and identified 67 tyrosine sulfopeptides, corresponding to 26 different proteins. This work discovered 23 new sulfoproteins with functions related to, for example, Ca2+-binding, glycan biosynthesis, and exocytosis. In addition, we report the first preliminary evidence for crosstalk between sulfation and phosphorylation in the Golgi, with implications for functional control.

Keywords:  DeltaMass; Golgi apparatus; MSFragger; PTM crosstalk; accurate mass; high-resolution mass spectrometry; open database search; orbitrap; sulfopeptide enrichment; sulfoproteomics; tyrosine sulfation

DOI:  https://doi.org/10.1021/acs.jproteome.3c00323
Nat Commun. 2023 Dec 18. 14(1): 8392

Proteomics-driven noninvasive screening of circulating serum protein panels for the early diagnosis of hepatocellular carcinoma.

Xiaohua Xing, Linsheng Cai, Jiahe Ouyang, Fei Wang, Zongman Li, Mingxin Liu, Yingchao Wang, Yang Zhou, En Hu, Changli Huang, Liming Wu, Jingfeng Liu, Xiaolong Liu.

Early diagnosis of hepatocellular carcinoma (HCC) lacks highly sensitive and specific protein biomarkers. Here, we describe a staged mass spectrometry (MS)-based discovery-verification-validation proteomics workflow to explore serum proteomic biomarkers for HCC early diagnosis in 1002 individuals. Machine learning model determined as P4 panel (HABP2, CD163, AFP and PIVKA-II) clearly distinguish HCC from liver cirrhosis (LC, AUC 0.979, sensitivity 0.925, specificity 0.915) and healthy individuals (HC, AUC 0.992, sensitivity 0.975, specificity 1.000) in an independent validation cohort, outperforming existing clinical prediction strategies. Furthermore, the P4 panel can accurately predict LC to HCC conversion (AUC 0.890, sensitivity 0.909, specificity 0.877) with predicting HCC at a median of 11.4 months prior to imaging in prospective external validation cohorts (No.: Keshen 2018_005_02 and NCT03588442). These results suggest that proteomics-driven serum biomarker discovery provides a valuable reference for the liquid biopsy, and has great potential to improve early diagnosis of HCC.

DOI: https://doi.org/10.1038/s41467-023-44255-2
J Vis Exp. 2023 Dec 01.

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry.

Jimmy Sung-Hei Tse, Ying-Hon Sze, Jimmy Ka-Wai Cheung, King-Kit Li, Thomas Chuen Lam.

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.

DOI: https://doi.org/10.3791/64617