The Circulating Proteome─Technological Developments, Current Challenges, and Future Trends

Journal of Proteome Research, Год журнала: 2024, Номер unknown

Опубликована: Окт. 31, 2024

Recent improvements in proteomics technologies have fundamentally altered our capacities to characterize human biology. There is an ever-growing interest using these novel methods for studying the circulating proteome, as blood offers accessible window into health. However, every methodological innovation and analytical progress calls reassessing existing approaches routines ensure that new data will add value greater biomedical research community avoid previous errors. As representatives of HUPO's Human Plasma Proteome Project (HPPP), we present 2024 survey current community, including latest build PeptideAtlas now comprises 4608 proteins detected 113 sets. We then discuss updates established methods, emerging technologies, investigations proteoforms, protein networks, extracellualr vesicles, antibodies microsamples. Finally, provide a prospective view tools studies proteins.

Язык: Английский

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Comprehensive Phenotyping of Extracellular Vesicles in Plasma of Healthy Humans – Insights Into Cellular Origin and Biological Variation

Journal of Extracellular Vesicles, Год журнала: 2025, Номер 14(1)

Опубликована: Янв. 1, 2025

ABSTRACT Despite immense interest in biomarker applications of extracellular vesicles (EVs) from blood, our understanding circulating EVs under physiological conditions healthy humans remains limited. Using imaging and multiplex bead‐based flow cytometry, we comprehensively quantified with respect to their cellular origin a large cohort blood donors. We assessed coefficients variations characterize biological variation explored demographic, clinical, lifestyle factors contributing observed variation. Cell‐specific EV subsets show wide range concentrations that do not correlate cell‐of‐origin suggesting steady‐state subset are regulated by complex mechanisms, which differ even for the same cell type. Interestingly, tetraspanin+ largely originate platelets lesser extent lymphocytes. Principal component analysis (PCA) association analyses demonstrate high inter‐individual across humans, only partly explained influence sex, menopausal status, age smoking on specific and/or subsets. No global subject's was detected. Our findings provide first comprehensive, quantitative data towards cell‐origin atlas plasma EVs, important implications clinical use as biomarkers.

Язык: Английский

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Effect of the 35 nm and 70 nm Size Exclusion Chromatography (SEC) Column and Plasma Storage Time on Separated Extracellular Vesicles

Current Issues in Molecular Biology, Год журнала: 2024, Номер 46(5), С. 4337 - 4357

Опубликована: Май 6, 2024

The technical difficulty of separating extracellular vesicles (EVs) from plasma proteins in human blood presents a significant hurdle EV research, particularly during nano ultra-high-performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) analysis, where detecting "vesicular" among abundant is challenging. Standardisation pressing issue prompting collaborative global efforts to address it. While the MISEV guidelines offer valuable recommendations, unanswered questions remain, regarding sample storage. We compared size exclusion chromatography (SEC) columns with pore sizes 35 nm and 70 identify fractions minimal contaminating highest concentration small EVs (sEVs). Following column selection, we explored potential differences quality quantity sEVs isolated platelet-free (PFP) after long-term storage at -80 °C (>2.5 years) freshly drawn blood. Our methodologically rigorous study indicates that prolonged storage, under correct processing conditions, does not compromise sEV quality. Both effectively vesicles, exhibiting higher abundance proteins. propose relatively rapid moderately efficient protocol for obtaining comparatively pure fraction plasma, facilitating clinical trials.

Язык: Английский

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A genome-wide association study identified PRKCB as a causal gene and therapeutic target for Mycobacterium avium complex disease

Cell Reports Medicine, Год журнала: 2025, Номер unknown, С. 101923 - 101923

Опубликована: Янв. 1, 2025

Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is a chronic progressive lung that increasing in incidence. Host genetic factors are associated with NTM-PD susceptibility. However, the heritability of not well understood. Here, we perform two-stage genome-wide association study (GWAS) and discover susceptibility locus at 16p21 NTM-PD, especially Mycobacterium avium complex (MAC) disease. As lead variant, rs194800 C allele augments protein kinase beta (PRKCB) gene expression associates severer NTM-PD. The functional studies show PRKCB exacerbates M. infection promotes intracellular survival macrophages by inhibiting phagosomal acidification. Mechanistically, interacts subunit G vacuolar-H+-ATPase (V-ATPase) vacuolar sorting-associated 16 homolog (VPS16), blocking fusion between lysosomes phagosomes. inhibitor has therapeutic potential against infection. These findings provide insights into etiology highlight as an attractive target for host-directed therapy MAC

Язык: Английский

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Extracellular vesicles as a source of biomarkers in hematological malignancies: looking toward clinical appilcations

Expert Review of Molecular Diagnostics, Год журнала: 2025, Номер unknown

Опубликована: Апрель 3, 2025

Extracellular vesicles are membranous particles released by cells in physiological and pathological conditions. Their cargo is heterogeneous since it includes different biomolecules such as nucleic acids proteins. Oncogenic alterations affect the composition of extracellular model their content during cancer evolution. This review provides an overview studies focused on source biomarkers hematological malignancies. A special insight into vesicles-derived tools for evaluating prognosis malignancies response to treatment given. a valuable However, translation from bench bedside challenged lack standardization preanalytical variables experimental workflow. The release standard operating procedures validation large cohort patients will help exploiting potential clinical setting.

Язык: Английский

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Novel PPT+SEC Workflow for High-Sensitivity Extracellular Vesicle Proteomics from Cell Media

Journal of Proteome Research, Год журнала: 2025, Номер unknown

Опубликована: Май 2, 2025

Size exclusion chromatography (SEC) is a well-established method for the isolation of extracellular vesicles (EVs), but large elution volumes necessitate concentration step prior to proteomics analysis. This can lead significant EV loss. Here we report an approach that enables EVs into just 80 μL, which directly compatible with analysis without need concentration. were characterized by transmission electron microscopy, Western blot, and nanoparticle tracking analysis, all confirmed presence small EVs. Proteomics was performed benchmarked against those isolated using automated UHPLC-SEC platform. The novel workflow identified more proteins markers, including 96 100 top exosomal from ExoCarta database, compared 91 samples UHPLC-SEC. When applied pancreatic cancer cell lines, demonstrated higher sensitivity previously reported markers cancer.

Язык: Английский

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Rapid and high-yield recovery of plasma-derived extracellular vesicles using modified chromatography with soluble protein depletion for biomarker discovery

Cell Communication and Signaling, Год журнала: 2025, Номер 23(1)

Опубликована: Май 30, 2025

Extracellular vesicles (EVs) are critical mediators of intercellular communication by transferring proteins, lipid and nucleic acids between cells. EVs in biofluids, particularly blood, have gathered significant interest as potential biomarkers for disease diagnosis. However, isolating from blood poses a challenge due to the high concentration plasma which obscure detection low abundant EV-associated proteins. Here, we optimized simplified efficient method plasma-derived combining size exclusion chromatography (SEC) with flow-through using Capto Core 700 beads. A brief incubation SEC-derived EV fractions beads (qEV + CC) enabled us isolate intact, high-purity reduced soluble protein contamination. As comparison, MagReSyn-based was not compatible elution intact after purification showed contamination Data-independent acquisition-based liquid chromatography-mass spectrometry isolated plasma-EVs qEV CC approach identified over 1,000 including an increased presence brain derived proteins markers linked neurodegenerative diseases, such amyloid precursor apolipoprotein E. These findings were further validated super-resolution microscopy at single resolution. Bioinformatic pathway network analyses revealed enrichment pathways involved RNA processing, cell adhesion synaptic function, highlighting molecules broad biomarker discovery. Our present EVs, providing valuable tool advancing EV-based development.

Язык: Английский

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A fast and sensitive size‐exclusion chromatography method for plasma extracellular vesicle proteomic analysis

PROTEOMICS, Год журнала: 2024, Номер 24(16)

Опубликована: Июнь 19, 2024

Abstract Extracellular vesicles (EVs) carry diverse biomolecules derived from their parental cells, making components excellent biomarker candidates. However, purifying EVs is a major hurdle in discovery since current methods require large amounts of samples, are time‐consuming and typically have poor reproducibility. Here we describe simple, fast, sensitive EV fractionation method using size exclusion chromatography (SEC) on fast protein liquid (FPLC) system. Our uses Superose 6 Increase 5/150, which has bed volume 2.9 mL. The FPLC system small column enable reproducible separation only 50 µL human plasma 15 min. To demonstrate the utility our method, used longitudinal samples group individuals who underwent intense exercise. A total 838 proteins were identified, which, 261 previously characterized as proteins, including classical markers, such cluster differentiation (CD)9 CD81. Quantitative analysis showed low technical variability with correlation coefficients greater than 0.9 between replicates. captured differences relevant involved response to physical activity. enables variability, will facilitate studies clinical cohorts.

Язык: Английский

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Zika Virus in Extracellular Vesicles: Insights from Integrated Proteomic and Metabolomic Dependent Regulation of B Cell and PI3K/AKT/mTOR Signaling Pathway

Опубликована: Авг. 12, 2024

Background; Cell-released extracellular vesicles (EVs) acting as 'metabolically and proteolytically active machines,' show potential in metabolomic proteomic analysis of serum EVs. Despite diverse challenges, post-isolation omics characterization EVs offers crucial insights for effective analysis; (2) Methods: The research, involved children with Congenital Zika Syndrome, utilizing mass spectrometry proteomics GC-MS metabolite identification. Vesicles were isolated using Izon qEV columns, quantified, characterized by NTA TEM. Data employed Cytoescape/String MetaboAnalyst, revealing variations metabolic profiles among groups through PCA volcano plots. Proteins Metabolite set enrichment provided biologically meaningful patterns to enriched metabolites; (3) Results: Using molecular exclusion chromatography, the characterized, size variations. Protein identified 13 significantly altered proteins, including upregulated (e.g., AOM8Q6 - IGLC7) downregulated Q8TD86 CALML6) ones. indicated involvement PI3K-AKT-mTOR pathway suggested a role Angiotensin inhibition CZS+. Upstream mTOR, Akt is central signaling molecule PI3K plays critical roles brain development well synaptic plasticity important Virus. study provides into mechanisms associated CZS; (4) Conclusions: pinpointed valuable possible biomarkers, specifically proteins metabolites, virus (ZIKV) infection. It stresses necessity broader investigations advanced techniques uncover targets, potentially advancing pharmacological strategies.

Язык: Английский

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Pancreatic β-cells package double C2-like domain beta protein into extracellular vesicles via tandem C2 domains

Frontiers in Endocrinology, Год журнала: 2024, Номер 15

Опубликована: Окт. 21, 2024

Introduction Double C2-like domain beta (DOC2B) is a vesicle priming protein critical for glucose-stimulated insulin secretion in β-cells. Individuals with type 1 diabetes (T1D) have lower levels of DOC2B their residual functional β-cell mass and platelets, phenotype also observed mouse model T1D. Thus, could provide important information on dys(function). Objective Our objective was to evaluate the secretome In addition soluble extracellular protein, we assessed localized within membrane-delimited nanoparticles – vesicles (EVs). Moreover, rat clonal β-cells, probed domains required sorting into EVs. Method Using Single Extracellular VEsicle Nanoscopy, quantified EVs derived from β-cells (human EndoC-βH1, INS-1 832/13, MIN6); two other cell types known regulate glucose homeostasis functionally utilize (skeletal muscle myotube L6-GLUT4myc human neuronal-like SH-SY5Y cells); islets sourced individuals no (ND). ND plasma, islets, lines were isolated either size exclusion chromatography or differential centrifugation. Isolated comprehensively characterized using dotblots, transmission electron microscopy, nanoparticle tracking analysis, immunoblotting. Results present 832/13 Compared cells myotubes, (EndoC-βH1, MIN6) produced significantly more (over whole lysates) higher compared myotubes; did not release appreciable DOC2B. Mechanistically, show that EV lumen; tandem C2 sufficient confer Discussion Clonal produce abundant culture, can be packaged EVs, small fraction excreted as protein. While DOC2B-laden are further studies will necessary determine if originating contributes plasma secretome.

Язык: Английский

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