Melinda Rezeli

Extracellular vesicles (EVs) hold promise as biomarkers for early disease detection, yet their nanoscale size and the protein-rich plasma background make enrichment essential. Conventional approaches such as ultracentrifugation and size-exclusion chromatography require ≥100 μL of input, hours of processing, and are poorly compatible with clinical workflows. We present a low-cost polymer-based acoustochromatography device that enables EV enrichment from 1 μL of blood plasma within 2–10 min─significantly faster than current gold-standard methods. A novel ultrasound-driven release mode increased recovery significantly compared to the previously reported acoustofluidic chromatography method. Comprehensive proteomic analysis confirmed highly pure EV fractions after acoustofluidic enrichment. Apolipoproteins were depleted to below 100 ng/mL, corresponding to >99.6% depletion as compared to neat plasma. This absolute residual concentration is lower than values reported for other EV isolation methods and enabled the detection of an additional 689 mostly EV-associated proteins above the plasma background. This low-cost, scalable platform unlocks proteomic profiling of ultralow-volume samples, paving the way for rapid and clinically compatible EV biomarker diagnostics.

Proteomic analysis resulting from the comparison of entinostat sensitive versus resistant cell lines

Proteomic analysis resulting from the comparison of SCLC cell lines showing synergistic or additive responses to entinostat and cisplatin therapy

Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin ( HTT ) gene. Although transcriptomic and proteomic changes have been characterized in patient-derived neurons, the contribution of post-translational modifications (PTMs), such as phosphorylation, remains poorly understood. Here, we present the first phosphoproteomic analysis by mass spectrometry (P-MS) of human induced neurons (iNs) directly reprogrammed from HD patient fibroblasts. We identified 177 phosphopeptides with significantly altered abundance in HD-iNs, mapping to phosphoproteins associated with key signaling pathways known to be affected in HD, such as splicing and autophagy. By integrating P-MS data with previously published proteomic and transcriptomic data from the same donors, we identified distinct subsets of ON-OFF phosphopeptides that exhibited a complete loss of phosphorylation in either HD- or Ctrl-iNs, without corresponding changes at the RNA or protein level. An exception was MXRA8, previously described in glial cells as a mediator of blood-brain barrier integrity and astrocyte-mediated neuroinflammation. This protein showed increased protein abundance despite the absence of phosphorylation in HD- iNs, suggesting a compensatory mechanism - a pattern also observed in human post mortem cortical HD tissue. Additionally, MXRA8 showed altered protein-protein interactions with lysosomal and metabolic regulators in HD-iNs, highlighting its potential role in autophagy impairment as well as in neurovascular dysfunction. These findings uncover a distinct layer of post-translational dysregulation in HD, suggesting that phospho-switch proteins such as MXRA8 may be candidate effectors of pathology and thus site-specific phosphorylation loss may contribute to impaired signaling and proteostasis in human HD neurons.

Background: Metastatic melanoma is a highly aggressive disease with poor survival rates despite recent therapeutic advancements with immunotherapy. The proteomic landscape of advanced melanoma remains poorly understood, especially regarding proteomic heterogeneity across metastases within patients. Methods: We collected 83 melanoma metastases from 19 different metastatic sites in 24 patients with advanced metastatic melanoma almost exclusively from the pre‐immunotherapy era, using semi‐rapid autopsies. The metastases were subjected to histopathological evaluation, RNA‐sequencing and mass spectrometry‐based proteomics for protein quantitation and non‐reference peptide (NRP) sequence detection using a proteogenomic data integration approach. Results: NRPs associated with mutations frequently occurred in proteins related to focal adhesion, vesicle‐mediated transport, MAPK signalling and immune response pathways across the cohort. Intrapatient heterogeneity was negligible when considering morphology and driver gene mutation status but was substantial at the proteogenomic level. This heterogeneity was not driven by metastasis location, albeit liver metastases exhibited distinct proteogenomic patterns, including upregulation of metabolic pathways. Cluster analysis outlined four proteomic clusters (C1–4) of the metastases, characterised by the upregulation of cell cycle and RNA‐splicing (C1), mitochondrial processes (C3), extracellular matrix (ECM) and immune pathways (C2) and ECM and vesicle‐mediated transport pathways (C4). Around two‐thirds of patients had metastases that had strongly distinct phenotypes. Patients in our cohort whose metastases were primarily assigned to clusters C1 and C3 exhibited shorter overall survival than patients whose metastases were categorised mainly into the C2 and C4 clusters. Conclusion: Our unique multi‐metastasis cohort captured the proteogenomic heterogeneity of immunotherapy‐naïve melanoma distant metastases, establishing a foundation for future studies aimed at identifying novel therapeutic targets to complement current immunotherapies. Key points: Comprehensive proteogenomic profiling of post‐mortem melanoma metastases, collected primarily before the immunotherapy era. Description of 1177 protein sequence variants predicted by RNA‐Seq and validated via mass spectrometry‐based proteomics. Empirical evidence of prominent intrapatient heterogeneity, driven by heterogeneous protein expression related to cell cycle‐ and mitochondrial processes, immune system and extracellular matrix organization.

Background: Melanoma is a highly heterogeneous disease, and a deeper molecular classification is essential for improving patient stratification and treatment approaches. Here, we describe the histopathology-driven proteogenomic landscape of 142 treatment-naïve metastatic melanoma samples to uncover molecular subtypes and clinically relevant biomarkers. Methods: We performed an integrative proteogenomic analysis to identify proteomic subtypes, assess the impact of BRAF V600 mutations, and study the molecular profiles and cellular composition of the tumor microenvironment. Clinical and histopathological data were used to support findings related to tissue morphology, disease progression, and patient outcomes. Results: Our analysis revealed five distinct proteomic subtypes that integrate immune and stromal microenvironment components and correlate with clinical and histopathological parameters. We demonstrated that BRAF V600-mutated melanomas exhibit biological heterogeneity, where an oncogene-induced senescence-like phenotype is associated with improved survival. This led to a proposed mortality risk-based stratification that may contribute to more personalized treatment strategies. Furthermore, tumor microenvironment composition strongly correlated with disease progression and patient outcomes, highlighting a histopathological connective tissue-to-tumor ratio assessment as a potential decision-making tool. We identified a melanoma-associated SAAV signature linked to extracellular matrix remodeling and SAAV-derived neoantigens as potential targets for anti-tumor immune responses. Conclusions: This study provides a comprehensive stratification of metastatic melanoma, integrating proteogenomic insights with histopathological features. The findings may aid in the development of tailored diagnostic and therapeutic strategies, improving patient management and outcomes.