Journal Of Proteomics[JOURNAL]
Sun
200 papers
Unraveling the mechanism of peptidases participation in the protein digestion of the whiteleg shrimp (Litopenaeus vannamei).
Estrella-Soliz C, Muhlia-Almazan A, Bojorquez-Velazquez E … +3 more , Rojo-Arreola L, Gonzalez-Rios H, Huerta-Ocampo JA
J Proteomics · 2025 Sep · PMID 40684932 · Publisher ↗
This study aims to identify and analyze the temporal variation in the activity and relative abundance of peptidases in the midgut gland of the Pacific whiteleg shrimp, Litopenaeus vannamei, during digestion. The dynamic...
This study aims to identify and analyze the temporal variation in the activity and relative abundance of peptidases in the midgut gland of the Pacific whiteleg shrimp, Litopenaeus vannamei, during digestion. The dynamic profiles of active peptidases throughout digestion were determined by zymogram profile analysis in the midgut gland of shrimp at different feeding times: preprandial, 1 h, and 3 h postprandial. Further proteomic analysis of midgut gland extracts confirmed the identity of different-class peptidases, as well as additional isoforms not previously reported, and changes in their relative abundance. Trypsins and chymotrypsins were the predominantly active peptidases throughout the digestion process. Cathepsin D was active during the preprandial time and 3 h after ingestion, whereas a metallopeptidase showed activity at preprandial time and 1 h postprandial. Additional trypsin isoforms of varying abundances were confirmed, while chymotrypsins and cathepsin L increased significantly, peaking 3 h postprandial. The results indicate significant changes in the relative abundance of new isoforms of trypsins, cathepsins, and metallopeptidases, as well as in the previously identified peptidases involved in shrimp food protein digestion over time. SIGNIFICANCE: The manuscript findings revealed the peptidases' potential participation in the digestive process of the Pacific whiteleg shrimp. The integrated results from different methods confirmed the identity and active state of some of the multiple peptidase classes in the shrimp midgut gland. Additional trypsin, cathepsin, and metallopeptidase isoforms and their abundance changes were detected before and after ingestion. These results provide additional information about the complexity and efficiency of the protein hydrolysis mechanism of L. vannamei.
Corrigendum to "Metabonomics analysis reveals the protective effect of nano‑selenium against heat stress of rainbow trout (Oncorhynchus mykiss)" [Journal of Proteomics 259 (2022)104545/ID: JPROT-104545].
Li L, Liu Z, Quan J … +3 more , Lu J, Zhao G, Sun J
J Proteomics · 2025 Sep · PMID 40683767 · Publisher ↗
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A perspective on integrating digital pathology, proteomics, clinical data and AI analytics in cancer research.
Guedes J, Woldmar N, Szasz AM … +8 more , Wieslander E, Pawłowski K, Horvatovich P, Malm J, Szadai L, Németh IB, Marko-Varga G, Gil J
J Proteomics · 2025 Sep · PMID 40680990 · Publisher ↗
Nearly 40 % of individuals will be diagnosed with cancer in their lifetime, translating to an estimated 20 million new cases annually. Despite remarkable therapeutic advances, only 15-20 % of patients achieve durable res...
Nearly 40 % of individuals will be diagnosed with cancer in their lifetime, translating to an estimated 20 million new cases annually. Despite remarkable therapeutic advances, only 15-20 % of patients achieve durable responses to immunotherapy, and the high cost of treatment (illustrated by immune checkpoint inhibitors like pembrolizumab and nivolumab, totaling roughly $191,000 per year) remains a formidable global challenge. The convergence of digital pathology, high-throughput molecular profiling, and advanced computational strategies has the potential to transform cancer research. By integrating high-resolution morphological data with proteomic, transcriptomic, and spatial molecular insights, we can elucidate the complex interplay between tumor cells and their microenvironment. In this perspective, we review how emerging techniques, from AI-driven image analysis to deep visual proteomics, can accelerate biomarker discovery, refine patient stratification, and ultimately improve clinical outcomes. We illustrate these principles with a case study in melanoma, where the integration of digital pathology and deep proteomic profiling uncovered a molecular signature predictive of recurrence in early-stage disease. As these technologies evolve, we foresee a future of precision oncology characterized by the seamless integration of morphological, clinical, and molecular data enabled by AI-driven analytics. SIGNIFICANCE: This perspective represents a pivotal step toward transforming cancer research by bridging the gap between traditional histopathological evaluation and modern molecular analytics. By integrating digital pathology with spatial proteomics and advanced AI-driven analytics, our approach provides a multidimensional view of tumor biology that captures both morphological nuances and molecular heterogeneity. This comprehensive framework not only enhances our understanding of the tumor microenvironment but also facilitates the discovery of robust biomarkers for disease recurrence and therapeutic response. Ultimately, our findings underscore the potential of precision oncology to tailor treatment strategies to individual patient profiles, thereby improving clinical outcomes and guiding the next generation of personalized cancer care.
Phosphoproteomics revealed salt stress tolerance mechanisms in the roots of sugar beet monomeric addition line M14.
Li H, Sui Z, Qin Z … +3 more , Chen S, Yu B, Li H
J Proteomics · 2025 Sep · PMID 40659243 · Publisher ↗
Soil salinity is a major abiotic stress that limits global crop production and food security. It is necessary to explore the mechanisms of salt stress tolerance in order to cultivate high-quality salt-tolerant varieties....
Soil salinity is a major abiotic stress that limits global crop production and food security. It is necessary to explore the mechanisms of salt stress tolerance in order to cultivate high-quality salt-tolerant varieties. Sugar beet M14 line exhibits capability of salt stress tolerance. Here we conducted a phosphoproteomic study of the sugar beet M14 roots treated with 200 mM and 400 mM NaCl. Using phosphopeptide enrichment and LC-MS/MS based phosphoproteomics techniques, 1427 differential abundant phosphoproteins (DAPPs) and 983 unique phosphoproteins were identified under 200 mM NaCl. 1234 DAPPs and 769 unique phosphopeptides were identified under 400 mM NaCl. The two datasets were significantly enriched for terms associated with protein kinases and transcription factors. Meanwhile, the biological pathways enriched between 200 mM and 400 mM NaCl containing signaling pathways, metabolism and transport were prominently represented, suggesting that distinct signaling cascades may converge in key biological processes (e.g., glycolysis, transport and reactive oxygen metabolism) to mediate the salt stress response and tolerance. Furthermore, unlike the DAPPs enriched in sugar beet M14 leaves, the DAPPs enriched in roots specifically participated in the plant-pathogen interaction and prenyltransferase pathways. Significance: Sugar beet is an important sugar-producing crop, and its roots are the primary organ usually affected by salt stress. However, the phosphoproteomics of sugar beet M14 roots under salt stress remains unclear. This study emphasizes the changes in the phosphorylation level of sugar beet M14 roots proteins under different salt concentrations. We analyzed the similarities and differences in function and participating pathways of the DAPPs and unique phosphoproteins obtained at different salt stresses. These findings not only provide important insights into the mechanisms mediated by phosphorylation modification in sugar beet M14 response to salt stress, but also will inform efforts toward improving crop yield and quality.
Rice proteome revealed antagonistic interactions between signaling components of cytokinin and ABA phytohormones during drought stress.
Gujjar RS, Worakan P, Chuekong W … +4 more , Sankhuan D, Chanapanchai S, Roytrakul S, Supaibulwatana K
J Proteomics · 2025 Sep · PMID 40651595 · Publisher ↗
Cytokinins are well-known for their multifaceted roles in fine-tuning the plant growth and development during abiotic stresses in plants. An optimized concentration of cytokinins is essential to positively-influence a ra...
Cytokinins are well-known for their multifaceted roles in fine-tuning the plant growth and development during abiotic stresses in plants. An optimized concentration of cytokinins is essential to positively-influence a range of stress-dependent signaling pathways. Our study focuses on how the alteration in cytokinin levels, post CPPU (N-2-(chloro-4-pyridyl)-N-phenyl urea) application, influences the protein components of drought susceptible rice cultivar, PTT1 (Pathumthani1), under water deficit stress. The proteome was investigated by shotgun proteomics and liquid chromatography-tandem mass spectrometry (LC-MS) at tillering and grain-filling stages of rice. The results indicated that the application of CPPU had an antagonistic effect of on the biosynthesis and signaling of ABA. Drought-stressed rice plants without CPPU treatment revealed enhanced abundance of negative regulator proteins of cytokinin signaling (ORR12 and ORR21) and cytokinin dehydrogenases (CKX2 and CKX3) which are antagonistic to cytokinin responses. By contrast, CPPU treated rice plants had reduced abundance of proteins implicated in ABA biosynthesis and signaling (ZEP, NCED4, PYL9, OSK1/SnRK1A, OSK4/SnRK1B and ABIL5) but showed greater abundance proteins implicated in cytokinin signaling and responses. Further, CPPU treatment induced the abundance of some proteins implicated in maintaining the osmotic balance. The results unravel the antagonistic nature of interaction between two major phytohormones, ABA and cytokinin.
Intercellular transfer of proteins and mitochondria - the contribution of proteomics.
Guzmán-Ortiz AL, Pérez-Figueroa E, Valle-Ríos R … +2 more , Martínez-González KM, Quezada H
J Proteomics · 2025 Sep · PMID 40645288 · Publisher ↗
One of the mechanisms of intercellular communication is the transfer of proteins and organelles among cells. This has been observed in diverse phylogenetic groups, and can be mediated by extracellular vesicles, like exos...
One of the mechanisms of intercellular communication is the transfer of proteins and organelles among cells. This has been observed in diverse phylogenetic groups, and can be mediated by extracellular vesicles, like exosomes or exophores, tunneling nanotubes, pores like plasmodesmata or processes like trogocytosis. The vast majority of studies in this field have used confocal microscopy and flow cytometry to detect proteins from donor cells in recipient cells. Proteomics has not been widely used, despite the fact that efficient tools are available for the labeling, enrichment and unbiased large-scale identification of the transferred proteins. Among these tools are trans-SILAC, affinity capture-MS/MS, BONCAT, TransitID and the use of cells from different species. In this review we describe illustrative examples of the intercellular transfer of proteins and mitochondria indicating the experimental methodologies used, both proteomics and non-proteomics, and emphasizing the capabilities of the mass spectrometry-based strategies.
Integrated proteomics and metabolomics analysis of chickpea seeds under aging condition.
Malviya R, Majumder S, Sharma P … +1 more , Gayen D
J Proteomics · 2025 Sep · PMID 40623522 · Publisher ↗
Seed aging in chickpeas during storage highlights significant economic and agricultural concerns. In this study, chickpea seeds were aged at 45 °C and 85 % humidity for 4 days, which affected both seeds' viability and vi...
Seed aging in chickpeas during storage highlights significant economic and agricultural concerns. In this study, chickpea seeds were aged at 45 °C and 85 % humidity for 4 days, which affected both seeds' viability and vigor. Comparative biochemical analyses showed a higher production of ROS and membrane damage in the aged seeds. Comparative proteomics and metabolomics analyses of seeds were conducted using Easy-nLC LC-MS/MS and GC-MS techniques. This examination identified 2605 proteins, 17654 peptides, and 68 metabolites between the control and aged seeds. A total of 118 statistically significant proteins were identified, with 98 proteins exhibiting differential abundance in response to the aging treatment. The in-silico functional and subcellular analysis revealed that these proteins and metabolites are related to germination, desiccation tolerance, phytohormones, fatty acid, amino acid metabolism, carbon assimilation pathways, and exhibited several locations inside the cell. The relative expression of 8 selected genes was verified using qRT-PCR to check the pattern with proteomics results. Temperature-induced lipocalin (CaTIL) is a protective protein that stabilizes the membrane and provides tolerance to aging, and was selected for molecular characterization. The subcellular localization of CaTIL was found to be in the plasma membrane of the onion cells.
Potential use of serum microvesicle indicators to distinguish different types of drug-induced liver injury.
Fu L, Zhang L, Huo G … +17 more , Qiu J, Qiao F, Huang L, Zhu X, Yu Z, Tang Y, Wu M, Chen B, Zheng C, Lv J, Shi L, Zhang X, Hwang S, Sun X, Li M, Shang Z, Gao Y
J Proteomics · 2025 Sep · PMID 40623521 · Publisher ↗
Drug-induced liver injury (DILI) may occur after the combination therapy of multiple drugs, which makes it difficult to identify the causative drug for liver injury in epidemiological research and clinical practice. Scut...
Drug-induced liver injury (DILI) may occur after the combination therapy of multiple drugs, which makes it difficult to identify the causative drug for liver injury in epidemiological research and clinical practice. Scutellaria baicalensis (SB), a widely used herb in traditional Chinese medicine, has been reported to cause liver injury when combined with antibiotics. However, diagnosing SB-drug induced liver injury (SB-DILI) and its distinction from antibiotic-induced DILI (AILI) is one of the significant challenges in modern clinical practice. There are still no effective indicators to distinguish between SB-DILI and AILI. Therefore, to discover and validate highly relevant indicators, we implemented a mass spectrometry workflow using label-free quantitative proteomics and Parallel Reaction Monitoring (PRM) in serum microvesicles (MVs) from 29 patients with SB-DILI, 28 patients with AILI, and 33 healthy volunteers (HVs). The results showed that a combined analysis of lysozyme (LYZ), component 4-binding protein α (C4BPA), and complement component 1r (C1R) from serum MVs yielded an area under the curve (AUC) ≥ 0.95, indicating that the combination analysis nearly fully distinguished between SB-DILI and AILI in these cohorts. This study underscores the potential of circulating MV indicators in differentiating between SB-DILI and AILI, offering significant implications for clinical diagnosis and therapeutic approaches for DILI. SIGNIFICANCE: Drug-induced liver injury (DILI) poses significant diagnostic challenges in clinical practice, particularly when caused by herbal-antibiotic combination therapies, where identifying the causative agent is critical yet elusive. This study addresses this unmet need by establishing the first serum microvesicle (MV)-based biomarker panel (LYZ, C4BPA, and C1R) capable of distinguishing Scutellaria baicalensis-induced DILI (SB-DILI) from antibiotic-induced DILI (AILI) with high accuracy (AUC ≥ 0.95). Leveraging label-free quantitative proteomics and rigorous validation via parallel reaction monitoring (PRM), our work advances the field of translational proteomics by demonstrating that MV-derived proteins reflect disease-specific pathophysiological processes, such as complement dysregulation and immune activation. Clinically, this panel addresses the rarity of SB-DILI and AILI (incidence <30/100,000) by providing an objective diagnostic tool to guide timely drug discontinuation, thereby reducing liver damage progression and optimizing therapeutic decisions. Methodologically, our workflow-combining MV isolation with targeted proteomics-sets a precedent for biomarker discovery in rare DILI subtypes. While preliminary, these findings lay the groundwork for multicenter validation studies to translate this innovation into clinical practice, ultimately improving precision medicine strategies for hepatotoxicity management.
Collecting and analysing pig mucosal scraping samples for mass spectrometry-based proteomics: A study on factors causing variation in proteomic data.
De Rijcke H, Staes A, De Smet S … +6 more , Heyndrickx M, Millet S, Meuris L, SImon D, Gevaert K, Van Poucke C
J Proteomics · 2025 Aug · PMID 40582614 · Publisher ↗
In this study, we evaluated the effects of experimenter handling and batch effects on proteomic analyses of mucosal scrapings in pigs. We analyzed mucosal samples from twelve piglets that were fed two distinct diets. Muc...
In this study, we evaluated the effects of experimenter handling and batch effects on proteomic analyses of mucosal scrapings in pigs. We analyzed mucosal samples from twelve piglets that were fed two distinct diets. Mucosal samples were obtained from a 60 cm segment collected from the jejunum, which was divided into six equal segments. Scrapings were collected by two experimenters, who each handled three of the six segments per pig. Both experimenters were given the exact same instructions for performing the mucosal scrapings. Samples were then randomized and processed in two separate groups on different days to examine batch effects. Protein extraction and digestion was carried out using Midi S-Traps (ProtiFi) and peptides were analyzed by DIA-MS. Data processing and statistical analysis were performed using DIA-NN and RStudio. Our analyses revealed no significant experimenter effects, but we observed small batch effects across datasets. Our findings emphasize the importance of standardized protocols to minimize experimenter-induced variability and the necessity of correcting for batch effects during data analysis. Our study provides a framework for minimizing unwanted variability in future proteomic studies, ensuring more reliable and reproducible findings. SIGNIFICANCE: With this study, we aimed to contribute to the improvement of the reliability and reproducibility of proteomic analyses in intestinal mucosal research. By systematically evaluating the effects of experimenter handling and batch processing on mucosal scraping samples, the study highlights key sources of variability that can influence proteomic outcomes. While experimenter effects were minimal, the identification of batch effects underscores the necessity of standardized protocols and appropriate corrections during data analysis. These findings provide a framework for minimizing unwanted variability in future studies, ultimately enhancing the accuracy of proteomic data interpretation and advancing research in intestinal health and disease.
The flippases Apt1 and Apt2 differentially influence extracellular vesicle cargo and polysaccharide secretion in Cryptococcus neoformans.
Castelli RF, de Oliveira HC, Santos MDM … +4 more , Camillo-Andrade AC, Dos Reis FCG, Carvalho PC, Rodrigues ML
J Proteomics · 2025 Aug · PMID 40543703 · Full text
In the fungus Cryptococcus neoformans, the aminophospholipid translocase 1 (Apt1) flippase plays roles in virulence, membrane architecture, and extracellular vesicle (EV) polysaccharide cargo. The effect of APT1 deletion...
In the fungus Cryptococcus neoformans, the aminophospholipid translocase 1 (Apt1) flippase plays roles in virulence, membrane architecture, and extracellular vesicle (EV) polysaccharide cargo. The effect of APT1 deletion on the fungal proteome is unknown, limiting the understanding of its functions in physiology. The APT gene family also includes APT2, whose functions in C. neoformans are virtually unknown. We investigated the impact of APT1 and APT2 deletion on EV formation in C. neoformans. The absence of Apt1, but not Apt2, led to a decreased concentration of the polysaccharide glucuronoxylomannan in EVs. We characterized the EV and cellular proteomes of C. neoformans mutants lacking APT1 and APT2, comparing them to the proteomes of wild-type (WT) cells. Paired comparisons revealed that WT and mutant EVs shared a significant part of their cargo but showed several strain-specific molecules and exhibited major differences in the abundance of various proteins. Conversely, the cellular proteomes of both mutants largely overlapped with WT (95.4 % shared proteins. Protein network analyses highlighted mutant-specific shifts: the apt1Δ/apt2Δ proteomes converged on secondary metabolite biosynthesis and RNA metabolism clusters, diverging from the predominance of translation in WT cells. These findings establish APT1 and APT2 as key regulators of EV composition in C. neoformans. SIGNIFICANCE: Our study reveals that the aminophospholipid translocase 1 (Apt1) and aminophospholipid translocase 2 (Apt2) play distinct roles in the physiology of Cryptococcus neoformans, particularly in the formation and composition of extracellular vesicles (EVs). By demonstrating that Apt1 deletion alters the proteomic landscape and reduces glucuronoxylomannan levels in EVs, while Apt2 deletion has no such effect, our findings provide critical insights into the functional divergence of these flippases. These insights underscore the potential of Apt1, but not necessarily Apt2, as therapeutic targets for developing novel antifungal strategies against this significant human pathogen.
Proteomic profiling of over 13,000 proteins across ten mouse tissues using multi-dimensional peptide pre-fractionation.
Liu X, Paulo JA
J Proteomics · 2025 Aug · PMID 40541929 · Full text
Proteome-wide abundance profiling across tissues can provide insight into the biological mechanisms underlying tissue-specific function, as well as potential related dysfunction and amelioration thereof. Here, we use sam...
Proteome-wide abundance profiling across tissues can provide insight into the biological mechanisms underlying tissue-specific function, as well as potential related dysfunction and amelioration thereof. Here, we use sample multiplexing to profile the proteomes of ten diverse mouse tissues using quantitative mass spectrometry-based sample multiplexing. Our optimized workflow, incorporating two-dimensional peptide pre-fractionation (which included both basic pH reversed-phase and strong ion exchange chromatography) enabled the quantification of over 13,000 proteins across brain, brown fat, heart, kidney, liver, lung, skeletal muscle, spleen, ovaries, and testes. Global analysis revealed distinct proteome profiles for each tissue, with clear clustering patterns reflecting functional similarities and differences. We highlighted the abundance of numerous tissue-specific proteins, exemplified by Synapsin-1 in brain, Uncoupling protein 1 in brown fat, and Zona pellucida proteins in reproductive tissues. Gene ontologies and pathway analyses of the most relatively abundant proteins in each tissue revealed enrichment patterns consistent with known physiological functions. For instance, brain tissue showed enrichment for synaptic components and neurotransmission processes, while liver tissue was enriched for metabolic pathways. This dataset serves as a valuable resource for mapping tissue-specific protein landscapes in mammals, offering potential insights into the molecular mechanisms of tissue function. SIGNIFICANCE: We present a proteome-wide analysis of ten diverse mouse tissues using an optimized TMTpro-based quantitative mass spectrometry workflow. This workflow is enhanced by two-dimensional peptide pre-fractionation with strong anion exchange partitioning followed by basic pH reversed-phase chromatography. By quantifying over 13,000 proteins, we provide an unprecedented dataset revealing distinct tissue-specific protein abundance profiles and their alignment with known physiological functions. This dataset as a resource offers valuable insights into the molecular underpinnings of tissue-specific biology and establishes a foundation for future research into physiological processes, disease mechanisms, and therapeutic development in mammals.
Quantitative proteomics analysis of radiation-induced rectal injury in rectal cancer patients undergoing radiotherapy.
Cai H, Lai Y, Zhu B … +6 more , Zeng J, Wang Y, Wu Y, Zhuang J, Liu X, Guan G
J Proteomics · 2025 Aug · PMID 40541928 · Publisher ↗
Radiation-induced rectal injury (RRI) affects perioperative treatment and the postoperative quality of life in patients with rectal cancer undergoing radiotherapy. This study aimed to clarify the molecular mechanisms of...
Radiation-induced rectal injury (RRI) affects perioperative treatment and the postoperative quality of life in patients with rectal cancer undergoing radiotherapy. This study aimed to clarify the molecular mechanisms of RRI and identify potential therapeutic targets. Hematoxylin-eosin and Masson's staining were utilized to evaluate RRI. Initially, 16 rectal samples were examined using data-independent acquisition proteomics to identify the differentially abundant proteins (DAPs). Subsequently, parallel reaction monitoring (PRM) was employed to validate DAP expression. Furthermore, DAP levels were assessed using enzyme-linked immunosorbent assay (ELISA) in 118 patients with rectal cancer. Pathologic examination revealed manifestations of RRI in rectal samples. A total of 1391 DAPs were identified, with 619 upregulated and 772 downregulated. Functional annotation revealed that these proteins are primarily involved in regulating actin cytoskeleton, metabolic pathways, and immune pathways. Enrichment analysis indicated significant enrichment of DAPs in pathways, including macrophage chemotaxis, neutrophil extracellular traps, and lipid metabolism. The expression of significantly upregulated DAPs (LBP, ITIH4, SERPINA3, FN1, PLG, HRG, FGA, and SAA1) in the relevant pathway was validated using PRM. ELISA revealed that the SAA1 level in the RRI group was significantly high. In conclusion, our study provides a proteomic profile of RRI, identifying biological markers and potential molecular regulatory mechanisms. SIGNIFICANCE: Radiation-induced intestinal injury (RII) significantly impacts the postoperative quality of life in patients undergoing pelvic radiotherapy. However, the current understanding of the mechanism behind RII remains unclear. In this study, Hematoxylin-eosin and Masson's staining were used to assess RRI. We employed data-independent acquisition proteomics analysis to characterize the proteomic profile associated with RII. Through this analysis, we identified differentially expressed proteins(DEPs) and potential molecular pathways implicated in RII. Parallel reaction monitoring and enzyme-linked immunosorbent assay further employed to validate the expression of DEPs. Our findings offer novel insights into the prevention and treatment strategies for RII, thereby potentially improving the clinical outcomes and quality of life for affected patients.
The melanoma MEGA-study: Integrating proteogenomics, digital pathology, and AI-analytics for precision oncology.
Guedes J, Szadai L, Woldmar N … +19 more , Jánosi ÁJ, Koroncziová K, Lengyel BM, Kelemen B, Boltas E, Gyulai R, Wieslander E, Pawłowski K, Horvatovich P, Betancourt L, Szasz AM, Vereb Z, Horvath P, Oskolás H, Appelqvist R, Malm J, Marko-Varga G, Németh IB, Gil J
J Proteomics · 2025 Aug · PMID 40532957 · Publisher ↗
Melanoma remains the most aggressive form of skin cancer, characterized by high metastatic potential, genetic heterogeneity, and resistance to conventional therapies. The Melanoma MEGA-Study is a multi-center initiative...
Melanoma remains the most aggressive form of skin cancer, characterized by high metastatic potential, genetic heterogeneity, and resistance to conventional therapies. The Melanoma MEGA-Study is a multi-center initiative designed to address these clinical challenges by integrating advanced proteogenomic profiling, clinical metadata, with AI-driven digital pathology and machine learning analytics, aiming to enhance personalized treatment strategies and improve patient outcomes. Between 2013 and 2022, a cohort of 1653 melanoma patients each contributed a primary tumor sample, with 361 providing 819 metastatic tumor samples. Clinical data collection for this cohort continued until May 2023. Comprehensive analyses using high-resolution mass spectrometry, optimized workflows for formalin-fixed paraffin-embedded tissues, and advanced digital pathology platforms enabled precise mapping of the tumor microenvironment, identification of metabolic reprogramming, and characterization of immune evasion signatures. The European Cancer Moonshot Lund Center's MEGA-Study, under the academic umbrella of Lund and Szeged universities, marks a significant advancement in its collaborative efforts with the National Institutes of Health (NIH) under the Cancer Moonshot partnership. This initiative exemplifies the center's dedication to pioneering cancer research and underscores the strength of its international collaborations. SIGNIFICANCE: The significance of this study lies in its pioneering integration of high-resolution proteomics, AI-driven digital pathology, and comprehensive clinical annotation to unravel the complex molecular landscape of melanoma. By leveraging a robust, population-based cohort of 1653 patients, including extensive analyses of both primary and metastatic tumor specimens, our approach provides unprecedented insights into the proteogenomic alterations that underpin tumor progression, immune evasion, and therapeutic resistance. The preliminary application of advanced mass spectrometry techniques to formalin-fixed paraffin-embedded tissues, combined with state-of-the-art digital pathology and machine learning, has enabled the identification of novel protein biomarkers and metabolic signatures that hold promise for refining patient stratification and informing personalized treatment strategies. This integrative framework not only deepens our understanding of melanoma biology but also establishes a scalable model for precision oncology that can be extended to other complex malignancies. Ultimately, our findings have the potential to transform clinical practice by facilitating earlier risk stratification, improving prognostication, and guiding the development of targeted therapeutic interventions for this highly aggressive cancer.
Proteomic analysis reveals differentially abundant proteins involved in post-pollination responses to heat stress in Brassica napus.
Hu X, Chen S, Ping X … +2 more , Siddique KHM, Cowling WA
J Proteomics · 2025 Aug · PMID 40518111 · Publisher ↗
Heat stress significantly reduces canola seed production during the post-pollination stages. This study explored changes in the proteome of flowers on the main stem of three Brassica napus cultivars exposed to transient...
Heat stress significantly reduces canola seed production during the post-pollination stages. This study explored changes in the proteome of flowers on the main stem of three Brassica napus cultivars exposed to transient heat stress after pollination. Flowers at the 2nd to 5th reproductive nodes on the main stem were collected on days 0, 1, 3 and 6 of heat stress and control treatments. The three cultivars, Alku, AV-Ruby, and YM11, exhibited varying degrees of heat sensitivity and resilience based on seed production in pods at these reproductive nodes. The seed yield per pod under heat stress was 75.3 % of the control in Alku and 64.2 % in YM11. However, AV-Ruby retained 93.5 % of its seed yield under heat stress, from which we conclude AV-Ruby was more resilient to heat stress during the post-pollination stage than Alku or YM11. There were 474 differentially abundant proteins (DAPs) identified across all cultivars and time points. Among the DAPs, two HSP20-like chaperones (A0A078I8F7, A0A078JBL3) and one HSP-related protein (A0A078JJT8) were consistently highly abundant under heat and were strong candidates as heat responsive proteins. Pathways related to maintaining membrane integrity were specifically enriched in AV-Ruby, and deserve further study for their potential involvement in heat tolerance. SIGNIFICANCE OF THE STUDY: Heat stress is a major factor threatening seed yield in cool season crops including oilseed rape, particularly during the post-pollination stages when pollination, fertilization, and embryo development are highly vulnerable to elevated temperatures. A comparative proteomic analysis was carried out to identify heat-responsive proteins during the post-pollination period. Among the DAPs, three were consistently associated with heat stress response. A range of biological processes, including protein folding and stress signalling, were involved in a general response to heat stress in all cultivars. Furthermore, pathways related to maintaining membrane integrity were specifically enhanced in a heat-resilient cultivar. These findings provide new insights into the heat response at the protein level and lay the groundwork for identifying potential molecular targets for breeding heat-tolerant oilseed rape cultivars.
Multi-omics reveals miR-181a-5p regulates PPAR-driven lipid metabolism in Oral squamous cell carcinoma: Insights from CRISPR/Cas9 knockout models.
Wang T, Liu Y, Wu X … +8 more , Wang X, Shi S, Song X, Ma Y, Zhang Z, Gao J, Sun R, Song G
J Proteomics · 2025 Aug · PMID 40490244 · Publisher ↗
Oral squamous cell carcinoma (OSCC) remains a therapeutic challenge due to its complex molecular landscape and metabolic adaptability. This study integrates proteomic and transcriptomic analyses to investigate the role o...
Oral squamous cell carcinoma (OSCC) remains a therapeutic challenge due to its complex molecular landscape and metabolic adaptability. This study integrates proteomic and transcriptomic analyses to investigate the role of miR-181a-5p in OSCC pathogenesis using CRISPR/Cas9-generated whole-body knockout (KO) mice. By inducing OSCC with the chemical carcinogen 4-nitroquinoline 1-oxide (4NQO), we identified significant dysregulation of lipid metabolism-associated proteins and tumor regulators in miR-181a-5p-KO tumors compared to wild-type controls. Quantitative proteomics revealed enrichment of the PPAR signaling pathway, with 12 key genes upregulated in KO mice, mechanistically linking miR-181a-5p deficiency to enhanced lipid droplet biogenesis and immunosuppressive microenvironments. Serum biomarker validation demonstrated elevated Cyfra21-1, SCC-Ag, and ISG20 levels in KO mice, correlating with tumor aggressiveness and radioresistance. Multi-omics integration further identified a diagnostic-prognostic protein signature with 89 % specificity for miR-181a-5p-deficient OSCC subtypes. These findings establish miR-181a-5p as a master regulator of PPAR-mediated metabolic reprogramming and immune evasion, offering novel proteome-driven insights into therapeutic targeting of lipid metabolism and biomarker discovery in OSCC. SIGNIFICANCE: This study integrates transcriptomic and proteomic analyses to elucidate the critical role of miR-181a-5p in regulating lipid metabolism via the PPAR signaling pathway during oral squamous cell carcinoma (OSCC) pathogenesis. Loss of miR-181a-5p enhances lipid metabolism, promoting membrane biosynthesis and metastasis. Multi-omics profiling identified a specific diagnostic-prognostic protein signature, highlighting CES3 and ISG20 as potential biomarkers for early diagnosis and therapeutic targeting in miR-181a-5p-deficient OSCC. The research establishes a foundation for miRNA-based liquid biopsy and PPAR-targeted nanotherapy. Mouse knockout models recapitulating human OSCC spatial biology validated miR-181a-5p's role in tumor initiation.
Cross and unique stress adaptation strategies of the polyextremophile Natranaerobius thermophilus to individual high salt, alkaline pH, and elevated temperature.
Xing Q, Tao X, Hu Q … +6 more , Guo X, Zhang Y, Mao X, Wang H, Li J, Zhao B
J Proteomics · 2025 Aug · PMID 40482852 · Publisher ↗
N. thermophilus is the first true anaerobic halophilic alkalithermophile. It employs a unique dual mechanism for hypersaline adaptation, utilizing both "compatible solutes" and "salt in" strategies. However, the molecula...
N. thermophilus is the first true anaerobic halophilic alkalithermophile. It employs a unique dual mechanism for hypersaline adaptation, utilizing both "compatible solutes" and "salt in" strategies. However, the molecular mechanisms underlying its responses to alkaline pH and thermal stress remain poorly characterized. An iTRAQ-based quantitative proteomics analysis revealed that N. thermophilus used a cross and unique adaptation strategies to three individual extreme stresses. This study fills gaps by elucidating previously unexplored alkaline-specific regulatory processes. It also provides the first comprehensive analysis of its thermal adaptation mechanisms. In response to high-salt and alkaline stress, the organism shifts its metabolism toward glycolysis and pyruvate-derived acetate synthesis, helping to meet increased ATP demands. Heat shock proteins are up-regulated during both alkaline and thermal adaptations, reflecting the "No free lunch" principle. Alkaline pH uniquely induces DNA repair proteins and S-adenosylmethionine biosynthesis enzymes, promoting genomic stability in proton-deficient environments. Besides, the compact genome and the positive correlation between GC content with growth temperature may be also a lineage-specific thermal adaptation of the halophilic and alkalithermophilic order Natranaerobiales. These findings illuminate the layered adaptation strategies that help address cross-stress challenges. Meanwhile, stress-specific reconfigurations enhance flexibility for survival in individual extremes. This work provides novel insights into the survival mechanisms of polyextremophiles, as well as advancing their potential biotechnological applications. SIGNIFICANCE: Halophilic alkalithermophile N. thermophilus exemplify life's capacity to thrive in environments where multiple physicochemical extremes intersect. However, the mechanisms underlying alkaline adaptation remain inadequately characterized, and our understanding of thermal adaptation is limited to genomic analyses. This study addresses critical gaps by disentangling the responses to hypersaline, alkalinity, and thermal stress, thereby elucidating how N. thermophilus organizes its survival strategies. This research reveals that N. thermophilus employs a strategy that combines conserved cross-stress mechanisms with unique stress adaptations to cope with the three distinct extreme stresses of high salinity, alkalinity, and temperature. By identifying the molecular modules through which these mechanisms operate, this research sets the stage for future applications in synthetic biology, particularly in the design of extremophile chassis for bioprocessing under multi-extreme conditions. These insights not only enhance our understanding of polyextremophiles but also pave the way for innovative biotechnological solutions.
Identification of crucial genes sustaining the curled leaf phenotype in perennial transgenic poplar via advanced proteomic and phosphoproteomic analyses.
Wu ZQ, Guan CN, Yang YB … +8 more , Zhang YX, Gai MY, Wang SY, Zhang XX, Wang YW, Xue J, Duan BH, Yang HL
J Proteomics · 2025 Aug · PMID 40472892 · Publisher ↗
Populus tomentosa hybrid poplar 741 is a superior tree species in northern China. Due to its rapid growth, high productivity, and range of available genetic tools, it has always been a focus of forestry research. The per...
Populus tomentosa hybrid poplar 741 is a superior tree species in northern China. Due to its rapid growth, high productivity, and range of available genetic tools, it has always been a focus of forestry research. The perennial genetically modified Populus 741, exhibiting sustained overexpression of PtoCYCD3;3, consistently shows adaxial curvature and pronounced surface wrinkling. The curvature of leaves holds great significance for forestry production systems. Moderate leaf curling can optimize the angle of light reception, thereby enhancing the efficiency of light absorption and photosynthetic performance, shortening the wood maturation cycle, and improving economic feasibility. Protein phosphorylation modification is a major regulatory mechanism in the cell cycle process. To investigate these morphological changes, TMT quantitative proteomics and phosphoproteomics were performed on leaves of transgenic and wild-type plants. Among 6005 identified proteins, 648 showed increased abundance, whereas 386 were reduced. In phosphoproteomics, 68 proteins exhibited differential phosphorylation, with 31 increasing and 37 decreasing. Quantitative proteomics identified significant changes in protein abundance associated with photosynthesis, phytohormones, and cell proliferation. Notably, histone deacetylase 6 (HDA6), ANGUSTIFOLIA (AN), and cellulose synthase-like (CSL) proteins associated with leaf curling were significantly upregulated in transgenic poplar. Phosphoproteomics revealed enrichment of proteins such as HERK1, DGK, OST1, and BIG, which are involved in brassinosteroid (BR), abscisic acid (ABA), and other phytohormone signaling pathways. These analyses demonstrated the impact of exogenous gene PtoCYCD3;3 integration on photosynthetic pathways, hormone signaling, and cell proliferation, highlighting its role in modulating leaf morphogenesis in perennial Populus 741. SIGNIFICANCE: Current assessments of the effects of exogenously introduced genes in Populus species are largely limited to short-term studies in annual or semiannual genetically modified specimens. In this study, we collected mature leaves from both perennial wild-type and transgenic 741 poplar trees and conducted comprehensive proteomic and phosphoproteomic analyses. The results not only revealed alterations in the abundance of multiple proteins associated with leaf curling but also elucidated key plant hormones and signaling pathways involved in leaf morphogenesis. These findings complement the signaling network involved in leaf morphogenesis and provide a novel perspective for studying perennial transgenic plants.
Unraveling the proteomic landscape of red-fleshed apples to identify regulators of anthocyanin accumulation.
Lautenbach J, Abbas Q, Brajkovic S … +4 more , Sieberer T, Neumüller M, Kuster B, Poppenberger B
J Proteomics · 2025 Aug · PMID 40472891 · Publisher ↗
Anthocyanins are colorful plant pigments with antioxidant properties, and a diet rich in these flavonoids bears health benefits. Therefore, a strong anthocyanin accumulation in edible plant parts is of significant intere...
Anthocyanins are colorful plant pigments with antioxidant properties, and a diet rich in these flavonoids bears health benefits. Therefore, a strong anthocyanin accumulation in edible plant parts is of significant interest, and in Malus domestica, the domesticated apple, certain red-fleshed apple varieties exhibit this trait. Enhanced anthocyanin accumulation in the flesh of apple fruits is attributed to the hyperactivation of the MYB transcription factor MdMYB10, which acts as a key regulator of anthocyanin biosynthesis by inducing the expression of multiple biosynthetic genes. While several studies have explored the underlying genetic mutations and resulting transcriptome changes, there is a lack of research on proteome alterations that cause the red-fleshed apple phenotype. To address this gap, a mass spectrometry-based proteomics approach was employed. Comparative proteomics identified differentially abundant proteins in young and mature fruits of the red-fleshed 'Bay13645' variety compared to the white-fleshed 'Gala'. Whereas several MYB transcription factors were enriched during early fruit development, they were no longer among the hyper-abundant proteins in ripe fruits of the red-fleshed genotype. In contrast, anthocyanin biosynthetic enzymes were enriched more strongly in ripe fruits of the red-fleshed cultivar, supporting previous results which had indicated developmental stage-specific differences in the control of the pigmentation process. The proteomic approach also identified novel regulatory factors and enzymes that may contribute to the red-fleshed apple phenotype, including a BAHD acyltransferase, Mal d proteins, and transcription factors of diverse families, and their potential relevance for the exhibition of this trait is discussed. SIGNIFICANCE: This study offers insights into the molecular mechanisms driving anthocyanin accumulation in red-fleshed apples. Utilizing a mass spectrometry-based proteomics strategy, the study reveals proteome alterations during fruit development that underlie the red-fleshed phenotype in Malus domestica. Notably, key enzymes of anthocyanin biosynthesis were markedly upregulated, underscoring their role in the pigmentation of the apple fruit pulp. Importantly, the study also identifies previously uncharacterized proteins, including a BAHD acyltransferase and a suite of transcription factors, shedding new light on the regulatory network orchestrating anthocyanin accumulation. These findings significantly expand our understanding of metabolic pathways that contribute to fruit pigmentation and open promising avenues for targeted crop improvement.
Development and application of a targeted phosphoproteomics method for analysing the mTOR pathway dynamics in zebrafish PAC2 cell line.
Huwa N, Schönenberger R, Groh KJ
J Proteomics · 2025 Aug · PMID 40466985 · Publisher ↗
The mechanistic target of rapamycin (mTOR) signalling pathway plays a crucial role in regulating cellular growth and proliferation. While extensively studied in mammals, the phosphorylation dynamics of this pathway in no...
The mechanistic target of rapamycin (mTOR) signalling pathway plays a crucial role in regulating cellular growth and proliferation. While extensively studied in mammals, the phosphorylation dynamics of this pathway in non-mammalian model organisms remain largely unexplored, often due to the scarcity of suitable antibodies to measure (phosphorylated) proteins of interest. To address this gap, we developed an antibody-independent targeted phosphoproteomics method applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify the abundance and phosphorylation levels of mTOR pathway-related proteins in zebrafish (Danio rerio), using the permanent cell line PAC2 as a model system. With optimized sample processing and data analysis strategies, we could successfully quantify 10 endogenous phosphosites and 15 endogenous proteins at different cell culture growth phases, revealing complex phosphorylation dynamics for both the upstream regulators (e.g., AKT, AMPK) and downstream effectors (e.g., eIF4EBP1, RPS6) of the mTOR pathway, which reflected transition from exponential growth to stationary subsistence. Our findings confirm the overall similarity of the mTOR pathway structure and functionality between zebrafish and mammals. Furthermore, this work demonstrates the high potential of the LC-MS/MS-based analytical approaches for studying phosphorylation-governed signalling in diverse organisms of interest, thus paving the way for further investigations in comparative physiology and toxicology across species. SIGNIFICANCE: We demonstrate the feasibility of using LC-MS/MS-based targeted phosphoproteomics to quantify protein phosphorylation dynamics of a specific pathway of interest - mTOR - in a non-mammalian model organism, zebrafish. This antibody-independent approach can enable the performance of further hypothesis-driven studies of phosphorylation-based signalling in diverse non-mammalian, non-model species. This tool could thus prove valuable for the fields of, e.g., comparative physiology and (eco)toxicology, where such investigations were previously limited due to the scarcity of suitable antibodies for specific proteins of interest in less frequently studied organisms. Moreover, thanks to the lower costs and higher throughput of targeted compared to global proteomics quantification methods, this approach can also be employed in studies aiming to validate the use of specific phosphosites as biomarkers of disease, stress or toxic chemical exposure in laboratory models or sentinel species in the environment, thus supporting future applications in toxicity testing or environmental monitoring.
Differentiating goose eggshells through proteomics: A case study from Xitou, North China.
Qi B, Dou H, Liu W … +3 more , Bennett EA, Rao H, Yang Y
J Proteomics · 2025 Aug · PMID 40460935 · Publisher ↗
Eggshells are significant cultural symbols linked to funeral customs and social development. Identifying their species is crucial in archaeological research. However, their small fragment size makes eggshells difficult t...
Eggshells are significant cultural symbols linked to funeral customs and social development. Identifying their species is crucial in archaeological research. However, their small fragment size makes eggshells difficult to accurately identify using traditional methods. Recent mass spectrometry advances have significantly enhanced eggshell species identification in archaeological research, but their success remains limited. For example, Anser cygnoides and Anser anser eggshells cannot yet be reliably distinguished. This paleoproteomic study uses Zooarchaeology by Mass Spectrometry and Liquid Chromatography-Tandem Mass Spectrometry to identify by proteotyping the species of eggshells from the Xitou site in China. We established a series of peptide markers and amino acid variants to distinguish A. cygnoides from A. anser, and with this method attribute three Xitou eggshell specimens to the graylag goose. Carbon isotope analysis and shell thickness measurements were conducted to understand the domestication of the geese associated with these eggshells. The isotope results indicate that these geese primarily ate C plants, and shell thickness results suggest they were possibly domesticated. These findings firstly provide direct molecular evidence of possibly domesticated graylag geese in China and offer new insights into poultry domestication and cultural practices. SIGNIFICANCE: Combing ZooMS and LC-MS/MS analysis, we have identified novel peptide markers to distinguish the eggshells of swan goose (Anser cygnoides) from those of graylag goose (Anser anser). Using a series of ZooMS markers and amino acid variations, the eggshells from Xitou site (north China) were identified to be derived from graylag goose. Further eggshells thickness and isotope analysis show that these geese were likely domesticated and primarily fed on C plants, offering valuable insights into early geese farming and sustainable practices during the Western Zhou period. Furthermore, the discovery of eggs as burial items highlights the important role that geese played in ancient Chinese culture and rituals.
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