Pub Date : 2025-06-27DOI: 10.1016/j.jprot.2025.105486
Hanne De Rijcke , An Staes , Stefaan De Smet , Marc Heyndrickx , Sam Millet , Leander Meuris , Devos SImon , Kris Gevaert , Christof Van Poucke
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.
{"title":"Collecting and analysing pig mucosal scraping samples for mass spectrometry-based proteomics: A study on factors causing variation in proteomic data","authors":"Hanne De Rijcke , An Staes , Stefaan De Smet , Marc Heyndrickx , Sam Millet , Leander Meuris , Devos SImon , Kris Gevaert , Christof Van Poucke","doi":"10.1016/j.jprot.2025.105486","DOIUrl":"10.1016/j.jprot.2025.105486","url":null,"abstract":"<div><div>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.</div></div><div><h3>Significance</h3><div>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.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"319 ","pages":"Article 105486"},"PeriodicalIF":2.8,"publicationDate":"2025-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144518894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-19DOI: 10.1016/j.jprot.2025.105483
Rafael F. Castelli , Haroldo C. de Oliveira , Marlon D.M. Santos , Amanda C. Camillo-Andrade , Flavia C.G. Dos Reis , Paulo C. Carvalho , Marcio L. Rodrigues
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.
{"title":"The flippases Apt1 and Apt2 differentially influence extracellular vesicle cargo and polysaccharide secretion in Cryptococcus neoformans","authors":"Rafael F. Castelli , Haroldo C. de Oliveira , Marlon D.M. Santos , Amanda C. Camillo-Andrade , Flavia C.G. Dos Reis , Paulo C. Carvalho , Marcio L. Rodrigues","doi":"10.1016/j.jprot.2025.105483","DOIUrl":"10.1016/j.jprot.2025.105483","url":null,"abstract":"<div><div>In the fungus <em>Cryptococcus neoformans</em>, the aminophospholipid translocase 1 (Apt1) flippase plays roles in virulence, membrane architecture, and extracellular vesicle (EV) polysaccharide cargo. The effect of <em>APT1</em> deletion on the fungal proteome is unknown, limiting the understanding of its functions in physiology. The <em>APT</em> gene family also includes <em>APT2</em>, whose functions in <em>C. neoformans</em> are virtually unknown. We investigated the impact of <em>APT1</em> and <em>APT2</em> deletion on EV formation in <em>C. neoformans</em>. 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 <em>C. neoformans</em> mutants lacking <em>APT1</em> and <em>APT2</em>, 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 <em>apt1Δ/apt2Δ</em> proteomes converged on secondary metabolite biosynthesis and RNA metabolism clusters, diverging from the predominance of translation in WT cells. These findings establish <em>APT1</em> and <em>APT2</em> as key regulators of EV composition in <em>C. neoformans</em>.</div></div><div><h3>Significance</h3><div>Our study reveals that the aminophospholipid translocase 1 (Apt1) and aminophospholipid translocase 2 (Apt2) play distinct roles in the physiology of <em>Cryptococcus neoformans</em>, 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.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"319 ","pages":"Article 105483"},"PeriodicalIF":2.8,"publicationDate":"2025-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144340235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-18DOI: 10.1016/j.jprot.2025.105485
Huajun Cai , Yanwei Lai , Bingwang Zhu , Jintao Zeng , Ye Wang , Yong Wu , Jinfu Zhuang , Xing Liu , Guoxian Guan
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.
{"title":"Quantitative proteomics analysis of radiation-induced rectal injury in rectal cancer patients undergoing radiotherapy","authors":"Huajun Cai , Yanwei Lai , Bingwang Zhu , Jintao Zeng , Ye Wang , Yong Wu , Jinfu Zhuang , Xing Liu , Guoxian Guan","doi":"10.1016/j.jprot.2025.105485","DOIUrl":"10.1016/j.jprot.2025.105485","url":null,"abstract":"<div><div>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.</div></div><div><h3>Significance</h3><div>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.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"319 ","pages":"Article 105485"},"PeriodicalIF":2.8,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144330172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-18DOI: 10.1016/j.jprot.2025.105484
Xinyue Liu, Joao A. Paulo
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.
{"title":"Proteomic profiling of over 13,000 proteins across ten mouse tissues using multi-dimensional peptide pre-fractionation","authors":"Xinyue Liu, Joao A. Paulo","doi":"10.1016/j.jprot.2025.105484","DOIUrl":"10.1016/j.jprot.2025.105484","url":null,"abstract":"<div><div>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.</div></div><div><h3>Significance</h3><div>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.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"319 ","pages":"Article 105484"},"PeriodicalIF":2.8,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144336671","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-16DOI: 10.1016/j.jprot.2025.105482
Jessica Guedes , Leticia Szadai , Nicole Woldmar , Ágnes Judit Jánosi , Klára Koroncziová , Blanka Míra Lengyel , Bella Kelemen , Eszter Boltas , Rolland Gyulai , Elisabet Wieslander , Krzysztof Pawłowski , Peter Horvatovich , Lazaro Betancourt , A. Marcell Szasz , Zoltan Vereb , Peter Horvath , Henriett Oskolás , Roger Appelqvist , Johan Malm , Gyorgy Marko-Varga , Jeovanis Gil
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.
{"title":"The melanoma MEGA-study: Integrating proteogenomics, digital pathology, and AI-analytics for precision oncology","authors":"Jessica Guedes , Leticia Szadai , Nicole Woldmar , Ágnes Judit Jánosi , Klára Koroncziová , Blanka Míra Lengyel , Bella Kelemen , Eszter Boltas , Rolland Gyulai , Elisabet Wieslander , Krzysztof Pawłowski , Peter Horvatovich , Lazaro Betancourt , A. Marcell Szasz , Zoltan Vereb , Peter Horvath , Henriett Oskolás , Roger Appelqvist , Johan Malm , Gyorgy Marko-Varga , Jeovanis Gil","doi":"10.1016/j.jprot.2025.105482","DOIUrl":"10.1016/j.jprot.2025.105482","url":null,"abstract":"<div><div>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.</div></div><div><h3>Significance</h3><div>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.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"319 ","pages":"Article 105482"},"PeriodicalIF":2.8,"publicationDate":"2025-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144314553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-13DOI: 10.1016/j.jprot.2025.105481
Xiaojie Hu , Sheng Chen , Xiaoke Ping , Kadambot H.M. Siddique , Wallace A. Cowling
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.
{"title":"Proteomic analysis reveals differentially abundant proteins involved in post-pollination responses to heat stress in Brassica napus","authors":"Xiaojie Hu , Sheng Chen , Xiaoke Ping , Kadambot H.M. Siddique , Wallace A. Cowling","doi":"10.1016/j.jprot.2025.105481","DOIUrl":"10.1016/j.jprot.2025.105481","url":null,"abstract":"<div><div>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 <em>Brassica napus</em> 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.</div></div><div><h3>Significance of the study</h3><div>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.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"319 ","pages":"Article 105481"},"PeriodicalIF":2.8,"publicationDate":"2025-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144302349","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-07DOI: 10.1016/j.jprot.2025.105480
Tian Wang , Yaqi Liu , Xuehai Wu , Xiaotang Wang , Shuxuan Shi , Xiaona Song , Yunhui Ma , Zhaorui Zhang , Jiping Gao , Rui Sun , Guohua Song
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.
{"title":"Multi-omics reveals miR-181a-5p regulates PPAR-driven lipid metabolism in Oral squamous cell carcinoma: Insights from CRISPR/Cas9 knockout models","authors":"Tian Wang , Yaqi Liu , Xuehai Wu , Xiaotang Wang , Shuxuan Shi , Xiaona Song , Yunhui Ma , Zhaorui Zhang , Jiping Gao , Rui Sun , Guohua Song","doi":"10.1016/j.jprot.2025.105480","DOIUrl":"10.1016/j.jprot.2025.105480","url":null,"abstract":"<div><div>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.</div></div><div><h3>Significance</h3><div>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.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"319 ","pages":"Article 105480"},"PeriodicalIF":2.8,"publicationDate":"2025-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144253740","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-06DOI: 10.1016/j.jprot.2025.105479
Qinghua Xing , Xinyi Tao , Qingping Hu , XiaoMeng Guo , Yingjie Zhang , Xinwei Mao , Haisheng Wang , Jun Li , Baisuo Zhao
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.
{"title":"Cross and unique stress adaptation strategies of the polyextremophile Natranaerobius thermophilus to individual high salt, alkaline pH, and elevated temperature","authors":"Qinghua Xing , Xinyi Tao , Qingping Hu , XiaoMeng Guo , Yingjie Zhang , Xinwei Mao , Haisheng Wang , Jun Li , Baisuo Zhao","doi":"10.1016/j.jprot.2025.105479","DOIUrl":"10.1016/j.jprot.2025.105479","url":null,"abstract":"<div><div><em>N. thermophilus</em> 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 <em>N. thermophilus</em> 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 <em>Natranaerobiales</em>. 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.</div></div><div><h3>Significance</h3><div>Halophilic alkalithermophile <em>N. thermophilus</em> 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 <em>N. thermophilus</em> organizes its survival strategies. This research reveals that <em>N. thermophilus</em> 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.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"319 ","pages":"Article 105479"},"PeriodicalIF":2.8,"publicationDate":"2025-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144243163","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-03DOI: 10.1016/j.jprot.2025.105470
Julia Lautenbach , Qussai Abbas , Sarah Brajkovic , Tobias Sieberer , Michael Neumüller , Bernhard Kuster , Brigitte Poppenberger
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.
{"title":"Unraveling the proteomic landscape of red-fleshed apples to identify regulators of anthocyanin accumulation","authors":"Julia Lautenbach , Qussai Abbas , Sarah Brajkovic , Tobias Sieberer , Michael Neumüller , Bernhard Kuster , Brigitte Poppenberger","doi":"10.1016/j.jprot.2025.105470","DOIUrl":"10.1016/j.jprot.2025.105470","url":null,"abstract":"<div><div>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 <em>Malus domestica</em>, 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.</div></div><div><h3>Significance</h3><div>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 <em>Malus domestica</em>. 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.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"319 ","pages":"Article 105470"},"PeriodicalIF":2.8,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144221333","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-03DOI: 10.1016/j.jprot.2025.105471
Zhao-qun Wu , Chao-nan Guan , Ye-Bo Yang , Yue-Xuan Zhang , Meng-Yu Gai , Shi-Yi Wang , Xiu-Xing Zhang , Yu-Wen Wang , Jing Xue , Bo-Hao Duan , Hai-Ling Yang
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.
{"title":"Identification of crucial genes sustaining the curled leaf phenotype in perennial transgenic poplar via advanced proteomic and phosphoproteomic analyses","authors":"Zhao-qun Wu , Chao-nan Guan , Ye-Bo Yang , Yue-Xuan Zhang , Meng-Yu Gai , Shi-Yi Wang , Xiu-Xing Zhang , Yu-Wen Wang , Jing Xue , Bo-Hao Duan , Hai-Ling Yang","doi":"10.1016/j.jprot.2025.105471","DOIUrl":"10.1016/j.jprot.2025.105471","url":null,"abstract":"<div><div><em>Populus tomentosa</em> hybrid poplar <em>741</em> 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 <em>Populus 741</em>, exhibiting sustained overexpression of <em>PtoCYCD3;3</em>, 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 <em>PtoCYCD3;3</em> integration on photosynthetic pathways, hormone signaling, and cell proliferation, highlighting its role in modulating leaf morphogenesis in perennial <em>Populus 741</em>.</div></div><div><h3>Significance</h3><div>Current assessments of the effects of exogenously introduced genes in <em>Populus</em> 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 <em>741</em> 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.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"319 ","pages":"Article 105471"},"PeriodicalIF":2.8,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144234410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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