Pub Date : 2025-08-18DOI: 10.1016/j.jprot.2025.105524
Zheng Fang , Yanjun Zhang , Xiaoxia Feng , Na Li , Liang Chen , Xianquan Zhan
Proteoforms represent the ultimate structural/functional forms of a gene product, defined by multiple factors, including amino acid sequences, post-translational modifications, spatial conformations, and interactions with other molecules. The human proteoform diversity significantly exceeds the number of human genes/transcripts, emphasizing the need for advanced analytical methods to characterize this complexity. Two-dimensional gel electrophoresis-liquid chromatography/mass spectrometry (2DE-LC/MS) and top-down MS (TD-MS) are complementary to detect, identify, and quantify the large-scale proteoforms. The emerging AI tools for structural biology such as AlphaFold 3 and D-I-TASSER will enable proteoformics to be high-throughput and precisely predict spatial conformations and molecular interactions. Integrating the large-scale experimental data derived from 2DE-LC/MS and TD-MS with AI-driven high-throughput structural analysis paves the way to deeply understand proteoform diversity and functionality. The combination of advanced 2DE-LC/MS, TD-MS, and AI-driven structural analysis represents a pivotal advancement in proteoformics. This integrated approach enables the comprehensive profiling of proteoforms, providing critical insights into their roles in health care. Such advancements hold promise for predictive, preventive, and personalized medicine, particularly through biomarker discovery and therapeutic target identification. Future developments in high-throughput capabilities and dynamic modeling are expected to address current challenges and further expand the applicability of proteoformics in biological and clinical research.
Significance
Proteoformics is the future of proteomics, whose two main complementary analytical approaches are 2DE-LC/MS and TD-MS. The AI-driven large-cale structural analysis enables to high-throughput and precisely analyze spatial conformations and molecular interactions of proteoforms, which helps to deeply understand proteoform diversity and functionality. Proteoformics holds transformative potential to uncover biomarkers, guide targeted therapies, and advance predictive diagnosis in the context of personalized medicine.
{"title":"Proteoformics: Current status and future perspectives","authors":"Zheng Fang , Yanjun Zhang , Xiaoxia Feng , Na Li , Liang Chen , Xianquan Zhan","doi":"10.1016/j.jprot.2025.105524","DOIUrl":"10.1016/j.jprot.2025.105524","url":null,"abstract":"<div><div>Proteoforms represent the ultimate structural/functional forms of a gene product, defined by multiple factors, including amino acid sequences, post-translational modifications, spatial conformations, and interactions with other molecules. The human proteoform diversity significantly exceeds the number of human genes/transcripts, emphasizing the need for advanced analytical methods to characterize this complexity. Two-dimensional gel electrophoresis-liquid chromatography/mass spectrometry (2DE-LC/MS) and top-down MS (TD-MS) are complementary to detect, identify, and quantify the large-scale proteoforms. The emerging AI tools for structural biology such as AlphaFold 3 and D-I-TASSER will enable proteoformics to be high-throughput and precisely predict spatial conformations and molecular interactions. Integrating the large-scale experimental data derived from 2DE-LC/MS and TD-MS with AI-driven high-throughput structural analysis paves the way to deeply understand proteoform diversity and functionality. The combination of advanced 2DE-LC/MS, TD-MS, and AI-driven structural analysis represents a pivotal advancement in proteoformics. This integrated approach enables the comprehensive profiling of proteoforms, providing critical insights into their roles in health care. Such advancements hold promise for predictive, preventive, and personalized medicine, particularly through biomarker discovery and therapeutic target identification. Future developments in high-throughput capabilities and dynamic modeling are expected to address current challenges and further expand the applicability of proteoformics in biological and clinical research.</div></div><div><h3>Significance</h3><div>Proteoformics is the future of proteomics, whose two main complementary analytical approaches are 2DE-LC/MS and TD-MS. The AI-driven large-cale structural analysis enables to high-throughput and precisely analyze spatial conformations and molecular interactions of proteoforms, which helps to deeply understand proteoform diversity and functionality. Proteoformics holds transformative potential to uncover biomarkers, guide targeted therapies, and advance predictive diagnosis in the context of personalized medicine.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105524"},"PeriodicalIF":2.8,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144894990","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 : 2025-08-18DOI: 10.1016/j.jprot.2025.105523
Maritza G. Verdugo-Molinares , Marco Ku-Centurion , Laura Cortes-Sanabria , Yadira O. Lugo-Melchor , Emilie Pinault , Luis Evangelista , Cesar O. Ramos-Garcia , Pierre Marquet , Zesergio Melo
Chronic kidney disease is a multifactorial entity characterized by decreased glomerular filtration rate (GFR). The last stage of the disease requires renal replacement therapy or kidney transplantation. As a disease with no treatment at earlier stages, and few biomarkers available, proteomics represent an excellent tool searching for new more efficient biomarkers. Urinary extracellular vesicles are an important source of information for kidney alterations, and their collection is not invasive. In this exploratory study, we worked on urine samples collected from patients at Centro Medico Nacional de Occidente in Guadalajara, Jalisco, and isolated urinary extracellular vesicles (uEVs) by ultracentrifugation. Our objective was to compare the Proteomic Profile of uEVs between Mexican patients with normal kidney function, end-stage renal disease, or kidney transplantation. High resolution mass spectrometry analysis reveals alterations in end-stage renal disease regarding the energy metabolism, cytoskeleton organization and cell motility. Proteomic alterations in transplant patients point towards the conservation of fibrotic process. Important proteins such as cystatins can be proposed as candidates for kidney transplant monitoring, while Gelsolin, a protein with an important role in assessing podocyte damage, stands out as a probable marker of chronic kidney disease. Data are available via ProteomeXchange with identifier PXD065380.
Significance
Chronic Kidney disease is a growing public health burden, increasing each year, and favored by major chronic diseases such as diabetes and hypertension. Although Mexico is one of the countries with the highest incidence of chronic kidney disease, proteomics studies involving Mexican patients had not yet been conducted. uEVs are features of particular interest to study the disease and discover biomarkers. We characterized the uEVs proteomic profile in Mexican patients, providing new insights into the pathogenesis of chronic kidney disease and kidney transplantation disorders. We identified promising biomarker candidates for transplant monitoring, and one as an early indicator of ESRD progression. uEVs may serve as a non-invasive platform for renal disease investigation, potentially offering non-invasive biomarkers for patient monitoring as well as mechanistic insights for future research into kidney pathophysiology.
{"title":"Potential kidney biomarkers in urinary extracellular vesicles from end stage renal disease and post-transplant patients","authors":"Maritza G. Verdugo-Molinares , Marco Ku-Centurion , Laura Cortes-Sanabria , Yadira O. Lugo-Melchor , Emilie Pinault , Luis Evangelista , Cesar O. Ramos-Garcia , Pierre Marquet , Zesergio Melo","doi":"10.1016/j.jprot.2025.105523","DOIUrl":"10.1016/j.jprot.2025.105523","url":null,"abstract":"<div><div>Chronic kidney disease is a multifactorial entity characterized by decreased glomerular filtration rate (GFR). The last stage of the disease requires renal replacement therapy or kidney transplantation. As a disease with no treatment at earlier stages, and few biomarkers available, proteomics represent an excellent tool searching for new more efficient biomarkers. Urinary extracellular vesicles are an important source of information for kidney alterations, and their collection is not invasive. In this exploratory study, we worked on urine samples collected from patients at Centro Medico Nacional de Occidente in Guadalajara, Jalisco, and isolated urinary extracellular vesicles (uEVs) by ultracentrifugation. Our objective was to compare the Proteomic Profile of uEVs between Mexican patients with normal kidney function, end-stage renal disease, or kidney transplantation. High resolution mass spectrometry analysis reveals alterations in end-stage renal disease regarding the energy metabolism, cytoskeleton organization and cell motility. Proteomic alterations in transplant patients point towards the conservation of fibrotic process. Important proteins such as cystatins can be proposed as candidates for kidney transplant monitoring, while Gelsolin, a protein with an important role in assessing podocyte damage, stands out as a probable marker of chronic kidney disease. Data are available via ProteomeXchange with identifier PXD065380.</div></div><div><h3>Significance</h3><div>Chronic Kidney disease is a growing public health burden, increasing each year, and favored by major chronic diseases such as diabetes and hypertension. Although Mexico is one of the countries with the highest incidence of chronic kidney disease, proteomics studies involving Mexican patients had not yet been conducted. uEVs are features of particular interest to study the disease and discover biomarkers. We characterized the uEVs proteomic profile in Mexican patients, providing new insights into the pathogenesis of chronic kidney disease and kidney transplantation disorders. We identified promising biomarker candidates for transplant monitoring, and one as an early indicator of ESRD progression. uEVs may serve as a non-invasive platform for renal disease investigation, potentially offering non-invasive biomarkers for patient monitoring as well as mechanistic insights for future research into kidney pathophysiology.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105523"},"PeriodicalIF":2.8,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144886149","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 : 2025-08-12DOI: 10.1016/j.jprot.2025.105522
Sergio Encarnación-Guevara , Jeovanis Gil
Lysine acetylation, once viewed primarily as a histone mark, is now recognized as a widespread regulator of protein function. Recent breakthroughs in chemical labeling, isotopic tagging workflows, and data-independent acquisition mass spectrometry enable precise, site-specific quantification of acetylation stoichiometry. This quantitative “acetylomics” approach reveals a “rheostat” model, where most acetylation sites exhibit low occupancy, acting as subtle modulators, while a subset of highly acetylated lysines (e.g., p53 C-terminus, AKT1, histones) serve as pivotal regulatory switches in gene expression, metabolism, and cell fate. Site-specific occupancy changes (e.g., p53, PKM2) increasingly serve as robust biomarkers for cancer diagnosis, prognosis, and therapeutic monitoring, often surpassing mRNA or total protein levels. Quantitative acetylation data now guide the development of targeted epigenetic therapies, including HDAC and p300/CBP inhibitors. Beyond oncology, acetylomics can pinpoint metabolic bottlenecks in heart failure, epigenetic deficits in neurodegenerative conditions, and inflammatory signaling nodes. With advances in high-throughput workflows, FFPE and liquid biopsy compatibility, and microfluidic platforms, acetylation stoichiometry is poised for clinical translation. We highlight both the promise and challenges of this emerging dimension of precision medicine, emphasizing the need for integrated multi-omics approaches and robust clinical validation to fully realize the potential of quantitative acetylomics in disease diagnosis and therapy.
Significance
Understanding the extent of acetylation occupancy in proteins, beyond simply determining presence or absence of acetylation, has profound implications for biology and medicine. This review emphasizes the importance of acetylation stoichiometry, connecting advanced proteomic technologies with translational science. We emphasize that quantifying site occupancy reveals which acetylation events truly modulate enzyme function. For instance, it can identify which acetylation events truly modulate enzyme activity or gene expression. Additionally, it can highlight molecular changes in diseases like cancer that are not apparent through qualitative analyses. These quantitative insights pave the way for clinical innovations, including novel biomarkers that stratify patients based on their acetylation profiles and targeted therapies that modulate acetylation levels. In summary, this work highlights the evolving landscape of protein acetylation research over the past two decades and its increasing influence on translational proteomics, celebrating milestones achieved by the global research community.
赖氨酸乙酰化，曾经主要被认为是一个组蛋白标记，现在被认为是一个广泛的蛋白质功能调节。最近在化学标记、同位素标记工作流程和数据独立采集质谱方面的突破使乙酰化化学计量学的精确、特定位点的量化成为可能。这种定量的“乙酰组学”方法揭示了一种“变阻器”模型，其中大多数乙酰化位点表现出低占用，作为微妙的调节剂，而高度乙酰化的赖氨酸子集（例如p53 c -端，AKT1，组蛋白）在基因表达，代谢和细胞命运中起关键的调节开关作用。位点特异性占用变化（如p53、PKM2）越来越多地作为癌症诊断、预后和治疗监测的强有力的生物标志物，通常超过mRNA或总蛋白水平。定量乙酰化数据现在指导靶向表观遗传治疗的发展，包括HDAC和p300/CBP抑制剂。除了肿瘤学，乙酰组学还可以精确定位心力衰竭的代谢瓶颈、神经退行性疾病的表观遗传缺陷和炎症信号节点。随着高通量工作流程、FFPE和液体活检相容性以及微流体平台的进步，乙酰化化学计量学已准备好用于临床翻译。我们强调了精准医学这一新兴维度的前景和挑战，强调需要整合多组学方法和强大的临床验证，以充分发挥定量乙酰组学在疾病诊断和治疗中的潜力。理解乙酰化在蛋白质中的占据程度，不仅仅是简单地确定乙酰化的存在与否，对生物学和医学具有深远的意义。这篇综述强调了乙酰化化学计量学的重要性，将先进的蛋白质组学技术与转化科学联系起来。我们强调，量化位点占用揭示哪些乙酰化事件真正调节酶的功能。例如，它可以识别哪些乙酰化事件真正调节酶活性或基因表达。此外，它还可以通过定性分析来突出癌症等疾病中不明显的分子变化。这些定量的见解为临床创新铺平了道路，包括基于乙酰化谱对患者进行分层的新型生物标志物和调节乙酰化水平的靶向治疗。总之，这项工作突出了过去二十年来蛋白质乙酰化研究的发展前景及其对翻译蛋白质组学的日益增长的影响，庆祝了全球研究界取得的里程碑。
{"title":"Expanding the landscape of lysine acetylation stoichiometry and clinical impact","authors":"Sergio Encarnación-Guevara , Jeovanis Gil","doi":"10.1016/j.jprot.2025.105522","DOIUrl":"10.1016/j.jprot.2025.105522","url":null,"abstract":"<div><div>Lysine acetylation, once viewed primarily as a histone mark, is now recognized as a widespread regulator of protein function. Recent breakthroughs in chemical labeling, isotopic tagging workflows, and data-independent acquisition mass spectrometry enable precise, site-specific quantification of acetylation stoichiometry. This quantitative “acetylomics” approach reveals a “rheostat” model, where most acetylation sites exhibit low occupancy, acting as subtle modulators, while a subset of highly acetylated lysines (e.g., p53 C-terminus, AKT1, histones) serve as pivotal regulatory switches in gene expression, metabolism, and cell fate. Site-specific occupancy changes (e.g., p53, PKM2) increasingly serve as robust biomarkers for cancer diagnosis, prognosis, and therapeutic monitoring, often surpassing mRNA or total protein levels. Quantitative acetylation data now guide the development of targeted epigenetic therapies, including HDAC and p300/CBP inhibitors. Beyond oncology, acetylomics can pinpoint metabolic bottlenecks in heart failure, epigenetic deficits in neurodegenerative conditions, and inflammatory signaling nodes. With advances in high-throughput workflows, FFPE and liquid biopsy compatibility, and microfluidic platforms, acetylation stoichiometry is poised for clinical translation. We highlight both the promise and challenges of this emerging dimension of precision medicine, emphasizing the need for integrated multi-omics approaches and robust clinical validation to fully realize the potential of quantitative acetylomics in disease diagnosis and therapy.</div></div><div><h3>Significance</h3><div>Understanding the extent of acetylation occupancy in proteins, beyond simply determining presence or absence of acetylation, has profound implications for biology and medicine. This review emphasizes the importance of acetylation stoichiometry, connecting advanced proteomic technologies with translational science. We emphasize that quantifying site occupancy reveals which acetylation events truly modulate enzyme function. For instance, it can identify which acetylation events truly modulate enzyme activity or gene expression. Additionally, it can highlight molecular changes in diseases like cancer that are not apparent through qualitative analyses. These quantitative insights pave the way for clinical innovations, including novel biomarkers that stratify patients based on their acetylation profiles and targeted therapies that modulate acetylation levels. In summary, this work highlights the evolving landscape of protein acetylation research over the past two decades and its increasing influence on translational proteomics, celebrating milestones achieved by the global research community.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105522"},"PeriodicalIF":2.8,"publicationDate":"2025-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144842547","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 : 2025-08-12DOI: 10.1016/j.jprot.2025.105521
Changheng Yang , Haiyang Li , Zijun Xu , Yaqiu Lin , Yong Wang , Lian Huang , Hua Xiang , Jiangjiang Zhu
Enhancing intramuscular fat (IMF) to improve the quality of livestock product has long been a goal in animal breeding. Recent studies have revealed a strong connection between malonylation and lipid metabolism, yet the function of malonylated proteins in ruminants largely unclear. In the present study, we identified the third day of goat intramuscular preadipocyte differentiation as a critical time point for lipid accumulation, with no significant alterations in malonylation levels. We identified 212 and 216 malonylated proteins on day 0 (d0) and day 3 (d3) of differentiation, respectively, enrichment in pathways such as glycolysis/gluconeogenesis, tight junctions, and actin cytoskeleton regulation. Our findings demonstrate the consistent presence of malonylation during preadipocyte differentiation, with minor quantitative variations, and highlight key malonylated proteins closely associated with lipid metabolism, including acetyl-CoA carboxylase (ACACA), translation control tumor protein 1 (TPT1), phosphoglycerate kinase 1 (PGK1), annexin A6 (ANXA6), and annexin A2 (ANXA2). Collectively, our study uncovers critical malonylated proteins during preadipocyte differentiation, establishing a foundation for exploring their roles in intramuscular fat deposition.
Significance
Currently, efforts are being made to improve meat quality by enhancing intramuscular fat (IMF) deposition, thereby promoting the development of the livestock industry. This study addresses a critical gap in our understanding of malonylation, a key post-translational modification, in livestock. By constructing the first malonylation protein modification map in goats and revealing dynamic changes during intramuscular preadipocyte differentiation, this research offers novel insights into the regulatory roles of malonylation in fat deposition. The findings not only advance the field of livestock proteomics but also provide a theoretical foundation for improving meat quality and exploring metabolic regulation in animals.
{"title":"Global profiling of protein lysine Malonylation during goat intramuscular Preadipocyte differentiation","authors":"Changheng Yang , Haiyang Li , Zijun Xu , Yaqiu Lin , Yong Wang , Lian Huang , Hua Xiang , Jiangjiang Zhu","doi":"10.1016/j.jprot.2025.105521","DOIUrl":"10.1016/j.jprot.2025.105521","url":null,"abstract":"<div><div>Enhancing intramuscular fat (IMF) to improve the quality of livestock product has long been a goal in animal breeding. Recent studies have revealed a strong connection between malonylation and lipid metabolism, yet the function of malonylated proteins in ruminants largely unclear. In the present study, we identified the third day of goat intramuscular preadipocyte differentiation as a critical time point for lipid accumulation, with no significant alterations in malonylation levels. We identified 212 and 216 malonylated proteins on day 0 (d0) and day 3 (d3) of differentiation, respectively, enrichment in pathways such as glycolysis/gluconeogenesis, tight junctions, and actin cytoskeleton regulation. Our findings demonstrate the consistent presence of malonylation during preadipocyte differentiation, with minor quantitative variations, and highlight key malonylated proteins closely associated with lipid metabolism, including acetyl-CoA carboxylase (ACACA), translation control tumor protein 1 (TPT1), phosphoglycerate kinase 1 (PGK1), annexin A6 (ANXA6), and annexin A2 (ANXA2). Collectively, our study uncovers critical malonylated proteins during preadipocyte differentiation, establishing a foundation for exploring their roles in intramuscular fat deposition.</div></div><div><h3>Significance</h3><div>Currently, efforts are being made to improve meat quality by enhancing intramuscular fat (IMF) deposition, thereby promoting the development of the livestock industry. This study addresses a critical gap in our understanding of malonylation, a key post-translational modification, in livestock. By constructing the first malonylation protein modification map in goats and revealing dynamic changes during intramuscular preadipocyte differentiation, this research offers novel insights into the regulatory roles of malonylation in fat deposition. The findings not only advance the field of livestock proteomics but also provide a theoretical foundation for improving meat quality and exploring metabolic regulation in animals.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105521"},"PeriodicalIF":2.8,"publicationDate":"2025-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144855619","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 : 2025-08-11DOI: 10.1016/j.jprot.2025.105520
Juan Carlos Vizuet-de-Rueda , Josaphat M. Montero-Vargas , Alberto C. López-Calleja , María Z.T. Manríquez-Ventura , Luis M. Teran
<div><div>Pollen aeroallergens cause up to 40 % of respiratory allergies and are challenging to control due to their widespread distribution in the environment. The pollen of <em>Ligustrum lucidum</em> (privet) is a significant source of inhalant allergens. However, despite its clinical relevance, the protein composition of <em>L. lucidum</em> pollen remains poorly characterized. Therefore, we employed an integrated proteomic and transcriptomic approach to explore its potential allergen composition, focusing on possible cross-reactivity with <em>Olea europea</em> (olive), a well-studied allergenic relative. Using LC-MS/MS-based proteomics and RNA-seq transcriptomics, we detected 13 of the 15 known olive-like allergens, demonstrating high cross-species conservation. Proteomic analysis identified nine homologous allergens, including Ole e 1, Ole e 2, Ole e 3, Ole e 5, Ole e 6, Ole e 9, Ole e 12, Ole e 13, and Ole e 14. Transcriptomic analysis revealed four additional putative allergens: Ole e 8, Ole e 10, Ole e 11, and Ole e 15. These proteins shared 74–95 % sequence identity with their olive counterparts and exhibited multiple isoforms. Our findings provide a set of <em>L. lucidum</em> pollen potential allergens and highlight the utility of multi-omics in allergen discovery. However, further clinical validation of these putative novel allergens is needed to assess their role in sensitization and cross-reactivity.</div></div><div><h3>Significance</h3><div>Privet (<em>Ligustrum</em>), a genus within the Oleaceae family, is biologically significant due to its role in triggering allergic respiratory diseases worldwide. As a close relative of olive (<em>Olea europaea</em>) and ash (<em>Fraxinus</em>), privet shares allergenic proteins that contribute to cross-reactivity among sensitized individuals. Climate change has been shown to extend their flowering period, increasing pollen exposure and exacerbating allergic symptoms. <em>Ligustrum</em> is widely used in urban landscaping due to its rapid growth, resistance to pollution, and adaptability to diverse soil conditions, which facilitates its global spread across North America, Europe, Asia, and South America. Notably, <em>L. lucidum</em> is a major sensitizing agent in Mexico City, where 37 % of allergic patients react to its pollen. The first identified allergen, Lig v 1, shares homology with Ole e 1 and Fra e 1. At the same time, Lig v 2 (profilin) mirrors Ole e 2, highlighting the molecular basis for cross-reactivity within the Oleaceae family. Recent proteomic studies have uncovered additional allergens, including enolase, β-1,3-glucanase, and ATP synthase subunits, further elucidating privet's allergenic potential. The absence of genomic data for <em>L. lucidum</em> has hindered research; however, advances in transcriptomic and proteomic approaches have enabled the identification of 13 of 15 known olive-like allergens in privet pollen, paving the way for improved diagnostics and targeted therapie
花粉致敏原导致高达40%的呼吸道过敏，由于其在环境中的广泛分布，很难控制。女贞的花粉是吸入性过敏原的重要来源。然而，尽管其临床相关性，露珠l.l lucidum花粉的蛋白质组成特征仍然很差。因此，我们采用了综合的蛋白质组学和转录组学方法来探索其潜在的过敏原组成，重点是与橄榄树（Olea europea）可能的交叉反应性，橄榄树是一种已经得到充分研究的过敏原亲缘关系。利用LC-MS/MS-based蛋白质组学和RNA-seq转录组学，我们检测到15个已知橄榄样过敏原中的13个，显示出高度的跨物种保守性。蛋白质组学分析鉴定出9个同源过敏原，包括Ole e 1、Ole e 2、Ole e 3、Ole e 5、Ole e 6、Ole e 9、Ole e 12、Ole e 13和Ole e 14。转录组学分析显示了另外四种可能的过敏原：Ole e8、Ole e10、Ole e11和Ole e15。这些蛋白与橄榄蛋白具有74 - 95%的序列一致性，并表现出多种同工型。我们的发现提供了一组露珠菌花粉潜在的过敏原，并强调了多组学在过敏原发现中的应用。然而，这些假定的新型过敏原需要进一步的临床验证来评估它们在致敏和交叉反应中的作用。鸢尾属（Ligustrum）是油科植物中的一个属，因其在全球范围内引发过敏性呼吸道疾病而具有重要的生物学意义。作为橄榄（Olea europaea）和白蜡树（Fraxinus）的近亲，女贞具有致敏个体之间交叉反应的致敏蛋白。气候变化延长了它们的花期，增加了花粉暴露，加剧了过敏症状。女贞草因其生长迅速、抗污染、适应多种土壤条件而被广泛应用于城市园林绿化中，这使得其在北美、欧洲、亚洲和南美等地广泛传播。值得注意的是，L. lucidum是墨西哥城的主要致敏剂，其中37%的过敏患者对其花粉有反应。第一个发现的过敏原ligv1与Ole e1和frae1具有同源性。与此同时，lig2 （profilin）与ole2相对应，突出了油科植物交叉反应的分子基础。最近的蛋白质组学研究发现了其他过敏原，包括烯醇化酶、β-1,3-葡聚糖酶和ATP合成酶亚基，进一步阐明了女贞的致敏潜力。缺乏lucidum的基因组数据阻碍了研究；然而，转录组学和蛋白质组学方法的进步已经能够在女贞花粉中鉴定出15种已知橄榄样过敏原中的13种，为改进诊断和靶向治疗铺平了道路。这强调了进一步调查女贞子过敏成分的必要性，特别是在气候变化和城市化放大其公共卫生影响的情况下，以及改进诊断和靶向治疗的潜力。
{"title":"Proteomic and transcriptomic analyses reveal new insights into allergens in Ligustrum lucidum pollen","authors":"Juan Carlos Vizuet-de-Rueda , Josaphat M. Montero-Vargas , Alberto C. López-Calleja , María Z.T. Manríquez-Ventura , Luis M. Teran","doi":"10.1016/j.jprot.2025.105520","DOIUrl":"10.1016/j.jprot.2025.105520","url":null,"abstract":"<div><div>Pollen aeroallergens cause up to 40 % of respiratory allergies and are challenging to control due to their widespread distribution in the environment. The pollen of <em>Ligustrum lucidum</em> (privet) is a significant source of inhalant allergens. However, despite its clinical relevance, the protein composition of <em>L. lucidum</em> pollen remains poorly characterized. Therefore, we employed an integrated proteomic and transcriptomic approach to explore its potential allergen composition, focusing on possible cross-reactivity with <em>Olea europea</em> (olive), a well-studied allergenic relative. Using LC-MS/MS-based proteomics and RNA-seq transcriptomics, we detected 13 of the 15 known olive-like allergens, demonstrating high cross-species conservation. Proteomic analysis identified nine homologous allergens, including Ole e 1, Ole e 2, Ole e 3, Ole e 5, Ole e 6, Ole e 9, Ole e 12, Ole e 13, and Ole e 14. Transcriptomic analysis revealed four additional putative allergens: Ole e 8, Ole e 10, Ole e 11, and Ole e 15. These proteins shared 74–95 % sequence identity with their olive counterparts and exhibited multiple isoforms. Our findings provide a set of <em>L. lucidum</em> pollen potential allergens and highlight the utility of multi-omics in allergen discovery. However, further clinical validation of these putative novel allergens is needed to assess their role in sensitization and cross-reactivity.</div></div><div><h3>Significance</h3><div>Privet (<em>Ligustrum</em>), a genus within the Oleaceae family, is biologically significant due to its role in triggering allergic respiratory diseases worldwide. As a close relative of olive (<em>Olea europaea</em>) and ash (<em>Fraxinus</em>), privet shares allergenic proteins that contribute to cross-reactivity among sensitized individuals. Climate change has been shown to extend their flowering period, increasing pollen exposure and exacerbating allergic symptoms. <em>Ligustrum</em> is widely used in urban landscaping due to its rapid growth, resistance to pollution, and adaptability to diverse soil conditions, which facilitates its global spread across North America, Europe, Asia, and South America. Notably, <em>L. lucidum</em> is a major sensitizing agent in Mexico City, where 37 % of allergic patients react to its pollen. The first identified allergen, Lig v 1, shares homology with Ole e 1 and Fra e 1. At the same time, Lig v 2 (profilin) mirrors Ole e 2, highlighting the molecular basis for cross-reactivity within the Oleaceae family. Recent proteomic studies have uncovered additional allergens, including enolase, β-1,3-glucanase, and ATP synthase subunits, further elucidating privet's allergenic potential. The absence of genomic data for <em>L. lucidum</em> has hindered research; however, advances in transcriptomic and proteomic approaches have enabled the identification of 13 of 15 known olive-like allergens in privet pollen, paving the way for improved diagnostics and targeted therapie","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105520"},"PeriodicalIF":2.8,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144842548","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 : 2025-08-05DOI: 10.1016/j.jprot.2025.105494
Jorge Noé García-Chávez , Robert Winkler
The rapid pace of shotgun proteomics data generation presents challenges for timely data analysis. In parallel, the scientific community is creating novel data interpretation tools, such as artificial intelligence, that have not yet been integrated into commercial software. Off-site data processing with free and open-source software (FOSS) enables the decentralization and scaling of informatics workflows. FOSS platforms also lower the costs of education and research. MASSyPupX is a FOSS mass spectrometry (MS) software collection that runs directly from a USB drive. Alternatively, setting up a MASSyPupX workstation or server provides a ready-to-use and reproducible MS analysis platform. Installed programming languages and libraries support the development of custom MS software and workflows. This paper demonstrates using MASSyPupX to convert and process raw shotgun proteomics data. Raw Thermo files were downloaded from ProteomeXchange and converted to the HUPO community format mzML. Data-dependent acquisition (DDA) data were evaluated with Comet, PeptideProphet, ProteinProphet, ProtyQuant, and the Trans-Proteomic Pipeline. Data-independent acquisition (DIA) shotgun proteomics data were analyzed with DIA-NN. Custom Bash, Python, and R scripts were used to post-process and visualize the results. The MASSyPupX project is hosted at https://codeberg.org/LabABI/MASSyPupX, and the current ISO can be downloaded from https://doi.org/10.5281/zenodo.14618430.
The MASSyPupX platform significantly advances shotgun proteomics data processing by offering a free and open-source software (FOSS) solution that is portable, scalable, and accessible. Operating directly from a USB drive or server, this Debian-based Linux distribution enables researchers to analyze data-dependent (DDA) and data- independent (DIA) acquisition proteomics data without installation, decentralizing workflows, reducing costs, and fostering collaboration and mass spectrometry data processing training. With pre-installed programming languages, libraries, and support for tools like Comet, PeptideProphet, DIA-NN, and ProtyQuant, MASSyPupX facilitates reproducible analyses, integrates cutting-edge computational techniques, and provides a user-friendly environment for education, research, and custom workflow development.
MASSyPupX democratizes access to advanced proteomics analysis, serving as a versatile tool for advancing biological and medical research through decentralized and cost-effective workflows.
{"title":"Off-site processing of data-dependent and data-independent acquisition shotgun proteomics data with MASSyPupX","authors":"Jorge Noé García-Chávez , Robert Winkler","doi":"10.1016/j.jprot.2025.105494","DOIUrl":"10.1016/j.jprot.2025.105494","url":null,"abstract":"<div><div>The rapid pace of shotgun proteomics data generation presents challenges for timely data analysis. In parallel, the scientific community is creating novel data interpretation tools, such as artificial intelligence, that have not yet been integrated into commercial software. Off-site data processing with free and open-source software (FOSS) enables the decentralization and scaling of informatics workflows. FOSS platforms also lower the costs of education and research. MASSyPupX is a FOSS mass spectrometry (MS) software collection that runs directly from a USB drive. Alternatively, setting up a MASSyPupX workstation or server provides a ready-to-use and reproducible MS analysis platform. Installed programming languages and libraries support the development of custom MS software and workflows. This paper demonstrates using MASSyPupX to convert and process raw shotgun proteomics data. Raw Thermo files were downloaded from ProteomeXchange and converted to the HUPO community format <span>mzML</span>. Data-dependent acquisition (DDA) data were evaluated with Comet, PeptideProphet, ProteinProphet, ProtyQuant, and the Trans-Proteomic Pipeline. Data-independent acquisition (DIA) shotgun proteomics data were analyzed with DIA-NN. Custom Bash, Python, and R scripts were used to post-process and visualize the results. The MASSyPupX project is hosted at <span><span>https://codeberg.org/LabABI/MASSyPupX</span><svg><path></path></svg></span>, and the current ISO can be downloaded from <span><span>https://doi.org/10.5281/zenodo.14618430</span><svg><path></path></svg></span>.</div><div>The MASSyPupX platform significantly advances shotgun proteomics data processing by offering a free and open-source software (FOSS) solution that is portable, scalable, and accessible. Operating directly from a USB drive or server, this Debian-based Linux distribution enables researchers to analyze data-dependent (DDA) and data- independent (DIA) acquisition proteomics data without installation, decentralizing workflows, reducing costs, and fostering collaboration and mass spectrometry data processing training. With pre-installed programming languages, libraries, and support for tools like Comet, PeptideProphet, DIA-NN, and ProtyQuant, MASSyPupX facilitates reproducible analyses, integrates cutting-edge computational techniques, and provides a user-friendly environment for education, research, and custom workflow development.</div><div>MASSyPupX democratizes access to advanced proteomics analysis, serving as a versatile tool for advancing biological and medical research through decentralized and cost-effective workflows.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105494"},"PeriodicalIF":2.8,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144775710","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 : 2025-07-31DOI: 10.1016/j.jprot.2025.105511
Diogo B. Lima , Max Ruwolt , Marlon D.M. Santos , Ke Pu , Fan Liu , Paulo C. Carvalho
We present Q2C, an open-source software designed to streamline mass spectrometer queue management and assess performance based on quality control metrics. Q2C provides a fast and user-friendly interface to visualize projects queues, manage analysis schedules and keep track of samples that were already processed. Our software includes analytical tools to ensure equipment calibration and provides comprehensive log documentation for machine maintenance, enhancing operational efficiency and reliability. Additionally, Q2C integrates with Google™ Cloud, allowing users to access and manage the software from different locations while keeping all data synchronized and seamlessly integrated across the system. For multi-user environments, Q2C implements a write-locking mechanism that checks for concurrent operations before saving data. When conflicts are detected, subsequent write requests are automatically queued to prevent data corruption, while the interface continuously refreshes to display the most current information from the cloud storage. Finally, Q2C, a demonstration video, and a user tutorial are freely available for academic use at https://github.com/diogobor/Q2C. Data are available from the ProteomeXchange consortium (identifier PXD055186).
Significance
Q2C addresses a critical gap in mass spectrometry facility management by unifying sample queue management with instrument performance monitoring. It ensures optimal instrument utilization, reduces turnaround times, and enhances data quality by dynamically prioritizing and routing samples based on analysis type and urgency. Unlike existing tools, Q2C integrates queue control and QC in a single platform, maximizing operational efficiency and reliability.
{"title":"Q2C: A software for managing mass spectrometry facilities","authors":"Diogo B. Lima , Max Ruwolt , Marlon D.M. Santos , Ke Pu , Fan Liu , Paulo C. Carvalho","doi":"10.1016/j.jprot.2025.105511","DOIUrl":"10.1016/j.jprot.2025.105511","url":null,"abstract":"<div><div>We present Q2C, an open-source software designed to streamline mass spectrometer queue management and assess performance based on quality control metrics. Q2C provides a fast and user-friendly interface to visualize projects queues, manage analysis schedules and keep track of samples that were already processed. Our software includes analytical tools to ensure equipment calibration and provides comprehensive log documentation for machine maintenance, enhancing operational efficiency and reliability. Additionally, Q2C integrates with Google™ Cloud, allowing users to access and manage the software from different locations while keeping all data synchronized and seamlessly integrated across the system. For multi-user environments, Q2C implements a write-locking mechanism that checks for concurrent operations before saving data. When conflicts are detected, subsequent write requests are automatically queued to prevent data corruption, while the interface continuously refreshes to display the most current information from the cloud storage. Finally, Q2C, a demonstration video, and a user tutorial are freely available for academic use at <span><span>https://github.com/diogobor/Q2C</span><svg><path></path></svg></span>. Data are available from the ProteomeXchange consortium (identifier PXD055186).</div></div><div><h3>Significance</h3><div>Q2C addresses a critical gap in mass spectrometry facility management by unifying sample queue management with instrument performance monitoring. It ensures optimal instrument utilization, reduces turnaround times, and enhances data quality by dynamically prioritizing and routing samples based on analysis type and urgency. Unlike existing tools, Q2C integrates queue control and QC in a single platform, maximizing operational efficiency and reliability.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105511"},"PeriodicalIF":2.8,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144768734","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 : 2025-07-29DOI: 10.1016/j.jprot.2025.105510
Qiaozhen Xu , Feng Tian , Xinyun Wang , Juanqi Lian , Xiaowei Zhang , Xiangmin Lin , Yanling Liu
Biofilms play a pivotal role in the survival and persistence of microorganisms, endowing them with heightened resistance to environmental stressors and antimicrobial agents. The EamB protein, which encodes an inner membrane transporter, acted as a negative regulator of biofilm formation, and the gene eamB deletion in the pathogen Aeromonas hydrophila LP-2 resulted in a significant increase in biofilm formation. Proteomic analysis revealed a total of 616 differentially abundant proteins between the ΔeamB and wild-type (WT) strains, with 308 downregulated and 308 upregulated. RT-qPCR was employed to verify the stability and accuracy of the proteomics data. Bioinformatic analysis indicated that EamB is involved in critical bacterial biological processes, including flagellar assembly, amino acid metabolism, and fatty acid degradation. Biofilm formation assays further revealed that supplementation with exogenous lysine significantly inhibited biofilm formation in the ΔeamB strain, conversely, exogenous cysteine and O-acetylserine obviously increased biofilm formation in the ΔeamB strain. These findings demonstrated that EamB may modulate bacterial biofilm formation in A. hydrophila through the regulation of amino acid metabolism. This finding provides novel insights into the regulatory mechanism underlying biofilm formation and highlights potential targets for the development of future antibacterial strategies.
Significance statement
This study elucidates the critical role of the eamB gene in Aeromonas hydrophila, a significant aquatic pathogen, by demonstrating its impact on biofilm formation and physiological traits. Through comparative proteomic analysis, we identified 616 differentially abundant proteins in the ΔeamB mutant, revealing its involvement in key metabolic pathways such as amino acid metabolism, flagellar assembly, and fatty acid degradation. Notably, eamB deletion enhanced biofilm formation, while exogenous amino acids like cysteine and O-acetylserine obviously increased biofilm formation in the ΔeamB strain. These findings highlight EamB as a regulator of biofilm formation, offering novel molecular insights into bacterial pathogenicity. This research advances our understanding of biofilm-associated antibiotic resistance and provides potential targets for developing strategies to mitigate infections caused by A. hydrophila in aquaculture and public health.
{"title":"Proteomics reveals the role of the EamB transporter from Aeromonas hydrophila LP-2 in biofilm formation","authors":"Qiaozhen Xu , Feng Tian , Xinyun Wang , Juanqi Lian , Xiaowei Zhang , Xiangmin Lin , Yanling Liu","doi":"10.1016/j.jprot.2025.105510","DOIUrl":"10.1016/j.jprot.2025.105510","url":null,"abstract":"<div><div>Biofilms play a pivotal role in the survival and persistence of microorganisms, endowing them with heightened resistance to environmental stressors and antimicrobial agents. The EamB protein, which encodes an inner membrane transporter, acted as a negative regulator of biofilm formation, and the gene <em>eamB</em> deletion in the pathogen <em>Aeromonas hydrophila</em> LP-2 resulted in a significant increase in biofilm formation. Proteomic analysis revealed a total of 616 differentially abundant proteins between the <em>ΔeamB</em> and wild-type (WT) strains, with 308 downregulated and 308 upregulated. RT-qPCR was employed to verify the stability and accuracy of the proteomics data. Bioinformatic analysis indicated that EamB is involved in critical bacterial biological processes, including flagellar assembly, amino acid metabolism, and fatty acid degradation. Biofilm formation assays further revealed that supplementation with exogenous lysine significantly inhibited biofilm formation in the <em>ΔeamB</em> strain, conversely, exogenous cysteine and <em>O</em>-acetylserine obviously increased biofilm formation in the <em>ΔeamB</em> strain. These findings demonstrated that EamB may modulate bacterial biofilm formation in <em>A. hydrophila</em> through the regulation of amino acid metabolism. This finding provides novel insights into the regulatory mechanism underlying biofilm formation and highlights potential targets for the development of future antibacterial strategies.</div></div><div><h3>Significance statement</h3><div>This study elucidates the critical role of the <em>eamB</em> gene in <em>Aeromonas hydrophila</em>, a significant aquatic pathogen, by demonstrating its impact on biofilm formation and physiological traits. Through comparative proteomic analysis, we identified 616 differentially abundant proteins in the <em>ΔeamB</em> mutant, revealing its involvement in key metabolic pathways such as amino acid metabolism, flagellar assembly, and fatty acid degradation. Notably, <em>eamB</em> deletion enhanced biofilm formation, while exogenous amino acids like cysteine and <em>O</em>-acetylserine obviously increased biofilm formation in the Δ<em>eamB</em> strain. These findings highlight EamB as a regulator of biofilm formation, offering novel molecular insights into bacterial pathogenicity. This research advances our understanding of biofilm-associated antibiotic resistance and provides potential targets for developing strategies to mitigate infections caused by <em>A. hydrophila</em> in aquaculture and public health.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105510"},"PeriodicalIF":2.8,"publicationDate":"2025-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144760414","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 : 2025-07-25DOI: 10.1016/j.jprot.2025.105507
John Oluwafemi Teibo , Roberta Maraninchi Silveira , Virginia Campos Silvestrini , Izadora Archiolli , Ana Paula Masson , Beatriz Pereira de Morais , Dayane Schmidt , Matheus Henrique dos Santos , Germano Aguiar Ferreira , Carolina Hassibe Thomé , Dominic Helm , Raja Sekhar Nirujogi , Dairo Renato Alessi , Virginia Picanço-Castro , Lucas Eduardo Botelho de Souza , Vitor Marcel Faça
Chimeric antigen receptor T-cell (CAR-T) therapy is at the forefront of the field of cell immunotherapy. In this study, we generated an anti-CD19 CAR-Jurkat T cell line using a locally produced second-generation anti-CD19 CAR construct, which allowed us to analyse early proteomic changes that are crucial for comprehending the signalling pathways and mechanism of action of this CAR-T cell. SILAC-heavy tagged Raji B-cells and anti-CD19 CAR-Jurkat T-cells were co-cultured for ten minutes. The proteomic profiles were acquired via DIA methodology on the Orbitrap Astral LC-MS/MS platform. The proteome was extensively covered, resulting in about 8800 protein identifications at 1 % FDR. The effector CAR-Jurkat cells showed proteomic changes involving antigen presentation by CD74. The target Raji B-cells exhibited more significant alterations. Effector proteins, namely CD247, CD28, DAP, LCK, p38 MAPK, and CASP3, were validated, as they have critical roles in antigen presentation, T-cell activation, and apoptosis. Pharmacological inhibition of LCK using Dasatinib further suggested its pivotal role in early CAR-T signalling. This study led us to identify proteins that function as molecular effectors of anti-CD19 CAR-T cell therapy during the initial phases of CAR-T-target cell engagement, advancing our knowledge of the mechanism and signalling pathways that will support CAR-T cell development.
Significance
Chimeric antigen receptor T-cell (CAR-T cell) therapy is state-of-the-art in cell and immunotherapy. Determining important players in cellular communication and signalling mediated by membranes and intracellular proteins requires understanding the connection between tumours and modified cells. We employed global proteomics in this study to better grasp the functional protein networks using a high-sensitivity mass spectrometric platform for protein identification and quantification. We identified proteins as molecular effectors of anti-CD19 CAR-T cell treatment during the early stages of CAR-T-target cell interaction. Our understanding of the mechanism and signalling pathways will promote the development of new CAR constructs and improve the efficacy and ability to overcome the resistance of this innovative cancer treatment strategy, which will advance the identification of adjuvant molecules for the regulation of CAR-T responses.
{"title":"Proteomics analysis reveal early event molecular effectors of anti-CD19 CAR-T cell therapy in hematological cancer","authors":"John Oluwafemi Teibo , Roberta Maraninchi Silveira , Virginia Campos Silvestrini , Izadora Archiolli , Ana Paula Masson , Beatriz Pereira de Morais , Dayane Schmidt , Matheus Henrique dos Santos , Germano Aguiar Ferreira , Carolina Hassibe Thomé , Dominic Helm , Raja Sekhar Nirujogi , Dairo Renato Alessi , Virginia Picanço-Castro , Lucas Eduardo Botelho de Souza , Vitor Marcel Faça","doi":"10.1016/j.jprot.2025.105507","DOIUrl":"10.1016/j.jprot.2025.105507","url":null,"abstract":"<div><div>Chimeric antigen receptor T-cell (CAR-T) therapy is at the forefront of the field of cell immunotherapy. In this study, we generated an anti-CD19 CAR-Jurkat T cell line using a locally produced second-generation anti-CD19 CAR construct, which allowed us to analyse early proteomic changes that are crucial for comprehending the signalling pathways and mechanism of action of this CAR-T cell. SILAC-heavy tagged Raji B-cells and anti-CD19 CAR-Jurkat T-cells were co-cultured for ten minutes. The proteomic profiles were acquired via DIA methodology on the Orbitrap Astral LC-MS/MS platform. The proteome was extensively covered, resulting in about 8800 protein identifications at 1 % FDR. The effector CAR-Jurkat cells showed proteomic changes involving antigen presentation by CD74. The target Raji B-cells exhibited more significant alterations. Effector proteins, namely CD247, CD28, DAP, LCK, p38 MAPK, and CASP3, were validated, as they have critical roles in antigen presentation, T-cell activation, and apoptosis. Pharmacological inhibition of LCK using Dasatinib further suggested its pivotal role in early CAR-T signalling. This study led us to identify proteins that function as molecular effectors of anti-CD19 CAR-T cell therapy during the initial phases of CAR-T-target cell engagement, advancing our knowledge of the mechanism and signalling pathways that will support CAR-T cell development.</div></div><div><h3>Significance</h3><div>Chimeric antigen receptor T-cell (CAR-T cell) therapy is state-of-the-art in cell and immunotherapy. Determining important players in cellular communication and signalling mediated by membranes and intracellular proteins requires understanding the connection between tumours and modified cells. We employed global proteomics in this study to better grasp the functional protein networks using a high-sensitivity mass spectrometric platform for protein identification and quantification. We identified proteins as molecular effectors of anti-CD19 CAR-T cell treatment during the early stages of CAR-T-target cell interaction. Our understanding of the mechanism and signalling pathways will promote the development of new CAR constructs and improve the efficacy and ability to overcome the resistance of this innovative cancer treatment strategy, which will advance the identification of adjuvant molecules for the regulation of CAR-T responses.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105507"},"PeriodicalIF":2.8,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144731928","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 : 2025-07-25DOI: 10.1016/j.jprot.2025.105509
Karina Araujo-Ruiz , Daniel Ignacio López-Flores , María Karla Martínez-Muné , Brenda Yomara García-Sánchez , Carlos J. Ramírez-Flores , Francisco Ernesto Sandoval-Rodríguez , Emmanuel Ríos-Castro , Mónica Edith Mondragón-Castelán , Sirenia González-Pozos , Ricardo Mondragón-Flores
The Toxoplasma gondii cytoskeleton is a highly organized structure essential for parasite motility, replication, and host cell invasion. To identify its components, a highly enriched fraction of tachyzoite cytoskeletons was obtained and quantitatively analyzed by mass spectrometry. We identified 623 proteins classified into 18 functional groups, including 30 IMC proteins, 34 cytoskeleton proteins, and 14 uncharacterized proteins. A comprehensive bioinformatic analysis was conducted to assess protein abundance (fmol), antigenicity, accessibility, interactome, and homology, with the aim of identifying immunogenic targets. Among the top vaccine candidates were -GRA12, IMC1, ROP8, and -IMC4, with ROP8 emerging as the most promising based on epitope prediction. Data are available via ProteomeXchange with identifier PXD063409.
Significance
Toxoplasma gondii represents one of the most virulent and successful parasites in human and veterinary pathogenesis. Since T. gondii is a highly dynamic parasite that depends on its cytoskeleton to invade and disseminate through tissues, knowledge of its cytoskeleton composition is essential for understanding the biological mechanisms involved in parasite-host interactions and for the design of pharmaceutical and vaccination strategies. Quantitative proteomic analysis of the T. gondii cytoskeleton provided new and extensive information on its composition and, through bioinformatics approaches, allowed us to suggest several candidate molecules for future immunoprotective design.
{"title":"Quantitative proteomic analysis of the Toxoplasma gondii cytoskeleton and bioinformatic identification of highly antigenic proteins","authors":"Karina Araujo-Ruiz , Daniel Ignacio López-Flores , María Karla Martínez-Muné , Brenda Yomara García-Sánchez , Carlos J. Ramírez-Flores , Francisco Ernesto Sandoval-Rodríguez , Emmanuel Ríos-Castro , Mónica Edith Mondragón-Castelán , Sirenia González-Pozos , Ricardo Mondragón-Flores","doi":"10.1016/j.jprot.2025.105509","DOIUrl":"10.1016/j.jprot.2025.105509","url":null,"abstract":"<div><div>The <em>Toxoplasma gondii</em> cytoskeleton is a highly organized structure essential for parasite motility, replication, and host cell invasion. To identify its components, a highly enriched fraction of tachyzoite cytoskeletons was obtained and quantitatively analyzed by mass spectrometry. We identified 623 proteins classified into 18 functional groups, including 30 IMC proteins, 34 cytoskeleton proteins, and 14 uncharacterized proteins. A comprehensive bioinformatic analysis was conducted to assess protein abundance (fmol), antigenicity, accessibility, interactome, and homology, with the aim of identifying immunogenic targets. Among the top vaccine candidates were -GRA12, IMC1, ROP8, and -IMC4, with ROP8 emerging as the most promising based on epitope prediction. Data are available <em>via</em> ProteomeXchange with identifier <span><span>PXD063409</span><svg><path></path></svg></span>.</div></div><div><h3>Significance</h3><div><em>Toxoplasma gondii</em> represents one of the most virulent and successful parasites in human and veterinary pathogenesis. Since <em>T. gondii</em> is a highly dynamic parasite that depends on its cytoskeleton to invade and disseminate through tissues, knowledge of its cytoskeleton composition is essential for understanding the biological mechanisms involved in parasite-host interactions and for the design of pharmaceutical and vaccination strategies. Quantitative proteomic analysis of the <em>T. gondii</em> cytoskeleton provided new and extensive information on its composition and, through bioinformatics approaches, allowed us to suggest several candidate molecules for future immunoprotective design.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105509"},"PeriodicalIF":2.8,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144722115","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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