Pub Date : 2026-01-20Epub Date: 2025-11-02DOI: 10.1016/j.jprot.2025.105553
Zhihui Feng , Biao Zhang , Yi Liu , Fei Yang , Yinping Lei , Songbai Liao , Xianliang Hou
Background
Bladder cancer (BC) is the most common malignancy of the urinary system. However, the median survival of patients with metastatic bladder cancer remains limited. Thus, there is an urgent imperative to develop novel biomarkers for BC-targeted therapies and to conduct in-depth investigations into BC pathogenesis leveraging multi-omics technologies.
Results
Our results revealed that proteins co-upregulated in both proteomic and phosphoproteomic analysis, such as SLC4A7 and MYO9B, demonstrated potential utility in distinguishing MIBC from NMIBC. Upregulation of CPT2 and palmitic acid in MIBC patients highlighted the dysregulation of physiological control mechanisms and enhanced pro-tumorigenic effects of lipid metabolic pathways.
Conclusions
Integrated multi-omics analysis reveals that key regulatory proteins such as SLC4A7 and MYO9B play pivotal roles in mediating the aggressive phenotypes of MIBC. Aberrant upregulation of CPT2 protein and metabolites like palmitic acid may drive malignant transformation from NMIBC to MIBC by promoting lipid metabolic reprogramming.
Significance
This study utilized LC-MS/MS to systematically profile the proteomic, phosphoproteomic, and metabolomic characteristics of MIBC, NMIBC, and adjacent noncancerous tissues, with the aim of identifying key molecules and metabolites driving bladder cancer progression. Our findings indicate that aberrant phosphorylation of regulatory proteins such as SLC4A7 and MYO9B may play a critical role in mediating the invasive phenotype of MIBC. In parallel, the upregulation of CPT2 and its associated metabolites (e.g., palmitic acid) suggests that lipid metabolic reprogramming, including enhanced β-oxidation and membrane phospholipid synthesis, may contribute to the malignant transition from NMIBC to MIBC. Overall, this study not only reveals potential molecules and metabolites driving bladder cancer progression but also provides a valuable reference for further exploration of pathways associated with bladder cancer invasiveness.
{"title":"Decoding bladder cancer aggressiveness: A proteomic, phosphoproteomic and metabolomic approach","authors":"Zhihui Feng , Biao Zhang , Yi Liu , Fei Yang , Yinping Lei , Songbai Liao , Xianliang Hou","doi":"10.1016/j.jprot.2025.105553","DOIUrl":"10.1016/j.jprot.2025.105553","url":null,"abstract":"<div><h3>Background</h3><div>Bladder cancer (BC) is the most common malignancy of the urinary system. However, the median survival of patients with metastatic bladder cancer remains limited. Thus, there is an urgent imperative to develop novel biomarkers for BC-targeted therapies and to conduct in-depth investigations into BC pathogenesis leveraging multi-omics technologies.</div></div><div><h3>Results</h3><div>Our results revealed that proteins co-upregulated in both proteomic and phosphoproteomic analysis, such as SLC4A7 and MYO9B, demonstrated potential utility in distinguishing MIBC from NMIBC. Upregulation of CPT2 and palmitic acid in MIBC patients highlighted the dysregulation of physiological control mechanisms and enhanced pro-tumorigenic effects of lipid metabolic pathways.</div></div><div><h3>Conclusions</h3><div>Integrated multi-omics analysis reveals that key regulatory proteins such as SLC4A7 and MYO9B play pivotal roles in mediating the aggressive phenotypes of MIBC. Aberrant upregulation of CPT2 protein and metabolites like palmitic acid may drive malignant transformation from NMIBC to MIBC by promoting lipid metabolic reprogramming.</div></div><div><h3>Significance</h3><div>This study utilized LC-MS/MS to systematically profile the proteomic, phosphoproteomic, and metabolomic characteristics of MIBC, NMIBC, and adjacent noncancerous tissues, with the aim of identifying key molecules and metabolites driving bladder cancer progression. Our findings indicate that aberrant phosphorylation of regulatory proteins such as SLC4A7 and MYO9B may play a critical role in mediating the invasive phenotype of MIBC. In parallel, the upregulation of CPT2 and its associated metabolites (e.g., palmitic acid) suggests that lipid metabolic reprogramming, including enhanced β-oxidation and membrane phospholipid synthesis, may contribute to the malignant transition from NMIBC to MIBC. Overall, this study not only reveals potential molecules and metabolites driving bladder cancer progression but also provides a valuable reference for further exploration of pathways associated with bladder cancer invasiveness.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105553"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145445248","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 : 2026-01-20Epub Date: 2025-11-17DOI: 10.1016/j.jprot.2025.105569
David Gomez-Zepeda , Felipe Cervantes-Hernández , Octavio Martínez , Neftalí Ochoa-Alejo , José Juan Ordaz-Ortiz
Chilli pepper (Capsicum annuum L., Solanaceae family), a horticultural crop native to the Americas, is esteemed for its flavour and bioactive compounds. To enhance our understanding of the molecular basis of chilli pepper fruit development and ripening, we characterised proteome dynamics from early fruit set to ripening in two Mexican cultivars. Protein profiles were analysed using label-free quantitative (LFQ) LC-MS proteomics with data-independent acquisition (DIA), resulting in the quantification of 421 proteins, predominantly involved in primary and secondary metabolism. The developmental stage was the primary factor driving proteome variation, with limited influence from cultivar differences. Clustering and relative quantification revealed intricate patterns of protein abundance across fruit maturation. Proteins associated with carotenoid and capsaicinoid biosynthesis exhibited temporal regulation consistent with their roles in determining fruit colour and pungency. This study provides insights into the molecular changes underlying chilli pepper fruit growth, development, and ripening and offers a proteomic overview for understanding some biochemical traits that define chilli pepper fruit characteristics.
Significance
Understanding the molecular basis of fruit growth, development, and ripening is crucial for enhancing crop quality and nutritional value. In chilli pepper, traits such as the production of valuable secondary metabolites, pigmentation, and pungency arise from finely regulated metabolic processes during fruit maturation. Here, we investigated these processes from a proteomics perspective, complemented with clustering and functional enrichment analyses. The findings reveal that developmental stage, rather than genotype, is the dominant factor shaping the fruit proteome, underscoring a conserved maturation program. Proteins involved in primary metabolism and specialised biosynthetic pathways, including those for carotenoids and capsaicinoids, show dynamic regulation consistent with their roles in fruit quality traits. These results contribute to the understanding of the molecular processes involved in fruit growth, development, and ripening and provide a proteomic framework for studying trait formation in Capsicum annuum and related Solanaceae crops.
{"title":"Proteome dynamics of chilli pepper fruits during development and ripening from two Mexican cultivars of Capsicum annuum L","authors":"David Gomez-Zepeda , Felipe Cervantes-Hernández , Octavio Martínez , Neftalí Ochoa-Alejo , José Juan Ordaz-Ortiz","doi":"10.1016/j.jprot.2025.105569","DOIUrl":"10.1016/j.jprot.2025.105569","url":null,"abstract":"<div><div>Chilli pepper (<em>Capsicum annuum</em> L., Solanaceae family), a horticultural crop native to the Americas, is esteemed for its flavour and bioactive compounds. To enhance our understanding of the molecular basis of chilli pepper fruit development and ripening, we characterised proteome dynamics from early fruit set to ripening in two Mexican cultivars. Protein profiles were analysed using label-free quantitative (LFQ) LC-MS proteomics with data-independent acquisition (DIA), resulting in the quantification of 421 proteins, predominantly involved in primary and secondary metabolism. The developmental stage was the primary factor driving proteome variation, with limited influence from cultivar differences. Clustering and relative quantification revealed intricate patterns of protein abundance across fruit maturation. Proteins associated with carotenoid and capsaicinoid biosynthesis exhibited temporal regulation consistent with their roles in determining fruit colour and pungency. This study provides insights into the molecular changes underlying chilli pepper fruit growth, development, and ripening and offers a proteomic overview for understanding some biochemical traits that define chilli pepper fruit characteristics.</div></div><div><h3>Significance</h3><div>Understanding the molecular basis of fruit growth, development, and ripening is crucial for enhancing crop quality and nutritional value. In chilli pepper, traits such as the production of valuable secondary metabolites, pigmentation, and pungency arise from finely regulated metabolic processes during fruit maturation. Here, we investigated these processes from a proteomics perspective, complemented with clustering and functional enrichment analyses. The findings reveal that developmental stage, rather than genotype, is the dominant factor shaping the fruit proteome, underscoring a conserved maturation program. Proteins involved in primary metabolism and specialised biosynthetic pathways, including those for carotenoids and capsaicinoids, show dynamic regulation consistent with their roles in fruit quality traits. These results contribute to the understanding of the molecular processes involved in fruit growth, development, and ripening and provide a proteomic framework for studying trait formation in <em>Capsicum annuum</em> and related Solanaceae crops.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105569"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145557108","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 : 2026-01-20Epub Date: 2025-11-11DOI: 10.1016/j.jprot.2025.105554
Jie Yu , Likang Xiao , Zhihua Yan , Xiaoyang Chen , Siyu Zhao , Congcong Yu , Yonghui Dong
Objective
Osteoarthritis (OA) is the most common degenerative joint disease mainly characterized by cartilage degradation, but the underlying molecular mechanisms remains unclear. This study aims to identify key genes, proteins, and pathways involved in OA progression using integrated transcriptomic and proteomic approaches.
Methods
Transcriptome and proteome profiles of lesioned (OA) and preserved (control) cartilage tissues were analyzed. Differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) were identified, followed by functional enrichment analysis. Key candidates were validated using qRT-PCR and western blotting. The role of ABI3BP in chondrocyte senescence and catabolism was investigated by siRNA knockdown, SA-β-gal staining, and immunofluorescence for senescence markers (P16, P21).
Results
Omics analysis revealed 24 significantly altered genes/proteins, including upregulated ABI3BP, TGFBI, ANOS1, S100A4, and TNFAIP6, and downregulated METTL7A. Enriched pathways included extracellular matrix (ECM) organization, ECM-receptor interaction, and phenylalanine metabolism. ABI3BP was notably elevated in OA cartilage. Its knockdown mitigated IL-1β-induced ECM degradation and reduced the level of senescence-associated markers, suggesting a protective effect against OA progression.
Conclusions
The results provided a comprehensive molecular profile of OA cartilage, highlighting ABI3BP as a regulator of chondrocyte senescence and ECM homeostasis. Targeting ABI3BP may offer a novel therapeutic strategy for OA treatment.
Significance of the study
This study provides multi-omics insights into the molecular mechanisms of osteoarthritis (OA), particularly focusing on cartilage degeneration. By integrating transcriptomic and proteomic analyses, we identified key dysregulated genes/proteins (ABI3BP, TGFBI, ANOS1, S100A4, TNFAIP6, and METTL7A) and highlighted their roles in extracellular matrix (ECM) disruption, cellular senescence, and inflammatory responses. Notably, ABI3BP was found to be upregulated in OA cartilage and functionally linked to chondrocyte catabolism and senescence. Its knockdown ameliorated IL-1β-induced damage, suggesting its potential as a novel therapeutic target for OA. These findings deepen our understanding of OA pathogenesis and pave the way for future targeted interventions.
{"title":"Integrated analysis of the transcriptome and proteome reveals that ABI3BP is involved in the pathogenesis of osteoarthritis","authors":"Jie Yu , Likang Xiao , Zhihua Yan , Xiaoyang Chen , Siyu Zhao , Congcong Yu , Yonghui Dong","doi":"10.1016/j.jprot.2025.105554","DOIUrl":"10.1016/j.jprot.2025.105554","url":null,"abstract":"<div><h3>Objective</h3><div>Osteoarthritis (OA) is the most common degenerative joint disease mainly characterized by cartilage degradation, but the underlying molecular mechanisms remains unclear. This study aims to identify key genes, proteins, and pathways involved in OA progression using integrated transcriptomic and proteomic approaches.</div></div><div><h3>Methods</h3><div>Transcriptome and proteome profiles of lesioned (OA) and preserved (control) cartilage tissues were analyzed. Differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) were identified, followed by functional enrichment analysis. Key candidates were validated using qRT-PCR and western blotting. The role of ABI3BP in chondrocyte senescence and catabolism was investigated by siRNA knockdown, SA-β-gal staining, and immunofluorescence for senescence markers (P16, P21).</div></div><div><h3>Results</h3><div>Omics analysis revealed 24 significantly altered genes/proteins, including upregulated ABI3BP, TGFBI, ANOS1, S100A4, and TNFAIP6, and downregulated METTL7A. Enriched pathways included extracellular matrix (ECM) organization, ECM-receptor interaction, and phenylalanine metabolism. ABI3BP was notably elevated in OA cartilage. Its knockdown mitigated IL-1β-induced ECM degradation and reduced the level of senescence-associated markers, suggesting a protective effect against OA progression.</div></div><div><h3>Conclusions</h3><div>The results provided a comprehensive molecular profile of OA cartilage, highlighting ABI3BP as a regulator of chondrocyte senescence and ECM homeostasis. Targeting ABI3BP may offer a novel therapeutic strategy for OA treatment.</div></div><div><h3>Significance of the study</h3><div>This study provides multi-omics insights into the molecular mechanisms of osteoarthritis (OA), particularly focusing on cartilage degeneration. By integrating transcriptomic and proteomic analyses, we identified key dysregulated genes/proteins (ABI3BP, TGFBI, ANOS1, S100A4, TNFAIP6, and METTL7A) and highlighted their roles in extracellular matrix (ECM) disruption, cellular senescence, and inflammatory responses. Notably, ABI3BP was found to be upregulated in OA cartilage and functionally linked to chondrocyte catabolism and senescence. Its knockdown ameliorated IL-1β-induced damage, suggesting its potential as a novel therapeutic target for OA. These findings deepen our understanding of OA pathogenesis and pave the way for future targeted interventions.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105554"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145513111","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 : 2026-01-20Epub Date: 2025-11-17DOI: 10.1016/j.jprot.2025.105570
Zeinab Ghasemishahrestani , Simone Santiago Carvalho de Oliveira , Rafaela dos Santos Moraes Francisco , Luis Felipe Costa Ramos , Renata Maria dos Santos , Gilberto Barbosa Domont , Nicolás A. Rey , André Luis Souza dos Santos , Fabio Cesar Sousa Nogueira , Marcos Dias Pereira
Cancer’s global burden highlights the urgent need for more effective therapies. Metal-based drugs, particularly Cu2-complexes, offer promising alternatives due to copper’s diverse biological functions. This study investigates the antitumor potential of two novel dinuclear Cu2-complexes, [Cu2(μ-CH3COO)(L)(OH2)]·2H2O (R9) and [Cu2(μ-OH)(HL)(OH2)]ClO4·2H2O (R10), in MCF-7 breast cancer cells. Both compounds exhibited greater cytotoxicity than cisplatin, with IC50 values of 1.01 ± 0.09 μM (R9) and 1.27 ± 0.14 μM (R10), while showing selectivity toward cancer cells, as indicated by higher IC50 values in healthy MCF10A cells. Treated MCF-7 cells showed increased granularity, mitochondrial membrane depolarization, and elevated reactive oxygen species. At IC50 concentrations, cell cycle analysis revealed Sub-G1 accumulation and DNA fragmentation (TUNEL assay), indicating apoptosis via intrinsic pathways, supported by caspase 9 activation. Label-free proteomics revealed distinct mechanisms for R10 compared to cisplatin. In R10-treated cells, key downregulated pathways included glycolysis, the TCA cycle, oxidative phosphorylation, PI3K–Akt signaling, and the ubiquitin–proteasome system. Apoptosis-related proteins such as structural proteins (ACTB, ACTG1, SPTAN1, TUBA4A), mitochondrial apoptotic factors (AIFM1), nuclear envelope components (LMNA), and stress-response regulators (JUN, EIF2S1) were dysregulated. Proteomics data is available via ProteomeXchange with identifier PXD064464. These findings support the potential of Cu2-complexes as effective antitumor agents with mechanisms distinct from cisplatin, offering superior efficacy through apoptosis induction.
Significance
Breast cancer remains one of the leading causes of cancer-related mortality among women, highlighting the need for more effective and selective therapeutic agents. While platinum-based drugs are widely used, their limitations call for novel alternatives. In this study, we demonstrate that two newly synthesized dinuclear copper(II) complexes, R9 and R10, exhibit strong cytotoxicity against MCF-7 breast cancer cells with higher selectivity compared to cisplatin. Through functional and proteomic analyses, we show that these compounds induce intrinsic apoptosis, disrupt cytoskeletal integrity, and modulate key signaling pathways such as PI3K-Akt and RAS-ERK. Our proteomic results reveal distinct molecular signatures for R10, underscoring its unique mechanism of action. Therefore, copper-based complexes represent promising candidates for breast cancer treatment, and proteomics provides critical insight into their therapeutic potential.
{"title":"Dinuclear Cu2+-complexes disrupt cellular pathways and rewire the breast cancer proteome","authors":"Zeinab Ghasemishahrestani , Simone Santiago Carvalho de Oliveira , Rafaela dos Santos Moraes Francisco , Luis Felipe Costa Ramos , Renata Maria dos Santos , Gilberto Barbosa Domont , Nicolás A. Rey , André Luis Souza dos Santos , Fabio Cesar Sousa Nogueira , Marcos Dias Pereira","doi":"10.1016/j.jprot.2025.105570","DOIUrl":"10.1016/j.jprot.2025.105570","url":null,"abstract":"<div><div>Cancer’s global burden highlights the urgent need for more effective therapies. Metal-based drugs, particularly Cu<sup>2</sup>-complexes, offer promising alternatives due to copper’s diverse biological functions. This study investigates the antitumor potential of two novel dinuclear Cu<sup>2</sup>-complexes, [Cu<sub>2</sub>(<em>μ</em>-CH<sub>3</sub>COO)(L)(OH<sub>2</sub>)]·2H<sub>2</sub>O (<strong>R9</strong>) and [Cu<sub>2</sub>(<em>μ</em>-OH)(HL)(OH<sub>2</sub>)]ClO<sub>4</sub>·2H<sub>2</sub>O (<strong>R10</strong>), in MCF-7 breast cancer cells. Both compounds exhibited greater cytotoxicity than cisplatin, with IC<sub>50</sub> values of 1.01 ± 0.09 μM (R9) and 1.27 ± 0.14 μM (<strong>R10</strong>), while showing selectivity toward cancer cells, as indicated by higher IC<sub>50</sub> values in healthy MCF10A cells. Treated MCF-7 cells showed increased granularity, mitochondrial membrane depolarization, and elevated reactive oxygen species. At IC<sub>50</sub> concentrations, cell cycle analysis revealed Sub-G1 accumulation and DNA fragmentation (TUNEL assay), indicating apoptosis via intrinsic pathways, supported by caspase 9 activation. Label-free proteomics revealed distinct mechanisms for <strong>R10</strong> compared to cisplatin. In <strong>R10</strong>-treated cells, key downregulated pathways included glycolysis, the TCA cycle, oxidative phosphorylation, PI3K–Akt signaling, and the ubiquitin–proteasome system. Apoptosis-related proteins such as structural proteins (ACTB, ACTG1, SPTAN1, TUBA4A), mitochondrial apoptotic factors (AIFM1), nuclear envelope components (LMNA), and stress-response regulators (JUN, EIF2S1) were dysregulated. Proteomics data is available via ProteomeXchange with identifier <span><span>PXD064464</span><svg><path></path></svg></span>. These findings support the potential of Cu<sup>2</sup>-complexes as effective antitumor agents with mechanisms distinct from cisplatin, offering superior efficacy through apoptosis induction.</div></div><div><h3>Significance</h3><div>Breast cancer remains one of the leading causes of cancer-related mortality among women, highlighting the need for more effective and selective therapeutic agents. While platinum-based drugs are widely used, their limitations call for novel alternatives. In this study, we demonstrate that two newly synthesized dinuclear copper(II) complexes, R9 and R10, exhibit strong cytotoxicity against MCF-7 breast cancer cells with higher selectivity compared to cisplatin. Through functional and proteomic analyses, we show that these compounds induce intrinsic apoptosis, disrupt cytoskeletal integrity, and modulate key signaling pathways such as PI3K-Akt and RAS-ERK. Our proteomic results reveal distinct molecular signatures for R10, underscoring its unique mechanism of action. Therefore, copper-based complexes represent promising candidates for breast cancer treatment, and proteomics provides critical insight into their therapeutic potential.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105570"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145557175","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 : 2026-01-20Epub Date: 2025-11-13DOI: 10.1016/j.jprot.2025.105567
Yulu Chen , Xun Zou , Lulu Wang , Yuan Gao , Hang Fu , Bin Liu , Haowen Jiang , Minjia Tan , Linhui Zhai
Proteomics research represents a critical area within modern biomedical science. Fixatives, as essential components in proteomic analysis, play a pivotal role in maintaining proteome stability and analytical accuracy, while also exerting a substantial influence on experimental outcomes. However, there remains a limited understanding of how various fixatives affect proteome stability. In this study, we conducted a systematic comparison of commonly used fixation agents—specifically formaldehyde, paraformaldehyde, methanol, and ethanol—to evaluate how they affect the stability of the cellular proteome. Our results show that different fixatives lead to notably different levels of proteome stability. Importantly, formaldehyde solution and paraformaldehyde produce cross-linking effects that can significantly reduce protein solubility and hinder the efficiency of enzymatic digestion. Additionally, we analyzed the proteomic profiles of both drug-treated and untreated samples under different fixation conditions to better understand how these conditions may affect the quality and interpretation of mass spectrometry data. These results provide a scientific basis for selecting the most appropriate fixative in proteomics research. Our study also contributes to improving the compatibility between fixatives and mass spectrometry technologies, offering dependable technical support for biological and biomedical research.
Significance
We conducted a systematic comparison of commonly used fixation agents—specifically formaldehyde, paraformaldehyde, methanol, and ethanol—to evaluate how they affect the stability of the cellular proteome. These results provide a scientific basis for selecting the most appropriate fixative in proteomics research. Our study also contributes to improving the compatibility between fixatives and mass spectrometry technologies, offering dependable technical support for biological and biomedical research.
{"title":"Systematic comparison of the effects of various fixatives on the stability of the cellular proteome","authors":"Yulu Chen , Xun Zou , Lulu Wang , Yuan Gao , Hang Fu , Bin Liu , Haowen Jiang , Minjia Tan , Linhui Zhai","doi":"10.1016/j.jprot.2025.105567","DOIUrl":"10.1016/j.jprot.2025.105567","url":null,"abstract":"<div><div>Proteomics research represents a critical area within modern biomedical science. Fixatives, as essential components in proteomic analysis, play a pivotal role in maintaining proteome stability and analytical accuracy, while also exerting a substantial influence on experimental outcomes. However, there remains a limited understanding of how various fixatives affect proteome stability. In this study, we conducted a systematic comparison of commonly used fixation agents—specifically formaldehyde, paraformaldehyde, methanol, and ethanol—to evaluate how they affect the stability of the cellular proteome. Our results show that different fixatives lead to notably different levels of proteome stability. Importantly, formaldehyde solution and paraformaldehyde produce cross-linking effects that can significantly reduce protein solubility and hinder the efficiency of enzymatic digestion. Additionally, we analyzed the proteomic profiles of both drug-treated and untreated samples under different fixation conditions to better understand how these conditions may affect the quality and interpretation of mass spectrometry data. These results provide a scientific basis for selecting the most appropriate fixative in proteomics research. Our study also contributes to improving the compatibility between fixatives and mass spectrometry technologies, offering dependable technical support for biological and biomedical research.</div></div><div><h3>Significance</h3><div>We conducted a systematic comparison of commonly used fixation agents—specifically formaldehyde, paraformaldehyde, methanol, and ethanol—to evaluate how they affect the stability of the cellular proteome. These results provide a scientific basis for selecting the most appropriate fixative in proteomics research. Our study also contributes to improving the compatibility between fixatives and mass spectrometry technologies, offering dependable technical support for biological and biomedical research.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105567"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145530416","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 : 2026-01-20Epub Date: 2025-11-21DOI: 10.1016/j.jprot.2025.105572
José Jesús Encinas-Arzate , Damaristelma De Jesús-Campos , Enrique Márquez-Ríos , Magdalena Hernández-Ortiz , Martha Beatriz Morales-Amparano , José Antonio López-Elías , Wilfrido Torres-Arreola , Sergio Encarnación-Guevara , Juan Carlos Ramírez-Suárez , María Gisela Carvallo-Ruiz , Esaú Bojórquez-Velázquez , José Ángel Huerta-Ocampo
Nutrient limitations alter microalgae metabolism and fatty acid biosynthesis, modifying the lipid profile and improving the chemical properties of the resulting biodiesel. This study investigated the effects of nitrate and silicate limitations on the proteomic and lipid profiles in Navicula incerta and its biodiesel potential. Proteomic analysis revealed 287 differentially abundant proteins (DAPs) out of 558 identified, with silicate-limited conditions showing the highest number of DAPs (212) and nitrogen-limited conditions showing 125 DAPs (log2FC > |0.57|, p-value <0.05). These changes underpin two distinct survival strategies: nitrogen limitation forced a broad metabolic shutdown, while silicate limitation prompted a strategic reallocation of resources, maintaining core biosynthesis and enhancing internal recycling. Both stresses induced a convergent lipid remodeling characterized by decreases in saturated fatty acids and increases in monounsaturated and polyunsaturated fatty acids. This shift optimizes membrane fluidity for stress resistance and enhances key biodiesel properties, confirming the potential of N. incerta as a sustainable source of high-quality biodiesel. These findings highlight the diatom's metabolic flexibility in optimizing resource allocation for survival, a trait that can be harnessed to tailor lipid profiles for advanced biofuel applications. Future work should focus on the impact, efficiency, and economic feasibility of cultivating N. incerta under nutrient stress.
Significance
Understanding the relationship between nitrogen and silicate concentrations and protein accumulation is crucial for harnessing the potential of microalgae in various industries and addressing environmental challenges. The proteomic analysis and lipid profiles obtained in N. incerta under nitrogen and silicate limitation demonstrate remarkable metabolic flexibility in response to nutrient limitations, optimizing resource allocation to ensure survival under nutrient stress. Nitrogen limitation drives the accumulation of carbohydrates and dry biomass, along with lipid mobilization and a shift toward unsaturated fatty acids, enhancing energy efficiency and membrane functionality. Silicate limitation, while having a milder effect, prioritizes resource conservation and the redistribution of existing reserves. These adaptive responses highlight the diatom's ability to maintain critical cellular functions under adverse conditions and reveal its potential for biodiesel production.
{"title":"Nitrate and silicate limitations induce metabolic reprogramming and lipid redistribution in Navicula incerta, enhancing biodiesel properties","authors":"José Jesús Encinas-Arzate , Damaristelma De Jesús-Campos , Enrique Márquez-Ríos , Magdalena Hernández-Ortiz , Martha Beatriz Morales-Amparano , José Antonio López-Elías , Wilfrido Torres-Arreola , Sergio Encarnación-Guevara , Juan Carlos Ramírez-Suárez , María Gisela Carvallo-Ruiz , Esaú Bojórquez-Velázquez , José Ángel Huerta-Ocampo","doi":"10.1016/j.jprot.2025.105572","DOIUrl":"10.1016/j.jprot.2025.105572","url":null,"abstract":"<div><div>Nutrient limitations alter microalgae metabolism and fatty acid biosynthesis, modifying the lipid profile and improving the chemical properties of the resulting biodiesel. This study investigated the effects of nitrate and silicate limitations on the proteomic and lipid profiles in <em>Navicula incerta</em> and its biodiesel potential. Proteomic analysis revealed 287 differentially abundant proteins (DAPs) out of 558 identified, with silicate-limited conditions showing the highest number of DAPs (212) and nitrogen-limited conditions showing 125 DAPs (log2FC > |0.57|, <em>p</em>-value <0.05). These changes underpin two distinct survival strategies: nitrogen limitation forced a broad metabolic shutdown, while silicate limitation prompted a strategic reallocation of resources, maintaining core biosynthesis and enhancing internal recycling. Both stresses induced a convergent lipid remodeling characterized by decreases in saturated fatty acids and increases in monounsaturated and polyunsaturated fatty acids. This shift optimizes membrane fluidity for stress resistance and enhances key biodiesel properties, confirming the potential of <em>N. incerta</em> as a sustainable source of high-quality biodiesel. These findings highlight the diatom's metabolic flexibility in optimizing resource allocation for survival, a trait that can be harnessed to tailor lipid profiles for advanced biofuel applications. Future work should focus on the impact, efficiency, and economic feasibility of cultivating <em>N. incerta</em> under nutrient stress.</div></div><div><h3>Significance</h3><div>Understanding the relationship between nitrogen and silicate concentrations and protein accumulation is crucial for harnessing the potential of microalgae in various industries and addressing environmental challenges. The proteomic analysis and lipid profiles obtained in <em>N. incerta</em> under nitrogen and silicate limitation demonstrate remarkable metabolic flexibility in response to nutrient limitations, optimizing resource allocation to ensure survival under nutrient stress. Nitrogen limitation drives the accumulation of carbohydrates and dry biomass, along with lipid mobilization and a shift toward unsaturated fatty acids, enhancing energy efficiency and membrane functionality. Silicate limitation, while having a milder effect, prioritizes resource conservation and the redistribution of existing reserves. These adaptive responses highlight the diatom's ability to maintain critical cellular functions under adverse conditions and reveal its potential for biodiesel production.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105572"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145587965","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 : 2026-01-20Epub Date: 2025-11-03DOI: 10.1016/j.jprot.2025.105555
Xiaofang Xie , Binghui Zhang , Xin Chen , Jiaqi Tang , Zhen Qiu , Chenghao Shen , Lingli Lian , Gang Gu , Xiangmin Lin , Farman Ali
Aeromonas hydrophila, an antibiotic-and multi-metal-resistant pathogen, threatens aquaculture, yet its adaptation to cadmium chloride (CdCl₂) remains poorly understood. A.hydrophilla was grown with CdCl₂ exposure, and data-independent acquisition (DIA)-based proteomics was employed to identify the 253 upregulated and 163 downregulated proteins. GO and KEGG enrichment analyses revealed upregulated proteins involved in sulfate metabolism, siderophore- and iron transport, and other transition metal transport processes, while downregulated proteins were linked to amino acid metabolism, the tricarboxylic acid (TCA) cycle, and fatty acid metabolism. Gene Set Enrichment Analysis (GSEA) indicated positive enrichment of ribosome, RNA degradation, sulfur metabolism, and riboflavin metabolism, and negative enrichment of valine, leucine, and isoleucine degradation, two-component system, and bacterial chemotaxis. CdCl₂ tolerance assays of four gene-deletion mutants confirmed proteomics predictions: ΔAHA_3605 (uncharacterized protein) and ΔAHA_1796 (GGDEF-domain deletion) were significantly more sensitive to CdCl₂, suggesting a role for cyclic di-GMP (c-di-GMP) signaling in cadmium resistance. Collectively, these results provide novel insights into the adaptive strategies of A. hydrophila, revealing key molecular determinants of CdCl₂ stress tolerance.
Significance
Cadmium contamination increasingly co-selects for antibiotic-resistant pathogens in aquaculture, yet the molecular basis of cadmium tolerance in Aeromonas hydrophila—one of the most problematic Gram-negative fish pathogens—has been unknown. Here, we provide the first comprehensive, DIA-based quantitative proteome map of A. hydrophila under CdCl₂ stress, uncovering a tightly orchestrated stress response that simultaneously remodels metal acquisition, down-regulates energy-intensive metabolism (TCA cycle, branched chain amino acid and fatty acid degradation), and activates sulfur-riboflavin-ribosome pathways to repair oxidative damage. Functional validation using gene-deletion mutants demonstrated that loss of AHA_3605 (uncharacterized) or AHA_1796 (GGDEF domain protein) significantly increased CdCl₂ sensitivity, implicating cyclic di-GMP signaling in cadmium resistance. These findings advance our mechanistic understanding of heavy-metal adaptation in aquatic pathogens and offer proteome informed targets to disrupt metal-driven co-selection of multidrug-resistant A. hydrophila in aquaculture ecosystems.
{"title":"Quantitative proteomic analysis of cadmium chloride tolerance mechanisms in Aeromonas hydrophila","authors":"Xiaofang Xie , Binghui Zhang , Xin Chen , Jiaqi Tang , Zhen Qiu , Chenghao Shen , Lingli Lian , Gang Gu , Xiangmin Lin , Farman Ali","doi":"10.1016/j.jprot.2025.105555","DOIUrl":"10.1016/j.jprot.2025.105555","url":null,"abstract":"<div><div><em>Aeromonas hydrophila</em>, an antibiotic-and multi-metal-resistant pathogen, threatens aquaculture, yet its adaptation to cadmium chloride (CdCl₂) remains poorly understood. <em>A.hydrophilla</em> was grown with CdCl₂ exposure, and data-independent acquisition (DIA)-based proteomics was employed to identify the 253 upregulated and 163 downregulated proteins. GO and KEGG enrichment analyses revealed upregulated proteins involved in sulfate metabolism, siderophore- and iron transport, and other transition metal transport processes, while downregulated proteins were linked to amino acid metabolism, the tricarboxylic acid (TCA) cycle, and fatty acid metabolism. Gene Set Enrichment Analysis (GSEA) indicated positive enrichment of ribosome, RNA degradation, sulfur metabolism, and riboflavin metabolism, and negative enrichment of valine, leucine, and isoleucine degradation, two-component system, and bacterial chemotaxis. CdCl₂ tolerance assays of four gene-deletion mutants confirmed proteomics predictions: <em>ΔAHA_3605</em> (uncharacterized protein) and <em>ΔAHA_1796</em> (GGDEF-domain deletion) were significantly more sensitive to CdCl₂, suggesting a role for cyclic di-GMP (<em>c</em>-di-GMP) signaling in cadmium resistance. Collectively, these results provide novel insights into the adaptive strategies of <em>A. hydrophila</em>, revealing key molecular determinants of CdCl₂ stress tolerance.</div></div><div><h3>Significance</h3><div>Cadmium contamination increasingly co-selects for antibiotic-resistant pathogens in aquaculture, yet the molecular basis of cadmium tolerance in <em>Aeromonas hydrophila</em>—one of the most problematic Gram-negative fish pathogens—has been unknown. Here, we provide the first comprehensive, DIA-based quantitative proteome map of <em>A. hydrophila</em> under CdCl₂ stress, uncovering a tightly orchestrated stress response that simultaneously remodels metal acquisition, down-regulates energy-intensive metabolism (TCA cycle, branched chain amino acid and fatty acid degradation), and activates sulfur-riboflavin-ribosome pathways to repair oxidative damage. Functional validation using gene-deletion mutants demonstrated that loss of <em>AHA_3605</em> (uncharacterized) or <em>AHA_1796</em> (GGDEF domain protein) significantly increased CdCl₂ sensitivity, implicating cyclic di-GMP signaling in cadmium resistance. These findings advance our mechanistic understanding of heavy-metal adaptation in aquatic pathogens and offer proteome informed targets to disrupt metal-driven co-selection of multidrug-resistant <em>A. hydrophila</em> in aquaculture ecosystems.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105555"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145452343","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 : 2026-01-20Epub Date: 2025-11-08DOI: 10.1016/j.jprot.2025.105556
Lin Wang , Wenmin Tian , Sen Wang , Yuhong Liu , Hongli Wang , Junjie Xiao , Zhongkuo Yu , Lixin Xie , Yang Chen
{"title":"Corrigendum to “Serum proteomics identifies biomarkers for predicting non-survivors in elderly COVID-19 patients” [Journal of Proteomics, volume 311 (2025) 105356].","authors":"Lin Wang , Wenmin Tian , Sen Wang , Yuhong Liu , Hongli Wang , Junjie Xiao , Zhongkuo Yu , Lixin Xie , Yang Chen","doi":"10.1016/j.jprot.2025.105556","DOIUrl":"10.1016/j.jprot.2025.105556","url":null,"abstract":"","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105556"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145482406","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 : 2026-01-20Epub Date: 2025-11-13DOI: 10.1016/j.jprot.2025.105566
Orlando Morales-Tarré , Xitlally Popa-Navarro , Alberto Paradela , Magdalena Hernández-Ortiz , Oscar Arrieta , Fernando Corrales , Sergio Encarnación-Guevara
Ubiquitination is a multifaceted post-translational modification that plays a crucial role in regulating the degradation of unnecessary cellular proteins and is involved in various cellular processes, including protein export via extracellular vesicles. We investigate how alterations in the intracellular levels of ubiquitinated proteins affect vesicle protein content in BEAS-2B cells. We increased the intracellular levels of ubiquitinated proteins by inhibiting proteasomal degradation with MG-132 and by blocking deubiquitinating enzymes using PR-619. Using centrifugation and ultracentrifugation, were isolate various vesicle types, specifically the largest vesicles (enriched in plasma membrane-derived microvesicles) and the smallest vesicles (enriched in endosomal exosomes). High-resolution mass spectrometry-based proteomics was utilized to quantify their protein content. The content of extracellular vesicles changed in response to both treatments, reflecting cellular changes and the export of stress signals. The increase in intracellular levels of ubiquitinated proteins induced metabolic stress in the cells, generally leading to a reduction in protein translation, an enhanced response to oxidative stress, changes in membrane transport, and alterations in cell-microenvironment interactions. The modifications observed in the vesicular proteome suggest that ubiquitination plays a significant role in regulating protein export. This regulation can be mastered for diagnostic purposes and for describing cells and tissues through liquid biopsies.
Significance
Ubiquitination is one of the most abundant post-translational modifications in cells, and its role, beyond marking proteins for degradation, is not fully understood. Characterizing the effect of this modification on protein export to extracellular vesicles can shed light on how a cell selects its contents to influence its microenvironment, send signals to distant tissues, or interact with the immune system. This is particularly relevant in the context of pathologies such as cancer, which hijacks the cellular vesicle-producing machinery and adapts it to its needs to influence the remodeling of its surroundings. Understanding how a cell regulates the specific contents of its vesicles may point the way toward the development of treatments or superior diagnostic and classification tools.
{"title":"The dynamics of ubiquitination and its role within the proteome of extracellular vesicles","authors":"Orlando Morales-Tarré , Xitlally Popa-Navarro , Alberto Paradela , Magdalena Hernández-Ortiz , Oscar Arrieta , Fernando Corrales , Sergio Encarnación-Guevara","doi":"10.1016/j.jprot.2025.105566","DOIUrl":"10.1016/j.jprot.2025.105566","url":null,"abstract":"<div><div>Ubiquitination is a multifaceted post-translational modification that plays a crucial role in regulating the degradation of unnecessary cellular proteins and is involved in various cellular processes, including protein export via extracellular vesicles. We investigate how alterations in the intracellular levels of ubiquitinated proteins affect vesicle protein content in BEAS-2B cells. We increased the intracellular levels of ubiquitinated proteins by inhibiting proteasomal degradation with MG-132 and by blocking deubiquitinating enzymes using PR-619. Using centrifugation and ultracentrifugation, were isolate various vesicle types, specifically the largest vesicles (enriched in plasma membrane-derived microvesicles) and the smallest vesicles (enriched in endosomal exosomes). High-resolution mass spectrometry-based proteomics was utilized to quantify their protein content. The content of extracellular vesicles changed in response to both treatments, reflecting cellular changes and the export of stress signals. The increase in intracellular levels of ubiquitinated proteins induced metabolic stress in the cells, generally leading to a reduction in protein translation, an enhanced response to oxidative stress, changes in membrane transport, and alterations in cell-microenvironment interactions. The modifications observed in the vesicular proteome suggest that ubiquitination plays a significant role in regulating protein export. This regulation can be mastered for diagnostic purposes and for describing cells and tissues through liquid biopsies.</div></div><div><h3>Significance</h3><div>Ubiquitination is one of the most abundant post-translational modifications in cells, and its role, beyond marking proteins for degradation, is not fully understood. Characterizing the effect of this modification on protein export to extracellular vesicles can shed light on how a cell selects its contents to influence its microenvironment, send signals to distant tissues, or interact with the immune system. This is particularly relevant in the context of pathologies such as cancer, which hijacks the cellular vesicle-producing machinery and adapts it to its needs to influence the remodeling of its surroundings. Understanding how a cell regulates the specific contents of its vesicles may point the way toward the development of treatments or superior diagnostic and classification tools.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105566"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145530566","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 : 2026-01-20Epub Date: 2025-10-17DOI: 10.1016/j.jprot.2025.105538
Jhenifer Yonara de Lima , Emanuella de Castro Andreassa , Marlon Dias Mariano Santos , Paulo Costa Carvalho , Tatiana de Arruda Campos Brasil de Souza
L-Asparaginase derived from Escherichia coli (EcA) is extensively employed in the treatment of acute lymphoblastic leukemia (ALL); however, immunogenic responses frequently result in therapeutic failure. This study aims to identify residues involved in EcA immunogenicity and to propose structural modifications that may reduce antigenic potential or facilitate the development of targeted therapeutic strategies to inhibit such interactions. To achieve this, we investigate the sites of interaction of HLA-DRB1*07:01 allele and EcA by crosslinking mass spectrometry (XL-MS) using recombinantly expressed proteins. Circular dichroism (CD) spectroscopy confirmed the proper folding of the expressed enzymes, ensuring their structural integrity. Additionally, XL-MS allowed us to experimentally determine the structure of HLA-DRB1*07:01, which was previously unknown. Structural analysis and sequence alignment revealed immunogenic epitopes on the enzyme surface, particularly near the active site and at K288, a highly reactive residue. Our findings highlight key immunogenic sites on EcA, particularly residues 53–58, 283–289, and K288, which represent promising targets for reducing immunogenicity while preserving enzymatic function. These proposed modifications should be experimentally validated to ensure enzyme activity is maintained while effectively mitigating immune responses.
Significance
Hypersensitivity to Escherichia coli-derived L-asparaginase (EcA) remains one of the main challenges in the treatment of acute lymphoblastic leukemia (ALL), often forcing patients to interrupt a therapy that could be lifesaving. In this study, we mapped at the structural level how EcA interacts with the HLA-DRB1*07:01 allele, one of the main genetic factors associated with these adverse reactions. By identifying specific regions of the enzyme that trigger the immune response, especially residue K288, we offer a foundation for targeted modifications that may reduce immunogenicity without affecting enzymatic activity. These findings provide a concrete path toward developing safer EcA variants and bring us closer to more effective and personalized treatments.
{"title":"Hypersensitivity and L-Asparaginase: A structural analysis of the interaction with HLA-DRB1*07:01","authors":"Jhenifer Yonara de Lima , Emanuella de Castro Andreassa , Marlon Dias Mariano Santos , Paulo Costa Carvalho , Tatiana de Arruda Campos Brasil de Souza","doi":"10.1016/j.jprot.2025.105538","DOIUrl":"10.1016/j.jprot.2025.105538","url":null,"abstract":"<div><div>L-Asparaginase derived from <em>Escherichia coli</em> (EcA) is extensively employed in the treatment of acute lymphoblastic leukemia (ALL); however, immunogenic responses frequently result in therapeutic failure. This study aims to identify residues involved in EcA immunogenicity and to propose structural modifications that may reduce antigenic potential or facilitate the development of targeted therapeutic strategies to inhibit such interactions. To achieve this, we investigate the sites of interaction of HLA-DRB1*07:01 allele and EcA by crosslinking mass spectrometry (XL-MS) using recombinantly expressed proteins. Circular dichroism (CD) spectroscopy confirmed the proper folding of the expressed enzymes, ensuring their structural integrity. Additionally, XL-MS allowed us to experimentally determine the structure of HLA-DRB1*07:01, which was previously unknown. Structural analysis and sequence alignment revealed immunogenic epitopes on the enzyme surface, particularly near the active site and at K288, a highly reactive residue. Our findings highlight key immunogenic sites on EcA, particularly residues 53–58, 283–289, and K288, which represent promising targets for reducing immunogenicity while preserving enzymatic function. These proposed modifications should be experimentally validated to ensure enzyme activity is maintained while effectively mitigating immune responses.</div></div><div><h3>Significance</h3><div>Hypersensitivity to <em>Escherichia coli</em>-derived L-asparaginase (EcA) remains one of the main challenges in the treatment of acute lymphoblastic leukemia (ALL), often forcing patients to interrupt a therapy that could be lifesaving. In this study, we mapped at the structural level how EcA interacts with the HLA-DRB1*07:01 allele, one of the main genetic factors associated with these adverse reactions. By identifying specific regions of the enzyme that trigger the immune response, especially residue K288, we offer a foundation for targeted modifications that may reduce immunogenicity without affecting enzymatic activity. These findings provide a concrete path toward developing safer EcA variants and bring us closer to more effective and personalized treatments.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105538"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145329551","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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