Pub Date : 2026-01-20DOI: 10.1016/j.jprot.2026.105601
Thais Mingatos de Toledo , Hellen Paula Valerio , Antônio Moreira Marques Neto , Simon Ngao Mule , Priscila Robertina dos Santos Donado , Claudia Blanes Angeli Pascale , Giuseppe Palmisano
Monoclonal antibodies are a class of biotherapeutic proteins that have been developed over the past decade, leading to improved standards of care for the treatment of multiple diseases. Multi-attribute methods have emerged as powerful tools for critical quality attributes (CQAs). They leverage high-resolution accurate mass spectrometry and automated computational pipelines to identify pre-established modifications using DDA. In this study, we describe the development of a mass spectrometry–based workflow capable of processing up to 96 samples simultaneously while monitoring a broad panel of PTMs. We evaluated microwave-assisted digestion under different buffers and pHs, assessing sequence coverage, missed cleavages, and the occurrence of chemical artifacts. Analyses were performed using both DDA and DIA. Raw data were processed in dependent-peptide search(DDA) and PTM-probing search(DIA), enabling PTM discovery without prior knowledge. Our results demonstrate that microwave-assisted digestion, combined with control of temperature and pH, provides a fast and reliable alternative for efficiently digesting biotherapeutic proteins. It achieves high sequence coverage while minimizing artificial PTM formation. We also show that DIA combined with MW digestion improved peptide identification, highlighting its potential for comprehensive characterization of antibodies. Among the tested buffers, sodium acetate under MW conditions was the most effective in reducing deamidation and oxidation levels.
Significance
This study presents a detailed and optimized protocol for microwave-assisted (MW) protein digestion, enabling simultaneous reduction and alkylation for antibody samples. The method is rapid and minimizes chemical artifacts typically introduced during sample preparation. By combining MW-assisted digestion with both data-dependent (DDA) and data-independent acquisition (DIA), we performed a comprehensive and unbiased multi-attribute analysis (MAM). Notably, the use of DIA alongside MW digestion allowed for higher reproducibility and more complete peptide and post-translational modification (PTM) detection compared to DDA alone. Compared to conventional overnight digestion, MW-assisted digestion significantly reduced deamidation levels, with evident influences of buffer composition and pH on PTM identification. Although the levels of protein oxidation persisted, indicating that further optimization is necessary, this approach substantially decreased other artifacts, particularly deamidation, highlighting its potential as a fast, reliable, and highly informative strategy for antibody characterization.
{"title":"Rapid high-throughput antibody analysis using microwave-assisted digestion","authors":"Thais Mingatos de Toledo , Hellen Paula Valerio , Antônio Moreira Marques Neto , Simon Ngao Mule , Priscila Robertina dos Santos Donado , Claudia Blanes Angeli Pascale , Giuseppe Palmisano","doi":"10.1016/j.jprot.2026.105601","DOIUrl":"10.1016/j.jprot.2026.105601","url":null,"abstract":"<div><div>Monoclonal antibodies are a class of biotherapeutic proteins that have been developed over the past decade, leading to improved standards of care for the treatment of multiple diseases. Multi-attribute methods have emerged as powerful tools for critical quality attributes (CQAs). They leverage high-resolution accurate mass spectrometry and automated computational pipelines to identify pre-established modifications using DDA. In this study, we describe the development of a mass spectrometry–based workflow capable of processing up to 96 samples simultaneously while monitoring a broad panel of PTMs. We evaluated microwave-assisted digestion under different buffers and pHs, assessing sequence coverage, missed cleavages, and the occurrence of chemical artifacts. Analyses were performed using both DDA and DIA. Raw data were processed in dependent-peptide search(DDA) and PTM-probing search(DIA), enabling PTM discovery without prior knowledge. Our results demonstrate that microwave-assisted digestion, combined with control of temperature and pH, provides a fast and reliable alternative for efficiently digesting biotherapeutic proteins. It achieves high sequence coverage while minimizing artificial PTM formation. We also show that DIA combined with MW digestion improved peptide identification, highlighting its potential for comprehensive characterization of antibodies. Among the tested buffers, sodium acetate under MW conditions was the most effective in reducing deamidation and oxidation levels.</div></div><div><h3>Significance</h3><div>This study presents a detailed and optimized protocol for microwave-assisted (MW) protein digestion, enabling simultaneous reduction and alkylation for antibody samples. The method is rapid and minimizes chemical artifacts typically introduced during sample preparation. By combining MW-assisted digestion with both data-dependent (DDA) and data-independent acquisition (DIA), we performed a comprehensive and unbiased multi-attribute analysis (MAM). Notably, the use of DIA alongside MW digestion allowed for higher reproducibility and more complete peptide and post-translational modification (PTM) detection compared to DDA alone. Compared to conventional overnight digestion, MW-assisted digestion significantly reduced deamidation levels, with evident influences of buffer composition and pH on PTM identification. Although the levels of protein oxidation persisted, indicating that further optimization is necessary, this approach substantially decreased other artifacts, particularly deamidation, highlighting its potential as a fast, reliable, and highly informative strategy for antibody characterization.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"326 ","pages":"Article 105601"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146030053","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}
Ovarian granulosa cells (Gc) play a vital role in follicle maturation and successful ovulation. Omentin-1 (ITLN1) is an adipokine involved in energy metabolism and insulin resistance; its expression has been demonstrated in the ovary and varies depending on the degree of pig fatness. However, its effect on the global proteome of Gc has not been previously investigated. It was hypothesized that ITLN1 affects the abundance of proteins involved in key processes occurring in Gc in pigs. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of Gc identified 208 significantly differentially abundant proteins (DAPs) in the Large White pigs, with 99 proteins upregulated and 109 downregulated. In fatter Meishan pigs, 42 statistically significant DAPs were identified, including 25 upregulated and 17 downregulated proteins. The identified DAPs were associated with the estrogen signaling pathway, cell cycle and DNA replication, protein synthesis, transport and maturation, cytoskeleton dynamics, cell signaling, and hormonal regulation. Notably, the number and identity of DAPs differed markedly between the two breeds, suggesting that ITLN1-mediated effects are modulated by fatness and breed-specific metabolic status. To further illustrate the observed differences, selected proteins were also analyzed using Western blotting and ELISA, which were consistent with the LC-MS/MS findings. The results indicate that ITLN1 has a modulatory influence on the porcine Gc proteome, which is dependent on fat content. This highlights the important role of ITLN1 in regulating ovarian functions.
Significance
This study provides a comprehensive proteomic analysis of porcine granulosa cells (Gc) after treatment with omentin-1 (ITLN1) in pigs with different fat content (Large White < Meishan). The identified differentially abundant proteins (DAPs) are intricately linked to critical biological pathways, including estrogen signaling, cell cycle regulation, DNA replication, protein synthesis and transport, cytoskeleton organization, and hormonal regulation. These findings enhance our understanding of the molecular mechanisms underpinning ovarian follicle development and breed-related reproductive traits in pigs. The insights gained could inform future strategies to improve fertility and reproductive efficiency in swine production, as well as provide a valuable resource for comparative studies on ovarian biology across species.
{"title":"The in vitro effect of omentin-1 on the global proteome of granulosa cells from normal weight Large White and fat Meishan pigs","authors":"Karolina Pich , Natalia Respekta-Długosz , Edyta Rytelewska , Bianka Świderska , Agata Malinowska , Nina Smolińska , Joëlle Dupont , Agnieszka Rak","doi":"10.1016/j.jprot.2026.105606","DOIUrl":"10.1016/j.jprot.2026.105606","url":null,"abstract":"<div><div>Ovarian granulosa cells (Gc) play a vital role in follicle maturation and successful ovulation. Omentin-1 (ITLN1) is an adipokine involved in energy metabolism and insulin resistance; its expression has been demonstrated in the ovary and varies depending on the degree of pig fatness. However, its effect on the global proteome of Gc has not been previously investigated. It was hypothesized that ITLN1 affects the abundance of proteins involved in key processes occurring in Gc in pigs. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of Gc identified 208 significantly differentially abundant proteins (DAPs) in the Large White pigs, with 99 proteins upregulated and 109 downregulated. In fatter Meishan pigs, 42 statistically significant DAPs were identified, including 25 upregulated and 17 downregulated proteins. The identified DAPs were associated with the estrogen signaling pathway, cell cycle and DNA replication, protein synthesis, transport and maturation, cytoskeleton dynamics, cell signaling, and hormonal regulation. Notably, the number and identity of DAPs differed markedly between the two breeds, suggesting that ITLN1-mediated effects are modulated by fatness and breed-specific metabolic status. To further illustrate the observed differences, selected proteins were also analyzed using Western blotting and ELISA, which were consistent with the LC-MS/MS findings. The results indicate that ITLN1 has a modulatory influence on the porcine Gc proteome, which is dependent on fat content. This highlights the important role of ITLN1 in regulating ovarian functions.</div></div><div><h3>Significance</h3><div>This study provides a comprehensive proteomic analysis of porcine granulosa cells (Gc) after treatment with omentin-1 (ITLN1) in pigs with different fat content (Large White < Meishan). The identified differentially abundant proteins (DAPs) are intricately linked to critical biological pathways, including estrogen signaling, cell cycle regulation, DNA replication, protein synthesis and transport, cytoskeleton organization, and hormonal regulation. These findings enhance our understanding of the molecular mechanisms underpinning ovarian follicle development and breed-related reproductive traits in pigs. The insights gained could inform future strategies to improve fertility and reproductive efficiency in swine production, as well as provide a valuable resource for comparative studies on ovarian biology across species.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"326 ","pages":"Article 105606"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146030022","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 : 2026-01-20DOI: 10.1016/j.jprot.2026.105607
Marcella N. Melo-Braga , Gabriela C. Magalhães , Filipe A. Silva , Frank Kjeldsen , Martin R. Larsen , Robson A.S. Santos , Thiago Verano-Braga
Angiotensin-(1–7) [Ang-(1–7)] is a heptapeptide of the renin-angiotensin system (RAS) with antitumoral effects reported in various tumoral cell lines, including the human lung adenocarcinoma A549 lineage. While previous studies have shown that Ang-(1–7) modulates MAPK and PI3K-AKT signaling, the precise molecular mechanisms involved remain incompletely understood. To investigate the signaling events of Ang-(1–7) in lung cancer-derived cells, we employed an integrated proteomic and phosphoproteomic approach in A549 cells. We analyzed early (minutes) and late (hours) molecular responses to Ang-(1–7) treatment. The treatment resulted in time-dependent modulation of multiple signaling pathways, including significant alterations in the MAPK, PI3K-AKT, and mTOR pathways at both the protein and phosphorylation levels. Notably, widespread early dephosphorylation events were observed, similar to the effects seen with other RAS peptides with antitumoral effects. Additionally, Ang-(1–7) promoted a long-lasting nuclear accumulation (up to 24 h) of the transcription factor FOXO1 indicating its activation. FOXO1 is known to regulate genes involved in apoptosis, cell cycle arrest, and oxidative stress, suggesting a role in mediating the peptide's antitumoral effects. The study provides new insights into the molecular basis of Ang-(1–7)’s antitumoral activity in A549 cells and reinforce its therapeutic potential in lung cancer. Raw data are available via ProteomeXchange with identifier PXD066687.
Significance
This study provides the first comprehensive, time-resolved proteomic and phosphoproteomic analysis of Angiotensin-(1–7) signaling in the lung cancer cell line A549. By capturing both early and late molecular events in A549 cells, we reveal that Ang-(1–7) modulates critical pathways involved in tumor progression, including MAPK, PI3K-AKT, and mTOR signaling. Importantly, we demonstrate the nuclear accumulation of FOXO1, a key transcription factor associated with tumor suppression, as part of the Ang-(1–7) response in A549 cells.
{"title":"Time-resolved proteomic and phosphoproteomic profiling of Angiotensin-(1–7) signaling in A549 cells","authors":"Marcella N. Melo-Braga , Gabriela C. Magalhães , Filipe A. Silva , Frank Kjeldsen , Martin R. Larsen , Robson A.S. Santos , Thiago Verano-Braga","doi":"10.1016/j.jprot.2026.105607","DOIUrl":"10.1016/j.jprot.2026.105607","url":null,"abstract":"<div><div>Angiotensin-(1–7) [Ang-(1–7)] is a heptapeptide of the renin-angiotensin system (RAS) with antitumoral effects reported in various tumoral cell lines, including the human lung adenocarcinoma A549 lineage. While previous studies have shown that Ang-(1–7) modulates MAPK and PI3K-AKT signaling, the precise molecular mechanisms involved remain incompletely understood. To investigate the signaling events of Ang-(1–7) in lung cancer-derived cells, we employed an integrated proteomic and phosphoproteomic approach in A549 cells. We analyzed early (minutes) and late (hours) molecular responses to Ang-(1–7) treatment. The treatment resulted in time-dependent modulation of multiple signaling pathways, including significant alterations in the MAPK, PI3K-AKT, and mTOR pathways at both the protein and phosphorylation levels. Notably, widespread early dephosphorylation events were observed, similar to the effects seen with other RAS peptides with antitumoral effects. Additionally, Ang-(1–7) promoted a long-lasting nuclear accumulation (up to 24 h) of the transcription factor FOXO1 indicating its activation. FOXO1 is known to regulate genes involved in apoptosis, cell cycle arrest, and oxidative stress, suggesting a role in mediating the peptide's antitumoral effects. The study provides new insights into the molecular basis of Ang-(1–7)’s antitumoral activity in A549 cells and reinforce its therapeutic potential in lung cancer. Raw data are available via ProteomeXchange with identifier <span><span>PXD066687</span><svg><path></path></svg></span>.</div></div><div><h3>Significance</h3><div>This study provides the first comprehensive, time-resolved proteomic and phosphoproteomic analysis of Angiotensin-(1–7) signaling in the lung cancer cell line A549. By capturing both early and late molecular events in A549 cells, we reveal that Ang-(1–7) modulates critical pathways involved in tumor progression, including MAPK, PI3K-AKT, and mTOR signaling. Importantly, we demonstrate the nuclear accumulation of FOXO1, a key transcription factor associated with tumor suppression, as part of the Ang-(1–7) response in A549 cells.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"326 ","pages":"Article 105607"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146025908","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 : 2026-01-19DOI: 10.1016/j.jprot.2026.105605
Fei Long , Xiaoyin Zeng , Fengyan Wang , Xufei Wang , Weijuan Shi , Yanting Lan , Jiahao Cheng , Chen Zhu , Yiqi Yang , Jing Xiao , Longbo Hu , Long Tan , Yuqiong Yang , Rongchang Chen , Zhenyu Liang , Tao Peng , Shaohua Lu
Analysis of proteins and other molecular components in induced sputum provides critical insights for the diagnosis, pathological assessment, and therapeutic monitoring of respiratory diseases. In this study, we collected three distinct types of induced sputum samples from patients with chronic obstructive pulmonary disease (COPD) and subjected them to proteomic and phosphoproteomic analysis using three different enzymatic digestion methods. We found that raw sputum samples yielded a higher number of uniquely identified proteins and phosphoproteins (1313 proteins and 1603 phosphorylation sites, corresponding to 782 phosphoproteins) and provided a more comprehensive characterization of COPD pathology. Furthermore, compared to in-gel digestion and in-solution digestion, the filter-aided sample preparation method increased protein identification by approximately 30% and yielded the highest number of unique protein identifications. Our study is the first to demonstrate that raw induced sputum can serve as a viable alternative source for liquid biopsy in respiratory diseases. We have also established the first methodological framework and dataset for proteomic and phosphoproteomic analysis of raw induced sputum, generating a preliminary map of the COPD sputum proteome and phosphoproteome. This novel proteomic and phosphoproteomic approach has untangled biologically relevant pathways in respiratory physiology, highlighting potential avenues for future research.
Significance
In this study, we aimed to investigate the feasibility of establishing and evaluating proteomic research methods using sputum samples from patients with chronic obstructive pulmonary disease (COPD). The ultimate goal was to develop analytical approaches suitable for sputum proteomics and phosphoproteomics and to preliminarily map the sputum proteome and phosphoproteome in COPD. It was found that raw sputum samples more comprehensively reflect the disease characteristics of COPD and are therefore more suitable for proteomic and phosphoproteomic studies of COPD. Among three mainstream enzymatic digestion methods, the Filter-Aided Sample Preparation (FASP) method demonstrated superior identification rates and was deemed most suitable for processing raw sputum samples. Furthermore, this study reports for the first time a draft map of the proteome and phosphoproteome of COPD sputum. This research provides valuable insights into sputum proteomic analysis and offers a useful resource for the study of respiratory diseases.
{"title":"Sputum proteomics and phosphoproteomics for improving chronic obstructive pulmonary disease knowledge","authors":"Fei Long , Xiaoyin Zeng , Fengyan Wang , Xufei Wang , Weijuan Shi , Yanting Lan , Jiahao Cheng , Chen Zhu , Yiqi Yang , Jing Xiao , Longbo Hu , Long Tan , Yuqiong Yang , Rongchang Chen , Zhenyu Liang , Tao Peng , Shaohua Lu","doi":"10.1016/j.jprot.2026.105605","DOIUrl":"10.1016/j.jprot.2026.105605","url":null,"abstract":"<div><div>Analysis of proteins and other molecular components in induced sputum provides critical insights for the diagnosis, pathological assessment, and therapeutic monitoring of respiratory diseases. In this study, we collected three distinct types of induced sputum samples from patients with chronic obstructive pulmonary disease (COPD) and subjected them to proteomic and phosphoproteomic analysis using three different enzymatic digestion methods. We found that raw sputum samples yielded a higher number of uniquely identified proteins and phosphoproteins (1313 proteins and 1603 phosphorylation sites, corresponding to 782 phosphoproteins) and provided a more comprehensive characterization of COPD pathology. Furthermore, compared to in-gel digestion and in-solution digestion, the filter-aided sample preparation method increased protein identification by approximately 30% and yielded the highest number of unique protein identifications. Our study is the first to demonstrate that raw induced sputum can serve as a viable alternative source for liquid biopsy in respiratory diseases. We have also established the first methodological framework and dataset for proteomic and phosphoproteomic analysis of raw induced sputum, generating a preliminary map of the COPD sputum proteome and phosphoproteome. This novel proteomic and phosphoproteomic approach has untangled biologically relevant pathways in respiratory physiology, highlighting potential avenues for future research.</div></div><div><h3>Significance</h3><div>In this study, we aimed to investigate the feasibility of establishing and evaluating proteomic research methods using sputum samples from patients with chronic obstructive pulmonary disease (COPD). The ultimate goal was to develop analytical approaches suitable for sputum proteomics and phosphoproteomics and to preliminarily map the sputum proteome and phosphoproteome in COPD. It was found that raw sputum samples more comprehensively reflect the disease characteristics of COPD and are therefore more suitable for proteomic and phosphoproteomic studies of COPD. Among three mainstream enzymatic digestion methods, the Filter-Aided Sample Preparation (FASP) method demonstrated superior identification rates and was deemed most suitable for processing raw sputum samples. Furthermore, this study reports for the first time a draft map of the proteome and phosphoproteome of COPD sputum. This research provides valuable insights into sputum proteomic analysis and offers a useful resource for the study of respiratory diseases.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"326 ","pages":"Article 105605"},"PeriodicalIF":2.8,"publicationDate":"2026-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146018988","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 : 2026-01-17DOI: 10.1016/j.jprot.2026.105603
Fatma Boukid
Barley protein is a multifunctional, sustainable plant-based ingredient with potential in food, nutraceutical, and industrial applications. This review synthesizes current knowledge on barley protein, emphasizing how proteomics and processing methods influence its composition, digestibility, and functional properties. Proteomic analyses reveal the distribution of major protein fractions, albumins, globulins, hordeins, and glutelins and their bioactive peptides, which exhibit antioxidant, antihypertensive, antidiabetic, and appetite-regulating activities. Protein concentrates and isolates offer improved digestibility and functional quality, though lysine remains limiting. Advanced techniques, including enzymatic hydrolysis, ultrasound-assisted extraction, and post-processing modifications, are evaluated for their impact on protein structure and functionality. Barley protein's potential applications in novel foods, micro- and nano-encapsulation, and targeted bioactive delivery are highlighted. By integrating proteomics insights with nutritional and technological perspectives, this work underscores the role of barley proteins in sustainable food systems.
Significance
This review synthesizes current knowledge on barley protein composition, emphasizing insights gained from proteomic analyses. By characterizing protein fractions, bioactive peptides, and allergenic determinants, proteomics enables a deeper understanding of barley's functional, nutritional, and health-related properties. The work highlights how extraction and processing influence protein quality and bioactivity, informing strategies for the development of novel plant-based foods. These insights provide a foundation for future research and industrial applications, advancing barley as a sustainable and functional protein source in human nutrition.
{"title":"Barley protein: From agricultural staple to sustainable protein solution","authors":"Fatma Boukid","doi":"10.1016/j.jprot.2026.105603","DOIUrl":"10.1016/j.jprot.2026.105603","url":null,"abstract":"<div><div>Barley protein is a multifunctional, sustainable plant-based ingredient with potential in food, nutraceutical, and industrial applications. This review synthesizes current knowledge on barley protein, emphasizing how proteomics and processing methods influence its composition, digestibility, and functional properties. Proteomic analyses reveal the distribution of major protein fractions, albumins, globulins, hordeins, and glutelins and their bioactive peptides, which exhibit antioxidant, antihypertensive, antidiabetic, and appetite-regulating activities. Protein concentrates and isolates offer improved digestibility and functional quality, though lysine remains limiting. Advanced techniques, including enzymatic hydrolysis, ultrasound-assisted extraction, and post-processing modifications, are evaluated for their impact on protein structure and functionality. Barley protein's potential applications in novel foods, micro- and nano-encapsulation, and targeted bioactive delivery are highlighted. By integrating proteomics insights with nutritional and technological perspectives, this work underscores the role of barley proteins in sustainable food systems.</div></div><div><h3>Significance</h3><div>This review synthesizes current knowledge on barley protein composition, emphasizing insights gained from proteomic analyses. By characterizing protein fractions, bioactive peptides, and allergenic determinants, proteomics enables a deeper understanding of barley's functional, nutritional, and health-related properties. The work highlights how extraction and processing influence protein quality and bioactivity, informing strategies for the development of novel plant-based foods. These insights provide a foundation for future research and industrial applications, advancing barley as a sustainable and functional protein source in human nutrition.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"326 ","pages":"Article 105603"},"PeriodicalIF":2.8,"publicationDate":"2026-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146003744","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}
Shotgun proteomics is widely used for comprehensive profiling of protein expression. However, this approach has inherent limitations in resolving “proteoforms”. Two-dimensional gel electrophoresis (2DE) has the potential to separate and visualize proteoforms effectively. To evaluate its utility, proteins extracted from soybean seed hypocotyls were analyzed by 2DE/LC-MS/MS. As a result, a total of 693 proteins were separated by 2DE, of which 302 were identified by LC-MS/MS. The dynamic range of protein abundance was approximately 102 to 104. This analysis revealed that the hypocotyls contain numerous proteoforms of proteins essential for seed physiology, including late embryogenesis abundant protein, glycinin, β-conglycinin, trypsin inhibitor, sucrose-binding protein, and glyceraldehyde-3-phosphate dehydrogenase. Furthermore, 2DE revealed that the expression of proteoforms of the major seed storage proteins, glycinin A2 and A3 subunits, and β-conglycinin β subunit in hypocotyls differed from that in cotyledons, suggesting distinct functional roles beyond nutrient storage during germination. Overall, the results demonstrate that 2DE is a valuable complementary technique to shotgun proteomics, providing proteoform-specific information that cannot be resolved by shotgun analysis alone.
Significance
Shotgun proteomics is widely used for comprehensive profiling of protein expression. However, this approach has inherent limitations when analyzing “proteoforms”. In the present study, we analyzed soybean seed hypocotyls using two-dimensional gel electrophoresis (2DE)-LC-MS/MS, demonstrating that 2DE is useful for effective proteoform analysis. 2DE provides proteoform-specific information that cannot be obtained from shotgun proteomics alone, making it a useful complementary method.
{"title":"Two-dimensional electrophoresis-based proteomics reveals soybean seed hypocotyl proteoforms","authors":"Jian-Zhong Tan , Hiroyuki Kagawa , Keiko Kizawa , Hisashi Hirano","doi":"10.1016/j.jprot.2026.105600","DOIUrl":"10.1016/j.jprot.2026.105600","url":null,"abstract":"<div><div>Shotgun proteomics is widely used for comprehensive profiling of protein expression. However, this approach has inherent limitations in resolving “proteoforms”. Two-dimensional gel electrophoresis (2DE) has the potential to separate and visualize proteoforms effectively. To evaluate its utility, proteins extracted from soybean seed hypocotyls were analyzed by 2DE/LC-MS/MS. As a result, a total of 693 proteins were separated by 2DE, of which 302 were identified by LC-MS/MS. The dynamic range of protein abundance was approximately 10<sup>2</sup> to 10<sup>4</sup>. This analysis revealed that the hypocotyls contain numerous proteoforms of proteins essential for seed physiology, including late embryogenesis abundant protein, glycinin, β-conglycinin, trypsin inhibitor, sucrose-binding protein, and glyceraldehyde-3-phosphate dehydrogenase. Furthermore, 2DE revealed that the expression of proteoforms of the major seed storage proteins, glycinin A2 and A3 subunits, and β-conglycinin β subunit in hypocotyls differed from that in cotyledons, suggesting distinct functional roles beyond nutrient storage during germination. Overall, the results demonstrate that 2DE is a valuable complementary technique to shotgun proteomics, providing proteoform-specific information that cannot be resolved by shotgun analysis alone.</div></div><div><h3>Significance</h3><div>Shotgun proteomics is widely used for comprehensive profiling of protein expression. However, this approach has inherent limitations when analyzing “proteoforms”. In the present study, we analyzed soybean seed hypocotyls using two-dimensional gel electrophoresis (2DE)-LC-MS/MS, demonstrating that 2DE is useful for effective proteoform analysis. 2DE provides proteoform-specific information that cannot be obtained from shotgun proteomics alone, making it a useful complementary method.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"326 ","pages":"Article 105600"},"PeriodicalIF":2.8,"publicationDate":"2026-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146003749","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 : 2026-01-13DOI: 10.1016/j.jprot.2026.105602
Roberta Pena da Paschoa , Lucas Rodrigues Xavier , Caio Cezar Guedes Corrêa , Karina da Silva Vieira , Daniel Dastan Rezabala Pacheco , Lucas do Espirito Santo Gomes , Carlos Eduardo Assis da Silva , Laura Eliza de Oliveira Alves , Vitor Batista Pinto , Claudete Santa-Catarina , Vanildo Silveira
The induction of somatic embryogenesis is controlled by various genes and proteins involved in hormonal pathways and stress responses, which act as key regulators of in vitro cellular reprogramming. In this study, we employed a temporal proteomic approach to investigate the underlying molecular mechanisms governing sugarcane (Saccharum spp.) embryogenic callus formation in response to 2,4-dichlorophenoxyacetic acid (2,4-D) during induction. Proteomic profiling revealed 996 differentially accumulated proteins (DAPs) across at least one pairwise comparison among time points (0, 7, 14 and 21 days) during callus induction. These DAPs were classified into different clusters on the basis of their accumulation profile. Proteins involved in embryogenesis, histone epigenetic regulation, hormone responses and protein post-translational modification accumulate during callus induction. The predicted interactions between the TOPLESS protein and auxin response proteins (SKP1, CUL1 and CAND1) are associated with increased accumulation of the histone deacetylase HDT2 protein, a regulator of chromatin condensation, during embryogenic callus initiation. Moreover, proteomic analysis revealed a temporal reduction in methylation cycle enzymes during callus induction, whereas global DNA methylation showed only a slight, non-significant increase, suggesting that additional regulatory layers are present. The identified protein dynamics provide valuable targets for refining somatic embryogenesis protocols and advancing their biotechnological applications in sugarcane.
Significance
Genetic engineering and plant cloning usually involve the induction of embryogenic competence using 2,4-dichlorophenoxyacetic acid (2,4-D). This study presents protein-protein interaction (PPI) networks regulated during the induction of sugarcane callus using 2,4-D, in addition to the morphological aspects of the explant during the process. Proteomic analysis of time series shows the regulation of protein kinases and transcriptional regulators TOPLESS, CUL1, SKP1, CAND1, and ARGONAUTE kinases, revealing mechanisms of activation of induction and multiplication of embryogenic callus. Furthermore, the possible interaction between GH3.8 and SnRK/SAPK kinases suggests a link between hormonal responses.
{"title":"Early regulatory networks driving somatic embryogenesis in Saccharum spp. L. revealed by time-resolved proteomics","authors":"Roberta Pena da Paschoa , Lucas Rodrigues Xavier , Caio Cezar Guedes Corrêa , Karina da Silva Vieira , Daniel Dastan Rezabala Pacheco , Lucas do Espirito Santo Gomes , Carlos Eduardo Assis da Silva , Laura Eliza de Oliveira Alves , Vitor Batista Pinto , Claudete Santa-Catarina , Vanildo Silveira","doi":"10.1016/j.jprot.2026.105602","DOIUrl":"10.1016/j.jprot.2026.105602","url":null,"abstract":"<div><div>The induction of somatic embryogenesis is controlled by various genes and proteins involved in hormonal pathways and stress responses, which act as key regulators of in vitro cellular reprogramming. In this study, we employed a temporal proteomic approach to investigate the underlying molecular mechanisms governing sugarcane (<em>Saccharum</em> spp.) embryogenic callus formation in response to 2,4-dichlorophenoxyacetic acid (2,4-D) during induction. Proteomic profiling revealed 996 differentially accumulated proteins (DAPs) across at least one pairwise comparison among time points (0, 7, 14 and 21 days) during callus induction. These DAPs were classified into different clusters on the basis of their accumulation profile. Proteins involved in embryogenesis, histone epigenetic regulation, hormone responses and protein post-translational modification accumulate during callus induction. The predicted interactions between the TOPLESS protein and auxin response proteins (SKP1, CUL1 and CAND1) are associated with increased accumulation of the histone deacetylase HDT2 protein, a regulator of chromatin condensation, during embryogenic callus initiation. Moreover, proteomic analysis revealed a temporal reduction in methylation cycle enzymes during callus induction, whereas global DNA methylation showed only a slight, non-significant increase, suggesting that additional regulatory layers are present. The identified protein dynamics provide valuable targets for refining somatic embryogenesis protocols and advancing their biotechnological applications in sugarcane.</div></div><div><h3>Significance</h3><div>Genetic engineering and plant cloning usually involve the induction of embryogenic competence using 2,4-dichlorophenoxyacetic acid (2,4-D). This study presents protein-protein interaction (PPI) networks regulated during the induction of sugarcane callus using 2,4-D, in addition to the morphological aspects of the explant during the process. Proteomic analysis of time series shows the regulation of protein kinases and transcriptional regulators TOPLESS, CUL1, SKP1, CAND1, and ARGONAUTE kinases, revealing mechanisms of activation of induction and multiplication of embryogenic callus. Furthermore, the possible interaction between GH3.8 and SnRK/SAPK kinases suggests a link between hormonal responses.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"326 ","pages":"Article 105602"},"PeriodicalIF":2.8,"publicationDate":"2026-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145986725","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}
<div><div>An aggressive and heterogeneous malignancy, referred to as triple-negative breast cancer, is characterised by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Treatment options remain limited, relying primarily on chemotherapy due to the lack of well-defined therapeutic targets, which is linked with poor prognosis and high recurrence rates. Proteomics and other high-throughput technologies have significantly advanced TNBC research by enabling the identification of protein-based biomarkers with potential applications in diagnosis, prognosis, and treatment. Through protein biomarkers that affect immune checkpoints, cell-surface glycoproteins, and regulators of tumor microenvironment interactions, key protein signatures from tumor tissue, serum, and exosomal proteomics have been found to have the potential to predict chemotherapy response and disease progression. To develop new therapeutic approaches, these biomarkers are being investigated. Combining proteomics with other omics technologies, such as transcriptomics and genomics, enables the development of precision medicine approaches and provides deeper insights into the pathophysiology of TNBC. Clinically validated and newly developed protein biomarkers for diagnosis, prognosis, and treatment interventions are described in this review. The molecular mechanistic aspects have also been discussed. These biomarkers have the potential to aid in the classification, risk stratification, and development of personalized treatment approaches for TNBC.</div></div><div><h3>Significance statement</h3><div>Triple-negative breast cancer (TNBC) remains a highly aggressive and heterogeneous form of breast cancer, with very few treatment strategies. This review synthesizes past discoveries, current clinical applications, and future opportunities of proteomics in TNBC, making it highly relevant to the theme of this Special Issue on “Past, Present and Future of Proteomics.” By consolidating evidence from human and other preclinical studies, it highlights how proteomic signatures have already transformed our understanding of TNBC biology and subtype classification, while also outlining their growing impact as diagnostic, prognostic, and therapeutic markers.</div><div>Importantly, the review emphasizes the translational shift enabled by next-generation proteomic technologies to redefine precision medicine for TNBC. It showcases how proteomics can facilitate personalized medicine, drug repurposing, and rational combination therapies, and describes novel avenues such as single-cell proteomics and integrative immunoproteogenomics that are driving the field forward.</div><div>Thus, this work not only consolidates what has been achieved but also provides perspectives on emerging technologies and innovative applications that could revolutionize biomarker discovery and clinical management of TNBC. It highlights proteomics as a critical pillar in shaping the futu
{"title":"Proteomic signatures in triple-negative breast cancer","authors":"Sohit Kashyap , Vishal Patidar , Anil Kumar , Prachi Sahu , Monisha Dhiman , Pardeep Garg , Aklank Jain , Anjana Munshi","doi":"10.1016/j.jprot.2026.105598","DOIUrl":"10.1016/j.jprot.2026.105598","url":null,"abstract":"<div><div>An aggressive and heterogeneous malignancy, referred to as triple-negative breast cancer, is characterised by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Treatment options remain limited, relying primarily on chemotherapy due to the lack of well-defined therapeutic targets, which is linked with poor prognosis and high recurrence rates. Proteomics and other high-throughput technologies have significantly advanced TNBC research by enabling the identification of protein-based biomarkers with potential applications in diagnosis, prognosis, and treatment. Through protein biomarkers that affect immune checkpoints, cell-surface glycoproteins, and regulators of tumor microenvironment interactions, key protein signatures from tumor tissue, serum, and exosomal proteomics have been found to have the potential to predict chemotherapy response and disease progression. To develop new therapeutic approaches, these biomarkers are being investigated. Combining proteomics with other omics technologies, such as transcriptomics and genomics, enables the development of precision medicine approaches and provides deeper insights into the pathophysiology of TNBC. Clinically validated and newly developed protein biomarkers for diagnosis, prognosis, and treatment interventions are described in this review. The molecular mechanistic aspects have also been discussed. These biomarkers have the potential to aid in the classification, risk stratification, and development of personalized treatment approaches for TNBC.</div></div><div><h3>Significance statement</h3><div>Triple-negative breast cancer (TNBC) remains a highly aggressive and heterogeneous form of breast cancer, with very few treatment strategies. This review synthesizes past discoveries, current clinical applications, and future opportunities of proteomics in TNBC, making it highly relevant to the theme of this Special Issue on “Past, Present and Future of Proteomics.” By consolidating evidence from human and other preclinical studies, it highlights how proteomic signatures have already transformed our understanding of TNBC biology and subtype classification, while also outlining their growing impact as diagnostic, prognostic, and therapeutic markers.</div><div>Importantly, the review emphasizes the translational shift enabled by next-generation proteomic technologies to redefine precision medicine for TNBC. It showcases how proteomics can facilitate personalized medicine, drug repurposing, and rational combination therapies, and describes novel avenues such as single-cell proteomics and integrative immunoproteogenomics that are driving the field forward.</div><div>Thus, this work not only consolidates what has been achieved but also provides perspectives on emerging technologies and innovative applications that could revolutionize biomarker discovery and clinical management of TNBC. It highlights proteomics as a critical pillar in shaping the futu","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"325 ","pages":"Article 105598"},"PeriodicalIF":2.8,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145927329","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 : 2025-12-31DOI: 10.1016/j.jprot.2025.105597
Murilo Salardani , Alison F.A. Chaves , Leonardo Cardili , Miyuki Uno , Solange M.T. Serrano , Roger Chammas , André Zelanis
Melanoma is an aggressive skin cancer with a high metastatic potential, influenced by both genetic and environmental factors. Proteases play a key role in shaping the tumor microenvironment and enabling transformed cells to actively colonize distant sites (metastasis). We performed proteomic mapping of protease cleavage sites in formalin-fixed paraffin-embedded tissue samples and profiled potentially active proteases in samples from melanoma patients with distinct prognostic outcomes. Although protein abundance alone did not indicate potential markers of disease progression, the observed cleaved fragments may serve for monitoring potentially active proteases in patient samples in targeted proteomics analysis. The findings provide valuable insights into melanoma biology and potential therapeutic prospects.
{"title":"Combining information on degradomics and gene expression data in prospecting metastatic melanoma proteolytic signatures","authors":"Murilo Salardani , Alison F.A. Chaves , Leonardo Cardili , Miyuki Uno , Solange M.T. Serrano , Roger Chammas , André Zelanis","doi":"10.1016/j.jprot.2025.105597","DOIUrl":"10.1016/j.jprot.2025.105597","url":null,"abstract":"<div><div>Melanoma is an aggressive skin cancer with a high metastatic potential, influenced by both genetic and environmental factors. Proteases play a key role in shaping the tumor microenvironment and enabling transformed cells to actively colonize distant sites (metastasis). We performed proteomic mapping of protease cleavage sites in formalin-fixed paraffin-embedded tissue samples and profiled potentially active proteases in samples from melanoma patients with distinct prognostic outcomes. Although protein abundance alone did not indicate potential markers of disease progression, the observed cleaved fragments may serve for monitoring potentially active proteases in patient samples in targeted proteomics analysis. The findings provide valuable insights into melanoma biology and potential therapeutic prospects.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"325 ","pages":"Article 105597"},"PeriodicalIF":2.8,"publicationDate":"2025-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145892564","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 : 2025-12-30DOI: 10.1016/j.jprot.2025.105589
Laura Plantera , Anna Didio , Uta Ceglarek , Ingo Bechmann
This study investigated the impact of tissue preservation methods on protein profiles analyzed by reversed-phase liquid chromatography-high-resolution mass spectrometry (LC-HRMS) using data-independent acquisition (DIA). Proteomic profiles from formalin-fixed, formalin-fixed and paraffin-embedded (FFPE), and fresh-frozen human brain tissues (cortex and hippocampus, n = 6) were compared, including an FFPE-specific protein extraction kit (n = 4).
Formalin-fixed samples more closely resembled fresh-frozen profiles than FFPE or FFPE-Kit samples, while still showing high correlation and overlap with FFPE tissues in principal component analyses. A core set of 1753 proteins was consistently detected across all sample preparation methods. A total of 35 proteins were identified exclusively in fresh-frozen samples, but without functional enrichment. Quantitative comparisons to the proteome of fresh-frozen tissue revealed an underrepresentation of cellular processes, energy metabolism, signaling, and transport related to protein properties such as length, location, and hydrophobicity. In contrast, neuronal development and phagosome-related pathways were overrepresented in fixed tissues.
In a pilot study comparing low (Braak 0-II, n = 4) and high (Braak IV-VI, n = 4) Alzheimer's disease (AD) stages using formalin-fixed samples, we identified 12 potential protein biomarkers, primarily nucleosomal proteins and carboxypeptidase M (CPM).
These findings suggest that formalin-fixed brain tissue provides reliable proteomic information, making it a valuable resource for neurodegenerative disease research.
Significance
Proteomics offers enormous potential for investigating the molecular regulation of the human brain. Valuable tissue samples are often preserved in formalin or additionally with paraffin for later analysis. The potential value of these preserved samples for proteomic analysis has already been recognized. However, tissue preservation poses a challenge for proteome analysis. Consequently, several studies have compared different protein extraction protocols for fixed samples. In addition, studies have been published comparing protein extraction from FFPE samples with fresh-frozen samples. To our knowledge, this is the first study to compare protein extraction across all three tissue preservation methods with subsequent functional analysis using samples obtained from the same donors, thereby eliminating inter-donor variability and enabling a direct comparison of preservation effects. This study validates a protein extraction protocol from formalin-fixed samples, laying the groundwork for future research into potential biomarkers in formalin-fixed samples.
本研究研究了组织保存方法对采用数据独立采集（DIA）的反相液相色谱-高分辨率质谱（LC-HRMS）分析的蛋白质谱的影响。比较福尔马林固定、福尔马林固定和石蜡包埋（FFPE）和新鲜冷冻人脑组织（皮质和海马，n = 6）的蛋白质组学图谱，包括FFPE特异性蛋白质提取试剂盒（n = 4）。福尔马林固定样品比FFPE或FFPE- kit样品更接近于新鲜冷冻样品，同时在主成分分析中仍与FFPE组织显示出高度的相关性和重叠。在所有样品制备方法中一致检测到1753个核心蛋白。在新鲜冷冻样品中鉴定出35种蛋白质，但没有功能富集。与新鲜冷冻组织的蛋白质组的定量比较揭示了与蛋白质特性（如长度、位置和疏水性）相关的细胞过程、能量代谢、信号传导和运输的代表性不足。相比之下，神经元发育和吞噬体相关途径在固定组织中被过度代表。在一项使用福尔马林固定样本比较低（Braak 0-II, n = 4）和高（Braak IV-VI, n = 4）阿尔茨海默病（AD）分期的初步研究中，我们确定了12种潜在的蛋白质生物标志物，主要是核小体蛋白和羧基肽酶M （CPM）。这些发现表明，福尔马林固定脑组织提供了可靠的蛋白质组学信息，使其成为神经退行性疾病研究的宝贵资源。意义蛋白质组学为研究人类大脑的分子调控提供了巨大的潜力。有价值的组织样本通常用福尔马林或石蜡保存，以备以后分析。这些保存的样品在蛋白质组学分析方面的潜在价值已经得到认可。然而，组织保存对蛋白质组学分析提出了挑战。因此，一些研究比较了固定样品的不同蛋白质提取方案。此外，已经发表的研究比较了从FFPE样品中提取的蛋白质与新鲜冷冻样品。据我们所知，这是第一项比较所有三种组织保存方法的蛋白质提取与随后使用来自同一供体的样品进行功能分析的研究，从而消除了供体间的差异，并能够直接比较保存效果。本研究验证了从福尔马林固定样品中提取蛋白质的方案，为未来研究福尔马林固定样品中潜在的生物标志物奠定了基础。
{"title":"Proteomic comparison of human brain tissue preservation methods","authors":"Laura Plantera , Anna Didio , Uta Ceglarek , Ingo Bechmann","doi":"10.1016/j.jprot.2025.105589","DOIUrl":"10.1016/j.jprot.2025.105589","url":null,"abstract":"<div><div>This study investigated the impact of tissue preservation methods on protein profiles analyzed by reversed-phase liquid chromatography-high-resolution mass spectrometry (LC-HRMS) using data-independent acquisition (DIA). Proteomic profiles from formalin-fixed, formalin-fixed and paraffin-embedded (FFPE), and fresh-frozen human brain tissues (cortex and hippocampus, <em>n</em> = 6) were compared, including an FFPE-specific protein extraction kit (<em>n</em> = 4).</div><div>Formalin-fixed samples more closely resembled fresh-frozen profiles than FFPE or FFPE-Kit samples, while still showing high correlation and overlap with FFPE tissues in principal component analyses. A core set of 1753 proteins was consistently detected across all sample preparation methods. A total of 35 proteins were identified exclusively in fresh-frozen samples, but without functional enrichment. Quantitative comparisons to the proteome of fresh-frozen tissue revealed an underrepresentation of cellular processes, energy metabolism, signaling, and transport related to protein properties such as length, location, and hydrophobicity. In contrast, neuronal development and phagosome-related pathways were overrepresented in fixed tissues.</div><div>In a pilot study comparing low (Braak 0-II, <em>n</em> = 4) and high (Braak IV-VI, n = 4) Alzheimer's disease (AD) stages using formalin-fixed samples, we identified 12 potential protein biomarkers, primarily nucleosomal proteins and carboxypeptidase M (CPM).</div><div>These findings suggest that formalin-fixed brain tissue provides reliable proteomic information, making it a valuable resource for neurodegenerative disease research.</div></div><div><h3>Significance</h3><div>Proteomics offers enormous potential for investigating the molecular regulation of the human brain. Valuable tissue samples are often preserved in formalin or additionally with paraffin for later analysis. The potential value of these preserved samples for proteomic analysis has already been recognized. However, tissue preservation poses a challenge for proteome analysis. Consequently, several studies have compared different protein extraction protocols for fixed samples. In addition, studies have been published comparing protein extraction from FFPE samples with fresh-frozen samples. To our knowledge, this is the first study to compare protein extraction across all three tissue preservation methods with subsequent functional analysis using samples obtained from the same donors, thereby eliminating inter-donor variability and enabling a direct comparison of preservation effects. This study validates a protein extraction protocol from formalin-fixed samples, laying the groundwork for future research into potential biomarkers in formalin-fixed samples.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"325 ","pages":"Article 105589"},"PeriodicalIF":2.8,"publicationDate":"2025-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145882649","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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