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.
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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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