Molecular & Cellular Proteomics最新文献

{"title":"Proteomic Landscape of Colorectal Cancer Derived Liver Metastasis Reveals Three Distinct Phenotypes With Specific Signaling and Enhanced Survival.","authors":"Paula Nissen, Nadezhda V Popova, Antonia Gocke, Daniel J Smit, Geoffrey Yuet Mun Wong, Matthew J McKay, Thomas J Hugh, Kerstin David, Hartmut Juhl, Hannah Voß, Jens U Marquardt, Björn Nashan, Hartmut Schlüter, Mark P Molloy, Manfred Jücker","doi":"10.1016/j.mcpro.2025.101026","DOIUrl":"10.1016/j.mcpro.2025.101026","url":null,"abstract":"<p><p>Colorectal carcinoma is a major global disease with the second highest mortality rate among carcinomas. The liver is the most common site for metastases which portends a poor prognosis. Nonetheless, considerable heterogeneity of colorectal cancer liver metastases (CRC-LM) exists, evidenced by varied recurrence and survival patterns in patients undergoing curative-intent resection. Our understanding of the basis for this biological heterogeneity is limited. We investigated this by proteomic analysis of 152 CRC-LM obtained from three different medical centers in Germany and Australia using mass spectrometry-based differential quantitative proteomics. The proteomics data of the individual cohorts were harmonized through batch-effect correction algorithms to build a large multicenter cohort. Applying ConsensusClusterPlus to the proteome data yielded three distinct CRC-LM phenotypes (referred to as CRLM-SD (splice-driven), CRLM-CA (complement-associated), and CRLM-OM (oxidative metabolic)). The CRLM-SD phenotype showed higher abundance of key regulators of alternative splicing as well as extracellular matrix proteins commonly associated with tumor cell growth. The CRLM-CA phenotype was characterized by a higher abundance of proteins involved in the classical pathway part of the complement system including the membrane attack complex proteins and those with antithrombotic activity. The CRLM-OM phenotype showed higher abundance of proteins involved in various metabolic pathways including amino acids and fatty acids metabolism, which correlated in the literature with advanced proliferation of metastases and increased recurrence. Patients classified as CRLM-OM had a significantly lower overall survival in comparison to CRLM-CA patients. Finally, we identified a set of prognosis-associated biomarkers for each group including EpCAM, CEACAM1, CEACAM5, and CEACAM6 for CRLM-SD, DCN, TIMP3, and OLFM4 for CRLM-CA and FMO3, CES2 and AGXT for CRLM-OM. In summary, the discovery of three proteomic subgroups associated with distinct signaling pathways and survival of the CRC-LM patients provides a novel classification for risk stratification, prognosis and potentially novel therapeutic targets in CRC-LM.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101026"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12335997/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144567546","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Secretagogin Downregulation Impairs Nerve Cell Migration in Hirschsprung Disease via Inhibition of the LEF-1/NCAM1 Axis.","authors":"Yun Zhou, Shuiqing Chi, Shuai Li, Zhibin Luo, Liying Rong, Mengxin Zhang, Yunshang Chen, Jialing Guo, Dehua Yang, Xi Zhang, Guoqing Cao, Shao-Tao Tang","doi":"10.1016/j.mcpro.2025.101032","DOIUrl":"10.1016/j.mcpro.2025.101032","url":null,"abstract":"<p><p>Hirschsprung disease (HSCR) is a common peripheral neurodevelopmental disorder and impaired enteric neural crest cell migration is one of the key factors. Secretagogin (SCGN) has been demonstrated to play a critical role in the rostral migratory stream during central nerve regeneration. However, there is a paucity of knowledge on the role of SCGN in enteric neural crest cell migration. Here, we revealed a significant downregulation of SCGN by protein profiles using tandem mass tag in HSCR lesion colon tissues. We identified decreased expression of SCGN could hinder cell migration in vitro and in vivo. Mechanistically, SCGN upregulated the transcription factor (lymphoid enhancer-binding factor 1 [LEF-1]), which directly activated the transcription of the cell adhesion molecule (neural cell adhesion molecule 1 [NCAM1]), thereby promoting cell migration. In conclusion, this study elucidates the role of SCGN in HSCR pathogenesis by demonstrating its involvement in affecting neural crest cell migration through the lymphoid enhancer-binding factor 1/neural cell adhesion molecule 1 axis. The findings could contribute to the diagnostic and therapeutic strategies for HSCR.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101032"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12359226/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144626626","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Accurate MS-Based Diagnostic Amyloid Typing Using Endogenously Normalized Protein Intensities in Formalin-Fixed Paraffin-Embedded Tissue.","authors":"Vanessa Hollfoth, Arslan Ali, Eyyub Bag, Philip Riemenschneider, Sven Mattern, Julia Luibrand, Mohamed Ali Jarboui, Kerstin Singer, Benjamin Goeppert, Mirita Franz-Wachtel, Martina Sauter, Shabnam Asadikomeleh, Tobias Feilen, Christian Hentschker, Silvia Ribback, Elke Hammer, Karsten Boldt, Frank Dombrowski, Oliver Schilling, Boris Macek, Marius Ueffing, Karin Klingel, Stephan Singer","doi":"10.1016/j.mcpro.2025.101040","DOIUrl":"10.1016/j.mcpro.2025.101040","url":null,"abstract":"<p><p>Amyloidoses are a group of diseases characterized by the pathological deposition of non-degradable misfolded protein fibrils. These include plasma cell neoplasms, chronic inflammatory conditions, and age-related disorders, among others. Precise identification of the fibril-forming, and thereby amyloidosis-type defining protein is crucial for prognosis and correct therapeutic intervention. While immunohistochemistry (IHC) is widely used for amyloid typing, it requires extensive interpretation expertise and can be limited by inconclusive staining results. Thus, mass spectrometry (MS), if available, has been proposed as the preferred method for amyloid typing by international specialized centers (United States and United Kingdom) using primarily spectral counts for quantification. Here, we introduce an alternative method of relative quantification to further enhance the accuracy and reliability of proteomic amyloid typing. We analyzed 62 formalin-fixed, paraffin-embedded (FFPE) tissue samples, primarily endomyocardial biopsies, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and employed internal normalization of iBAQ values of amyloid-related proteins relative to serum amyloid P component (APCS) for amyloidosis typing. The APCS method demonstrated robust performance across multiple LC-MS/MS platforms and achieved complete concordance with clear cut IHC typed amyloidosis cases. More importantly, it resolved unclear amyloid cases with inconclusive staining results. Additionally, for samples without a distinct fibril-forming protein identified in the standard procedure, de novo sequencing uncovered immunoglobulin light chain components, enabling the diagnosis of rare AL-amyloidosis subtypes. Finally, we established machine learning approach (XGBoost) achieving 94% accuracy by using ∼160 amyloid-related proteins as input variables. In summary, the iBAQ APCS normalization method extended by de novo sequencing allows robust, accurate, and reliable diagnostic amyloid typing, and can be complemented by an AI-based classification. Careful reviewing of each histological sample and the clinical context, nevertheless, remains indispensable for accurate interpretation.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101040"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12396419/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144699037","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MSstatsTMT Improves Accuracy of Thermal Proteome Profiling.","authors":"Amanda M Figueroa-Navedo, Rohan Kapre, Tushita Gupta, Yingrong Xu, Clifford G Phaneuf, Pierre M Jean Beltran, Liang Xue, Alexander R Ivanov, Olga Vitek","doi":"10.1016/j.mcpro.2025.100999","DOIUrl":"10.1016/j.mcpro.2025.100999","url":null,"abstract":"<p><p>Thermal proteome profiling investigates protein-protein, protein-nucleic acid, or protein-drug interactions, and the impact of metabolite binding and post-translational modifications on these interactions. The experiments quantitatively characterize biological samples treated with small molecules versus controls and subjected to timed exposures to multiple temperatures. Typically, each enzymatically digested sample is labeled with a tandem mass tag (TMT), where each TMT channel corresponds to a specific temperature treatment, and profiled using liquid chromatography coupled with mass spectrometry in data-dependent data acquisition mode. The resulting mass spectra are processed with computational tools to identify and quantify proteins and filter out noise. Protein-drug interactions are detected by fitting curves to the protein-level reporter ion abundances across the temperatures. Interacting proteins are identified by shifts in the fitted curves between treated samples and controls. In this article, we focus on data processing and curve fitting in thermal proteome profiling. We review the statistical methods currently used for thermal proteome profiling and demonstrate that such methods can yield substantially different results. We advocate for the statistical analysis strategy implemented in the open-source R package MSstatsTMT, as it does not require subjective pre-filtering of the data or curve fitting and appropriately represents all the sources of variation. It supports experimental designs that trade-off temperatures for a larger number of biological replicates and handles multiple drug concentrations or pools of samples treated with multiple temperatures, thus increasing the sensitivity of the results. We demonstrate these advantages of MSstatsTMT as compared to the currently used alternatives in a series of simulated and experimental datasets, which include conventional thermal proteome profiling and its OnePot counterpart that pools the samples treated at multiple temperatures into one sample and incorporates multiple doses of a drug. The suggested MSstatsTMT-based workflow is documented in publicly available and fully reproducible R vignettes.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100999"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12335990/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144180265","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"EGFR Phosphorylates and Associates with EFNB1 to Regulate Cell Adhesion to Fibronectin.","authors":"Ana I Osornio-Hernández, François J M Chartier, Tim L Schuehle, Sara L Banerjee, Sabine Elowe, Patrick Laprise, Andrew Freywald, Mélanie Laurin, Nicolas Bisson","doi":"10.1016/j.mcpro.2025.101027","DOIUrl":"10.1016/j.mcpro.2025.101027","url":null,"abstract":"<p><p>Ephrin-Bs (EFNB1-3) are ligands for members of the largest subfamily of receptor tyrosine kinases (RTKs) in humans, the EPH receptors. Interestingly, ephrin-Bs are transmembrane proteins that may also act as receptors themselves upon EPH binding, activating so-called reverse signaling pathways that are critical for multiple cellular processes. Although a number of ephrin-B signaling effectors have been identified, the molecular mechanisms underlying ephrin-B-driven cellular processes remain unresolved, suggesting that multiple signaling effectors are yet to be discovered. Here, we employed proximity labeling proteomics to delineate the proximity network of EFNB1 in steady state and under active reverse signaling conditions. This allowed us to identify 90 uncharacterized EFNB1 proximity partners, from which we could distinguish three main groups: EPH receptor stimulation-dependent, stimulation-independent, and negatively modulated by EPH receptor stimulation. We further investigated the functional relationship between EFNB1 and one of the candidates identified, the epidermal growth factor receptor (EGFR). We found that EFNB1 and EGFR associate in cells and showed that the formation of this complex relies on EFNB1's PDZ-binding motif (PBM). Strikingly, we demonstrate that EGFR directly phosphorylates tyrosine residues within EFNB1's PBM, which results in the disruption of the EFNB1-EGFR complex. Furthermore, we show that the EFNB1-EGFR association is required for EFNB1-dependent cell adhesion to fibronectin. Taken together, our results shed light on a functional relationship between EFNB1 and EGFR.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101027"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345308/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144575911","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"mTORC2 Regulates Non-homologous End Joining Through Modulating the Temporal Dynamics of 53BP1.","authors":"Chunqing Wang, Hao Wang, Yunqiu Wang, Mingming Xiao, Xiaoxuan Song, Xiaofei Wang, Xiaofang Zhang, Kexing Jin, Dongqing Zhao, Hanxiang Chen, Ning Zhang, Ruibing Chen","doi":"10.1016/j.mcpro.2025.101035","DOIUrl":"10.1016/j.mcpro.2025.101035","url":null,"abstract":"<p><p>DNA damage repair is a critical biological process that maintains genomic integrity, and its dysregulation is closely related to tumorigenesis. To reveal the roles of mammalian target of rapamycin complex 2 (mTORC2) in DNA damage response (DDR), we investigated the temporal changes of cellular protein phosphorylation in mTORC2 deficient renal cancer cells in response to DNA double-strand break (DSB) induced by ionizing radiation (IR) using quantitative phosphoproteomics. The results showed that knockdown of Rictor, a specific component of mTORC2, induced profound changes in the dynamics of protein phosphorylation in response to IR. Intriguingly, the phosphorylation levels of multiple signaling molecules from the non-homologous end joining (NHEJ) pathway were affected by Rictor. Mechanistic study revealed that mTORC2 could regulate the spatiotemporal dynamics of p53 binding protein 1 (53BP1) in DDR. Rictor knockdown changed the phosphorylation of 53BP1 at multiple Ser/Thr sites. The efficiency of NHEJ was significantly reduced in Rictor deficient cells, and the maintenance of 53BP1 nuclear foci induced by IR was prolonged. Furthermore, mTORC2 modulated DSB repair through protein kinase B (PKB/Akt) and cyclin-dependent kinase 1 (CDK1). Finally, Rictor knockdown conferred hypersensitivity to IR and chemotherapeutic treatment in renal cancer cells, implying the potential use of the combination of mTORC2 inhibition with genotoxic therapy for renal cancer treatment.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101035"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12356323/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144659633","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dissecting Branch-Specific Unfolded Protein Response Activation in Drug-Tolerant BRAF-Mutant Melanoma Using Data-Independent Acquisition Mass Spectrometry.","authors":"Lea A Barny, Jake N Hermanson, Sarah K Garcia, Philip E Stauffer, Lars Plate","doi":"10.1016/j.mcpro.2025.101036","DOIUrl":"10.1016/j.mcpro.2025.101036","url":null,"abstract":"<p><p>Cells rely on the Unfolded Protein Response (UPR) to maintain ER protein homeostasis (proteostasis) when faced with elevated levels of misfolded and aggregated proteins. The UPR is comprised of three main branches-ATF6, IRE1, and PERK-that coordinate the synthesis of proteins involved in folding, trafficking, and degradation of nascent proteins to restore ER function. Dysregulation of the UPR is linked to numerous diseases, including neurodegenerative disorders, cancer, and diabetes. Despite its importance, identifying UPR targets has been challenging due to their heterogeneous induction, which varies by cell type and tissue. Additionally, defining the magnitude and range of UPR-regulated genes is difficult because of intricate temporal regulation, feedback between UPR branches, and extensive cross-talk with other stress-signaling pathways. To comprehensively identify UPR-regulated proteins and determine their branch specificity, we developed a data-independent acquisition (DIA) liquid-chromatography mass spectrometry (LC-MS) pipeline. Our optimized workflow improved identifications of low-abundant UPR proteins and leveraged an automated SP3-based protocol on the Biomek i5 liquid handler for label-free peptide preparation. Using engineered stable cell lines that enable selective pharmacological activation of each UPR branch without triggering global UPR activation, we identified branch-specific UPR proteomic targets. These targets were subsequently applied to investigate proteomic changes in multiple BRAF-mutant melanoma cell lines treated with a BRAF inhibitor (PLX4720, i.e., vemurafenib). Our findings revealed differential regulation of the XBP1s branch of the UPR in the BRAF-mutant melanoma cell lines after PLX4720 treatment, likely due to calcium activation, suggesting that the UPR plays a role as a non-genetic mechanism of drug tolerance in melanoma. In conclusion, the validated branch-specific UPR proteomic targets identified in this study provide a robust framework for investigating this pathway across different cell types, drug treatments, and disease conditions in a high-throughput manner.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101036"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12362115/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144649820","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Signatures of the Systemic Effects of a Snake Venom and Antivenom: Multiomics Profiling of the Kidney Pathology.","authors":"Alison F A Chaves, Bianca C S C de Barros, Miguel Cosenza-Contreras, Mariana S L C Morone, Ana T A Sachetto, Niko Pinter, Marlene Schmid, Marcelo L Santoro, Oliver Schilling, Solange M T Serrano","doi":"10.1016/j.mcpro.2025.101023","DOIUrl":"10.1016/j.mcpro.2025.101023","url":null,"abstract":"<p><p>Animal venoms comprise many toxins that work in concert to break apart the robust homeostatic systems of prey organisms. Conversely, prey organisms actively antagonize each step of envenoming, which displays a complex kinetics involving important changes at molecular, cell, tissue, and organism levels. In this study we explored the mammalian host response to envenoming using proteomics/N-terminomics and phosphoproteomics approaches to evaluate the in vivo effects of Bothrops jararaca venom in the mouse kidney after injection in the thigh muscle (1.6 mg/kg), mimicking a snakebite, and the impact of anti-Bothrops antivenom injected 1 h later (1.6 mg/kg; i.v. tail). For proteomics/N-terminomics, proteins were TMT-labeled, to allow for specific (tryptic) and semi-specific searches of MS/MS spectra to assess both global proteome and degradome. We quantified >7000 proteins, and prominent changes were observed in the kidney tissue, where protein differential abundance was identified after 3, 6, and 24 h, including markers of acute-phase response and injury. Likewise, the N-terminomic analysis revealed a significant impact of venom progressing from 3 h to 24 h, resulting in dysregulated proteolysis and indicating the activation of host proteases. The protease fingerprint matched legumain and cathepsin profiles. Venom toxins also promoted alteration in the dynamics of phosphorylation, with the activation of kinases. Under the conditions tested, antivenom administration (i) did not reduce the number of differentially abundant proteins and inflammation markers, (ii) partially attenuated the generation of proteolytic products in envenomed animals, and (iii) directly perturbed the phosphorylation signaling in control animals. Taken together, our findings underscore for the first time the mouse renal response to a protease-rich venom, revealed by the dynamic alteration in protein abundance, protease targets, and phosphorylation events, providing new facets of snake venom and antivenom systemic effects, which are important for the development of new therapies.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101023"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12336009/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144528914","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proteome Profiling of Cerebrospinal Fluid and Machine Learning Reveal Protein Classifiers of Two Forms of Alzheimer's Disease Characterized by Increased or Not Altered Levels of Tau.","authors":"Elisabetta Scalia, Matteo Calligaris, Margot Lo Pinto, Salvatore Castelbuono, Matilda Iemmolo, Vincenzina Lo Re, Giulia Bivona, Tommaso Piccoli, Giulio Ghersi, Simone Dario Scilabra","doi":"10.1016/j.mcpro.2025.101025","DOIUrl":"10.1016/j.mcpro.2025.101025","url":null,"abstract":"<p><p>Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder that presents with heterogeneous clinical and pathological features, necessitating improved biomarkers for accurate diagnosis and patient stratification. In this study, we applied a data-independent acquisition-based proteomics workflow to cerebrospinal fluid (CSF) samples from 138 individuals, including AD patients with high (Aβ+/tau+) or normal (Aβ+/tau-) CSF tau levels, and non-AD controls. Analysis using an Astral mass spectrometer enabled unprecedented proteome depth, identifying 2661 proteins with high data completeness. Comparative proteomic profiling revealed distinct protein signatures for Aβ+/tau+ and Aβ+/tau- subtypes. These findings were validated using an independent internal cohort and further corroborated with publicly available datasets from larger external AD cohorts, demonstrating the robustness and reproducibility of our results. Using machine learning, we identified a panel of 15 protein classifiers that accurately distinguished the two AD subtypes and controls across datasets. Notably, several of these proteins were elevated in the preclinical stage, underscoring their potential utility for early diagnosis and stratification. Together, our results demonstrate the power of data-independent acquisition proteomics on the Astral platform, combined with machine learning, to uncover subtype-specific biomarkers of AD and support the development of personalized diagnostic strategies.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101025"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12335992/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144553950","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Methanolic Urea-Enhanced Protein Extraction Enabling the Largest Bacterial Phosphorylation Resource.","authors":"Pei-Shan Wu, Ting-An Chen, Bo-Yu Chen, Yasushi Ishihama, Miao-Hsia Lin","doi":"10.1016/j.mcpro.2025.101019","DOIUrl":"10.1016/j.mcpro.2025.101019","url":null,"abstract":"<p><p>Mass spectrometry (MS)-based phosphoproteomics analysis is a powerful approach for elucidating the regulatory roles of protein phosphorylation across all domains of life. However, bacterial phosphoproteomics still faces significant technical challenges due to the extremely low substoichiometry of phosphorylation evens and the structural complexity of bacterial cell envelopes, which impede efficient cell lysis, protein recovery, and purity. To address these obstacles, we developed Methanolic Urea-enhanced Protein Extraction (MUPE), a streamlined, detergent-free, solvent-based method that leverages the amphiphilic nature of methanol and the chaotropic properties of urea to enhance protein yield and lysis efficiency. Furthermore, MUPE seamlessly integrates with liquid-liquid extraction, enabling efficient protein purification without requiring sample transfer and complex manipulations. This workflow significantly improves phosphoproteome coverage and quantitative accuracy across Gram-positive and Gram-negative bacteria while minimizing sample input requirements. Our datasets substantially expand the known landscape of bacterial O-phosphorylation, revealing distinct phosphorylation preferences within bacterial signaling networks. Application of MUPE to Listeria monocytogenes under bile insult revealed extensive phosphorylation changes independent of protein expression, highlighting phosphorylation as a rapid and dynamic regulatory mechanism. Collectively, MUPE provides a robust and scalable platform for bacterial phosphoproteomic studies, advancing our understanding of phosphosignaling in the context of bacterial physiology and pathogenesis.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101019"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345307/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144506830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}