Molecular & Cellular Proteomics最新文献

{"title":"AlphaCross-XL: A Seamless Tool for Automated and Proteome-Scale Mapping of Cross-Linked Peptides Onto 3D Protein Structures.","authors":"Sanjyot Vinayak Shenoy, Deeptarup Biswas, Arthur Zalevsky, Audrey Kishishita, Ayushi Verma, Ishan Upadhay, Yi He, Andrej Sali, Rosa Viner, Kamal Mandal, Sanjeeva Srivastava, Arun P Wiita","doi":"10.1016/j.mcpro.2025.101057","DOIUrl":"10.1016/j.mcpro.2025.101057","url":null,"abstract":"<p><p>Crosslinking mass spectrometry (XL-MS) is an exciting proteomics technology to capture native protein conformations in real time within biological systems. Historically, however, implementation of this technology has typically been limited to single purified recombinant proteins or in vitro-assembled protein complexes. These limitations are associated with inherent challenges in XL-MS analysis, including extremely low abundance of crosslinked (XL) peptides and complex deconvolution of XL peptide-derived spectral data. However, impressive recent developments in computation and instrumentation have now made it feasible to address biological questions using proteome-wide XL-MS analysis. Although some XL mapping software tools exist, these require manual input of specific Protein Data Bank structures at the single protein level and do not function at the high-throughput scale required to analyze datasets derived from thousands of proteins. To address this need, we therefore sought to develop a strategy enabling automated mapping of XL peptides onto the 3D structures of proteins, at a proteome-wide scale. Herein, we describe AlphaCross-XL, a first-in-class seamless computational tool for automated mapping of XL peptides onto the protein structures for intraprotein crosslinks and loop links. The AlphaCross-XL software first retrieves protein structures from the AlphaFold Protein Structure Database and maps all the identified crosslinks onto the 3D structure. It also calculates the Euclidian distance between the crosslinked residues and reports the violated and satisfied crosslink distances based on a user-defined distance threshold, which is visually discriminated by color in PyMOL. Last, the tool also supports further validation of user-submitted protein structures, which can include any computer-predicted protein structure and experimentally derived protein structures (i.e., from Protein Data Bank). AlphaCross-XL is available at https://github.com/sanjyotshenoy/alphacross-xl.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101057"},"PeriodicalIF":5.5,"publicationDate":"2025-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961653","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}

{"title":"Global Proteomics Reveals Distinct Muscle Adaptations to Menstrual Cycle Phase-Based Sprint Interval Training in Endurance-Trained Females.","authors":"Julie Kissow, Kamine Julie Jacobsen, Søren Jessen, Laura Bachmann Thomsen, Júlia Prats Quesada, Jens Bangsbo, Atul Shahaji Deshmukh, Morten Hostrup","doi":"10.1016/j.mcpro.2025.101053","DOIUrl":"10.1016/j.mcpro.2025.101053","url":null,"abstract":"<p><p>Advances in mass-spectrometry (MS)-based technologies have leveraged our understanding of protein-wide adaptations in human skeletal muscle in response to exercise. However, there is a lack of such data in females, particularly pertaining to already trained females and menstrual cycle phase-based sprint interval training (SIT) despite its efficacy and popularity. Here, we present a comprehensive global proteome analysis of skeletal muscle adaptations to high-frequency SIT during different menstrual cycle phases in endurance-trained females. We randomized 49 eumenorrheic females to either high-frequency SIT in the follicular (FB) or luteal phase (LB) over one menstrual cycle comprising eight sessions of 6 × 30-s all-out efforts. MS-proteomics, covering 4155 proteins after filtering, revealed notable differences in muscle adaptations to phase-based SIT. LB suppressed mitochondrial pathways of the tricarboxylic acid cycle and electron transport chain while enriching ribosomal complexes. Conversely, FB enriched filament organization and skeletal system development. Mitochondrial repression during LB was linked to reduced V˙ O_2max, whereas exercise capacity improved in FB only. Our findings show that menstrual cycle phase-based high-frequency SIT induces distinct protein-wide muscle adaptations and affects phenotype in endurance-trained eumenorrheic females. NCT04136457.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101053"},"PeriodicalIF":5.5,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144862335","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}

{"title":"AMPylation Regulates 5'-3' Exonuclease PLD3 Processing.","authors":"Laura Hoffmann, Eva-Maria Eckl, Marleen Bérouti, Michael Pries, Aron Koller, Charlotte Guhl, Ute A Hellmich, Veit Hornung, Wei Xiang, Lucas T Jae, Pavel Kielkowski","doi":"10.1016/j.mcpro.2025.101051","DOIUrl":"10.1016/j.mcpro.2025.101051","url":null,"abstract":"<p><p>The 5'-3' exonuclease phospholipase D3 (PLD3) is a single-pass transmembrane protein undergoing sequential post-translational modifications by N-glycosylation, AMPylation, and proteolytic cleavage. The substrates of PLD3 5'-3' exonuclease activity are single-stranded DNAs and RNAs, which act as ligands for Toll-like receptors and trigger a downstream proinflammatory response. Although PLD3 has primarily been studied in immune cells, recent findings indicate its enrichment in neurons, where it plays a role in regulating axonal fitness in Alzheimer's disease. However, the regulatory mechanisms governing the proteolytic processing of PLD3 into its catalytically active soluble form and its functional roles in both immune and neuronal cells remain unclear. Here, we describe the functional implications of PLD3 AMPylation, its direct interaction with the protein adenylyltransferase (FICD), and changes in PLD3 processing in Parkinson's disease patient-derived neurons. We identified PLD3 AMPylation sites within the protein's soluble region and showed that mutation of these sites hampers PLD3 activation and its catalytic activity. Overexpression of FICD AMP transferase accelerates PLD3 degradation and induces cellular stress response. Together, our findings demonstrate a critical role of AMPylation in PLD3 processing and regulation of its catalytic activity and provide new insights into the protein's transport and localization to lysosomes. The observation that PLD3 regulation in Parkinson's disease-derived neurons is altered compared with healthy neurons further highlights its role in neurodegenerative diseases.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101051"},"PeriodicalIF":5.5,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12494813/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144859361","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":"Identification of Post-translationally Modified MHC Class I-Associated Peptides as Potential Cancer Immunotherapeutic Targets.","authors":"Keira E Mahoney, Larry Reser, Maria Virginia Ruiz Cuevas, Jennifer G Abelin, Jeffrey Shabanowitz, Donald F Hunt, Stacy A Malaker","doi":"10.1016/j.mcpro.2025.100971","DOIUrl":"10.1016/j.mcpro.2025.100971","url":null,"abstract":"<p><p>Over the past 3 decades, the Hunt laboratory has developed advancements in mass spectrometry-based technologies to enable the identification of peptides bound to major histocompatibility complex (MHC) molecules. The MHC class I processing pathway is responsible for presenting these peptides to circulating cytotoxic T cells, allowing them to recognize and eliminate malignant cells, many of which have aberrant signaling. Professor Hunt hypothesized that due to the dysregulation in phosphorylation in cancer that abnormal phosphopeptides could be presented by this pathway, and went on to demonstrate that this was, in fact, the case. Thereafter, the laboratory continued to sequence MHC-associated phosphopeptides and contributed several improved methods for their enrichment, detection, and sequencing. This article summarizes the most recent advancements in identification of modified MHC-associated peptides and includes the cumulative list of phosphopeptides sequenced by the Hunt lab. Further, many other post-translational modifications (PTMs) were found to modify MHC peptides, including O-GlcNAcylation, methylation, and kynurenine; in total, we present here a list of 2450 MHC-associated PTM peptides. Many of these were disease-specific and found across several patients, thus highlighting their potential as cancer immunotherapy targets. We are sharing this list with the field in hopes that it might be used in investigating this potential. Overall, the Hunt lab's contributions have significantly advanced our understanding of antigen presentation and dysregulation of PTMs, supporting modern immunotherapy and vaccine development efforts.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100971"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12362676/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144028356","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":"Nucleolar Proteomics Revealed the Regulation of RNA Exosome Localization by MTR4.","authors":"Yaqian Zhang, Guangzhen Jiang, Ke Wang, Minjie Hong, Xinya Huang, Xiangyang Chen, Xuezhu Feng, Shouhong Guang","doi":"10.1016/j.mcpro.2025.101031","DOIUrl":"10.1016/j.mcpro.2025.101031","url":null,"abstract":"<p><p>The nucleolus is the largest membrane-less organelle within the nucleus and plays critical roles in regulating the cell cycle, senescence, and stress responses. The RNA exosome is a multiprotein ribonucleolytic complex involved in RNA processing and degradation in the cytoplasm, the nucleus, and the nucleolus. Previous studies have shown that the subcellular localization of the RNA exosome is crucial for its function. However, the mechanism that regulates its spatial distribution remains largely unexplored. In this study, we identified the nuclear RNA helicase MTR4 as a regulator of the RNA exosome localization through nucleolar quantitative proteomics technology. Immunostaining and fluorescence tagging confirmed that the depletion of MTR4 resulted in the translocation of the RNA exosome subunits from the nucleolus to the nucleoplasm. Notably, the translocation is specifically regulated by MTR4 and does not depend on other cofactors of the MTR4-containing Trf4/5-Air1/2-Mtr4 polyadenylation, poly(A) exosome-targeting, and nuclear exosome targeting complexes. The nucleolar accumulation of exosome subunits mutually depends on other exosome subunits. Additionally, actinomycin D treatment, which inhibits transcription, induced the RNA exosome to translocate from the nucleolus to the nucleoplasm, likely through the regulation of nucleolar MTR4 levels. These findings uncover a regulatory mechanism that modulates the localization of the RNA exosome within the nucleolus.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101031"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12356310/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144619082","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 Single-Step Protein Extraction for Lung Extracellular Matrix Proteomics Enabled by the Photocleavable Surfactant Azo and timsTOF Pro.","authors":"Anna G Towler, Andrew J Perciaccante, Timothy J Aballo, Yanlong Zhu, Fei Wang, Sarah Lloyd, Kuniko Kadoya, Yupeng He, Yu Tian, Ying Ge","doi":"10.1016/j.mcpro.2025.100950","DOIUrl":"10.1016/j.mcpro.2025.100950","url":null,"abstract":"<p><p>The extracellular matrix (ECM) is a dynamic, complex network of proteins, collectively known as the \"matrisome,\" which not only provides essential structural support to cells and tissues but also regulates critical cellular processes. Dysregulation of ECM is implicated in many diseases, underscoring the need to characterize the matrisome to better understand disease mechanisms. We have previously developed a dual-step protocol enabled by the photocleavable surfactant Azo for the extraction of ECM proteins from tissue using pH-neutral decellularization followed by solubilization by Azo. While effective for characterization of ECM proteins, such a dual-step protocol requires two extracts per sample, limiting the throughput and complicating the comparison of protein quantitation across different extraction conditions. Here, we develop a single-step Azo-enabled protein extraction for the solubilization of ECM proteins from lung tissue to improve the throughput for studies with large sample sizes. Using this method, we identified 324 ECM proteins, including 137 core ECM and 187 ECM-associated proteins. Core ECM proteins including elastin, fibronectin, and fibrillar collagens were reproducibly identified and quantified. We observed a 94.6% overlap in the ECM proteins identified between the single-step and the dual-step Azo extracts, indicating the single-step Azo extraction achieves ECM protein coverage comparable to the dual-step extraction. Overall, we have demonstrated that this single-step Azo extraction is not only highly efficient but also comprehensive for ECM protein identification and quantification, making it a powerful method for ECM proteomics, especially for studies with large sample sizes.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100950"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143663953","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}

{"title":"Deep Exploration of the Immunopeptidome of a Pancreatic Cancer Cell Line: Implications for Clinical Immunopeptidomics and Immunotherapy.","authors":"Mohammadreza Dorvash, Patricia T Illing, Nathan P Croft, Sri H Ramarathinam, Anthony W Purcell","doi":"10.1016/j.mcpro.2025.101030","DOIUrl":"10.1016/j.mcpro.2025.101030","url":null,"abstract":"<p><p>Pancreatic ductal adenocarcinoma (PDAC), with its devastating prognosis and limited treatment options, demands innovative therapeutic strategies. T-cell-based immunotherapy has shown promise for many cancers, including PDAC, but is limited by our knowledge of the breadth of cancer-specific T-cell epitopes available. Thus, a comprehensive assessment of the immunopeptidome of PDAC is essential to pave the way for the effective design of immunotherapy and related interventions. In this study, we immunoaffinity purified Human Leukocyte Antigen (HLA) class I-bound peptides from the Panc1 cell line grown in the absence and presence of cytokine stimulation. These peptides were subjected to an off-line high-pH reversed-phase (HPH-RP) fractionation prior to data acquisition. We demonstrate that HPH-RP fractionation followed by data-dependent acquisition (DDA) is a relatively simple and reliable technique that expands the depth of coverage of the PDAC immunopeptidome, allowing the identification of over 22,500 canonical HLA-bound peptides. In addition, the complementary separation by HPH-RP improved the identification confidence, particularly in the case of co-fragmenting precursors in data-independent acquisition (DIA) workflows. This strategy facilitated the identification of a high number of cancer-testis antigen (CTA)-derived immunopeptides. However, given that fractionation is typically associated with an adsorptive loss, it is impractical to apply HPH-RP on often minuscule clinical specimens and biopsies. Thus, we explored the feasibility of immunopeptidome analysis with cellular inputs as low as 1 million cells (equivalent to approximately 1 mg of tissue) using either a ZenoSWATH DIA interpreted using a spectral library derived from the HPH-RP strategy, or an optimized DDA workflow on the SCIEX ZenoToF 7600 system. Both approaches enabled robust detection of CTA-derived and other potentially clinically actionable immunopeptides even at the lowest cellular inputs. We discuss the relative merits of both acquisition strategies and how they can form the basis for future clinical translational immunopeptidomics approaches to screen tumor antigen presentation in low cellular input PDAC biopsies and provide new opportunities for target identification in immune-based therapies. Data are available via ProteomeXchange with identifiers PXD054360 and PXD054417.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101030"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12362687/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144608830","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":"Multi-omics Characterization of Acquired Olaparib Resistance in BRCA1 and BRCA2 Mutant Breast Cancer Cell Lines.","authors":"Holda A Anagho-Mattanovich, Meeli Mullari, Matthias Anagho-Mattanovich, Hayoung Cho, Anna-Kathrine Pedersen, Oana Palasca, Jesper V Olsen, Marie Locard-Paulet, Michael L Nielsen","doi":"10.1016/j.mcpro.2025.101034","DOIUrl":"10.1016/j.mcpro.2025.101034","url":null,"abstract":"<p><p>Poly (ADP-ribose) polymerase inhibitors (PARPi) are widely used as targeted therapies against breast cancers with BRCA mutations. However, the development of resistance to PARPi poses a significant challenge for long-term efficacy of these therapies, warranting further understanding of mechanisms of PARPi resistance. Here, we generated and characterized Olaparib resistance in BRCA1/2 mutant breast cancer cell lines MDAMB436 and HCC1428 using a systems-level multi-omics approach, including transcriptome, proteome, phosphoproteome, and ADP-ribosylation analysis. Our analyses revealed that resistance development strongly correlated with protein expression changes, while modest effects on phosphorylation- and ADP-ribosylation-dependent signaling pathways were observed. We found that BRCA1 expression was reestablished in OR MDAMB436 cell lines, while PARP1 expression was decreased. In OR HCC1428 cell lines, the BRCA2 mutation was not reverted. However, we observed increased expression of Fanconi anemia group D2 (FANCD2), histone parylation factor 1 (HPF1), and Nicotinamide phosphoribosyltransferase (NAMPT) in various cell lines, suggesting increased replication fork protection, and changes in the ADPr pathway and adaptation of metabolic pathways as resistance mechanisms. Our findings provide valuable insights into the complex landscape of PARPi resistance, offering potential targets for further investigation and therapeutic intervention.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101034"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12355081/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144649897","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":"Driving Therapeutic Innovation in Neurodegenerative Disease With Hydrogen Deuterium eXchange Mass Spectrometry.","authors":"Andrea Pierangelini, Benedikt M Kessler, Darragh P O'Brien","doi":"10.1016/j.mcpro.2025.101017","DOIUrl":"10.1016/j.mcpro.2025.101017","url":null,"abstract":"<p><p>Human neurodegenerative conditions such as Parkinson's and Alzheimer's disease are characterized by the formation and deposition of toxic protein species which exacerbate neuronal dysfunction, impacting the structure and function of the healthy brain. Deciphering the mechanisms underlying protein (mis)folding and aggregation is not only essential for a more coherent view of neurodegeneration, but also crucial for the development of novel therapeutics targeting this family of disorders. Key pathological drivers of neurodegeneration, such as alpha-synuclein and tau proteins, have traditionally proved extremely challenging to characterize structurally due to their intrinsic and widespread structural plasticity. Hydrogen-Deuterium eXchange Mass Spectrometry has emerged as a powerful tool to help circumvent this, owing to its ability to capture protein intrinsic disorder in solution, in addition to the transient structural conformations that typify protein aggregation pathways. This review brings together the most recent research where Hydrogen-Deuterium eXchange Mass Spectrometry has shed light on mechanisms of neurodegeneration. We highlight how the technique has been successfully integrated into therapeutic development workflows targeting some of the most prevalent neurodegenerative diseases.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101017"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12311602/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144369072","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":"Deciphering the Proteomic Profile of the Parasite Fasciola hepatica After Initial Vertebrate Host Infection Reveals New Insights Into Its Early Stages.","authors":"Marta López-García, Krystyna Cwiklinski, David Becerro-Recio, María Teresa Ruiz-Campillo, Verónica Molina-Hernández, José Pérez-Arévalo, Álvaro Martínez-Moreno, Javier González-Miguel, Mar Siles-Lucas","doi":"10.1016/j.mcpro.2025.101005","DOIUrl":"10.1016/j.mcpro.2025.101005","url":null,"abstract":"<p><p>The migration of the trematode parasite Fasciola hepatica within its vertebrate host following infection by ingestion of the metacercariae represents a critical event in its establishment and survival. The early stages of infection, during which F. hepatica crosses the intestinal barrier and advances to the liver through the peritoneum, initiate changes in the parasite that drive its development from a free-living state on pasture to an obligate blood-feeding parasite. Using an in vivo mouse model, this study explores the proteomic changes in the parasite as it crosses the intestinal barrier and migrates to the peritoneal cavity (24 h post infection (p.i.)) and liver parenchyma (8 days p.i.). This model was coupled with SWATH-MS, enabling a comparative evaluation of parasite protein abundance during the early stages of infection. We identified a total of 1180 F. hepatica proteins from three developmental time points: newly excysted juveniles (FhNEJ) at 3 h post excystment in vitro, and parasites collected in vivo at 24 h and 8 days p.i., separated into two different parasite compartments (somatic and tegumental). These extracts exhibited differentially expressed proteins (DEPs) across the analyzed time points, with 274 and 463 differentially expressed proteins identified in parasites obtained at 24 h and 8 days p.i., respectively. Our findings further highlight the adaptations F. hepatica undergoes within the first week of infection, including a shift toward anaerobic metabolic pathways, induction of signal transduction pathways involved in growth, and enrichment of crucial cysteine peptidases associated with feeding and immunomodulation. This study represents the first in-depth proteome analysis of parasites recovered 8 days into infection, adding to the wealth of molecular data available for Fasciola spp. to enhance our understanding of early host-parasite interactions. These data are crucial for the development of future in vitro models of fasciolosis and for identifying vaccine candidates targeting the early parasite stages.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101005"},"PeriodicalIF":5.5,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12390924/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144199618","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}