Journal of proteomics最新文献

{"title":"The protein cargo of extracellular vesicles. Recent advances in lung cancer research","authors":"Orlando Morales-Tarré ,&nbsp;Xitlally Popa Navarro ,&nbsp;Sergio Encarnación-Guevara","doi":"10.1016/j.jprot.2025.105557","DOIUrl":"10.1016/j.jprot.2025.105557","url":null,"abstract":"<div><div>Extracellular vesicles (EVs) are membranous microparticles produced by all living cells, from bacteria to humans. In recent years, interest in these vesicles has increased because they are linked to various biological functions, such as coagulation, the removal of undegraded proteins, intercellular signaling, stimulation and inactivation of T lymphocytes, and the transfer of antigens to APC. Since these vesicles carry specific characteristics of the parental cells, their characterization is a valuable opportunity to understand, through a liquid biopsy, the mechanisms involved in different pathologies, such as cancer. Lung cancer, is known to increase the production of extracellular vesicles, making their study particularly relevant in this group of diseases where biopsy collection is extremely difficult. Most research has centered on characterizing the nucleic acid content of vesicles due to their ability to alter gene expression in recipient cells. In contrast, the protein content of extracellular vesicles has been studied less extensively. Here, we describe the mechanisms of production for the main classes of extracellular vesicles and delve into their protein content. We also analyze recent progress in the proteomic characterization of extracellular vesicles in lung cancer research and explore their potential in diagnosis and the mechanisms underlying the disease's development.</div></div><div><h3>Significance</h3><div>This article highlights the advancements that the field of proteomics has brought to the characterization of extracellular vesicles derived from lung cancer. Given that lung cancer is the leading cause of cancer-related deaths, it is crucial to molecularly characterize this disease within the current landscape of biomedical research. We aim to draw the reader´s attention to the benefits of analyzing extracellular vesicles, as their protein content holds promise as an alternative liquid biopsy to achieve these objectives. By discussing existing findings and the challenges that remain, we hope to motivate researchers to utilize proteomics tools to develop new methodologies that can be consistently applied in clinical research and personalized patient care.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105557"},"PeriodicalIF":2.8,"publicationDate":"2025-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145482310","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":"Quantitative proteomic analysis of cadmium chloride tolerance mechanisms in Aeromonas hydrophila","authors":"Xiaofang Xie ,&nbsp;Binghui Zhang ,&nbsp;Xin Chen ,&nbsp;Jiaqi Tang ,&nbsp;Zhen Qiu ,&nbsp;Chenghao Shen ,&nbsp;Lingli Lian ,&nbsp;Gang Gu ,&nbsp;Xiangmin Lin ,&nbsp;Farman Ali","doi":"10.1016/j.jprot.2025.105555","DOIUrl":"10.1016/j.jprot.2025.105555","url":null,"abstract":"<div><div><em>Aeromonas hydrophila</em>, an antibiotic-and multi-metal-resistant pathogen, threatens aquaculture, yet its adaptation to cadmium chloride (CdCl₂) remains poorly understood. <em>A.hydrophilla</em> was grown with CdCl₂ exposure, and data-independent acquisition (DIA)-based proteomics was employed to identify the 253 upregulated and 163 downregulated proteins. GO and KEGG enrichment analyses revealed upregulated proteins involved in sulfate metabolism, siderophore- and iron transport, and other transition metal transport processes, while downregulated proteins were linked to amino acid metabolism, the tricarboxylic acid (TCA) cycle, and fatty acid metabolism. Gene Set Enrichment Analysis (GSEA) indicated positive enrichment of ribosome, RNA degradation, sulfur metabolism, and riboflavin metabolism, and negative enrichment of valine, leucine, and isoleucine degradation, two-component system, and bacterial chemotaxis. CdCl₂ tolerance assays of four gene-deletion mutants confirmed proteomics predictions: <em>ΔAHA_3605</em> (uncharacterized protein) and <em>ΔAHA_1796</em> (GGDEF-domain deletion) were significantly more sensitive to CdCl₂, suggesting a role for cyclic di-GMP (<em>c</em>-di-GMP) signaling in cadmium resistance. Collectively, these results provide novel insights into the adaptive strategies of <em>A. hydrophila</em>, revealing key molecular determinants of CdCl₂ stress tolerance.</div></div><div><h3>Significance</h3><div>Cadmium contamination increasingly co-selects for antibiotic-resistant pathogens in aquaculture, yet the molecular basis of cadmium tolerance in <em>Aeromonas hydrophila</em>—one of the most problematic Gram-negative fish pathogens—has been unknown. Here, we provide the first comprehensive, DIA-based quantitative proteome map of <em>A. hydrophila</em> under CdCl₂ stress, uncovering a tightly orchestrated stress response that simultaneously remodels metal acquisition, down-regulates energy-intensive metabolism (TCA cycle, branched chain amino acid and fatty acid degradation), and activates sulfur-riboflavin-ribosome pathways to repair oxidative damage. Functional validation using gene-deletion mutants demonstrated that loss of <em>AHA_3605</em> (uncharacterized) or <em>AHA_1796</em> (GGDEF domain protein) significantly increased CdCl₂ sensitivity, implicating cyclic di-GMP signaling in cadmium resistance. These findings advance our mechanistic understanding of heavy-metal adaptation in aquatic pathogens and offer proteome informed targets to disrupt metal-driven co-selection of multidrug-resistant <em>A. hydrophila</em> in aquaculture ecosystems.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105555"},"PeriodicalIF":2.8,"publicationDate":"2025-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145452343","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Decoding bladder cancer aggressiveness: A proteomic, phosphoproteomic and metabolomic approach","authors":"Zhihui Feng ,&nbsp;Biao Zhang ,&nbsp;Yi Liu ,&nbsp;Fei Yang ,&nbsp;Yinping Lei ,&nbsp;Songbai Liao ,&nbsp;Xianliang Hou","doi":"10.1016/j.jprot.2025.105553","DOIUrl":"10.1016/j.jprot.2025.105553","url":null,"abstract":"<div><h3>Background</h3><div>Bladder cancer (BC) is the most common malignancy of the urinary system. However, the median survival of patients with metastatic bladder cancer remains limited. Thus, there is an urgent imperative to develop novel biomarkers for BC-targeted therapies and to conduct in-depth investigations into BC pathogenesis leveraging multi-omics technologies.</div></div><div><h3>Results</h3><div>Our results revealed that proteins co-upregulated in both proteomic and phosphoproteomic analysis, such as SLC4A7 and MYO9B, demonstrated potential utility in distinguishing MIBC from NMIBC. Upregulation of CPT2 and palmitic acid in MIBC patients highlighted the dysregulation of physiological control mechanisms and enhanced pro-tumorigenic effects of lipid metabolic pathways.</div></div><div><h3>Conclusions</h3><div>Integrated multi-omics analysis reveals that key regulatory proteins such as SLC4A7 and MYO9B play pivotal roles in mediating the aggressive phenotypes of MIBC. Aberrant upregulation of CPT2 protein and metabolites like palmitic acid may drive malignant transformation from NMIBC to MIBC by promoting lipid metabolic reprogramming.</div></div><div><h3>Significance</h3><div>This study utilized LC-MS/MS to systematically profile the proteomic, phosphoproteomic, and metabolomic characteristics of MIBC, NMIBC, and adjacent noncancerous tissues, with the aim of identifying key molecules and metabolites driving bladder cancer progression. Our findings indicate that aberrant phosphorylation of regulatory proteins such as SLC4A7 and MYO9B may play a critical role in mediating the invasive phenotype of MIBC. In parallel, the upregulation of CPT2 and its associated metabolites (e.g., palmitic acid) suggests that lipid metabolic reprogramming, including enhanced β-oxidation and membrane phospholipid synthesis, may contribute to the malignant transition from NMIBC to MIBC. Overall, this study not only reveals potential molecules and metabolites driving bladder cancer progression but also provides a valuable reference for further exploration of pathways associated with bladder cancer invasiveness.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105553"},"PeriodicalIF":2.8,"publicationDate":"2025-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145445248","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative quantitative proteomics of tumoral and peritumoral tissues for distinguishing human glioblastoma from low-grade astrocytoma","authors":"Zhigang Xiong ,&nbsp;Chaoxi Li ,&nbsp;Qiangqiang Han ,&nbsp;Yanting Su ,&nbsp;Liming Cheng ,&nbsp;Hao-Long Zeng","doi":"10.1016/j.jprot.2025.105547","DOIUrl":"10.1016/j.jprot.2025.105547","url":null,"abstract":"&lt;div&gt;&lt;div&gt;Glioblastoma multiforme (GBM) and low-grade astrocytoma (LGA) are diffuse gliomas with distinct biological features and prognoses. However, the molecular mechanisms driving their differences are not fully understood. In this study, we performed a multi-dataset analysis integrating in-house quantitative proteomics data with high-quality external datasets to identify GBM-specific proteomic alterations between tumoral and peritumoral tissues relative to LGA. Our analysis revealed more pronounced proteomic differences between intra- and peritumoral tissues in GBM than in LGA. Proteins specifically dysregulated in GBM were predominantly linked to upregulated RNA splicing and spliceosome signaling. Through multi-dataset integration, we identified 73 GBM-specific upregulated proteins enriched in processes such as transcriptional regulation, hypoxia and necrosis, cytoskeleton organization, extracellular matrix remodeling, and immune response. Conversely, the 129 GBM-specific downregulated proteins were mainly involved in tumor suppression, G-protein signaling, calcium signaling, and neuronal gap junctions. Using these signature proteins, we developed a risk model based on CDK2, HDAC1, IGFBP2, SLC6A1, and TNR, which significantly predicted overall survival in glioma patients. This study delineates the proteomic landscape of GBM in comparison to LGA and offers a valuable resource for future mechanistic and clinical investigations.&lt;/div&gt;&lt;/div&gt;&lt;div&gt;&lt;h3&gt;Significance&lt;/h3&gt;&lt;div&gt;Diffuse gliomas are a heterogeneous brain tumor that include both low-grade and high-grade variants, each characterized by distinct morphological and biological features. Glioblastoma multiforme (GBM) is the most common primary high-grade and malignant brain tumor, with a median survival of less than 15 months despite aggressive treatment, including surgery, radiotherapy, and chemotherapy. In contrast, low-grade astrocytoma (LGA) exhibits indolent growth and a less aggressive clinical course, resulting in significantly longer patient survival compared to GBM. The molecular mechanisms underlying the biological differences between LGA and GBM are incompletely understood, and there is an urgent need for new molecular biomarkers to enhance diagnosis and therapeutic options. In this study, we conducted a comprehensive multi-dataset analysis by integrating our in-house quantitative proteomics data with several high-quality external datasets. We analyzed tumor and peritumoral tissue samples from human GBM and LGA, and further identified the GBM-specifically regulated proteomic signatures and signaling pathways, which is crucial for understanding the molecular mechanisms driving the aggressive behavior of GBM and could serve as a foundation for developing novel diagnostic biomarkers and therapeutic targets. Future research should focus on validating these GBM-specific proteins in larger cohorts and exploring their functional roles in GBM progression. Additionally, integrating multi-omic","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"322 ","pages":"Article 105547"},"PeriodicalIF":2.8,"publicationDate":"2025-10-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145401380","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":"Differences between protein cargo in outer membrane vesicles released from Mannheimia haemolytica A2 in the presence and absence of bovine lactoferrin","authors":"Christian Avalos-Gómez ,&nbsp;Mario Alejandro Aguilar-Chaparro ,&nbsp;Emmanuel Ríos-Castro ,&nbsp;Ernesto Marin-Flamand ,&nbsp;Ana Elvia Sánchez-Mendoza ,&nbsp;Mireya de la Garza","doi":"10.1016/j.jprot.2025.105550","DOIUrl":"10.1016/j.jprot.2025.105550","url":null,"abstract":"<div><div>Pneumonic mannheimiosis (PM) is a respiratory disease that causes significant economic losses in the ruminant livestock industry. Due to the lack of effective commercial products and the growing problem of bacterial antibiotic resistance, recent studies have focused on identifying new therapeutic strategies. <em>Mannheimia haemolytica</em>, the primary bacterial pathogen in ovine PM, naturally releases outer membrane vesicles (OMVs) whose protein cargo varies depending on environmental conditions. Lactoferrin (LF) is a glycoprotein of the mammalian innate immune system with bacteriostatic and bactericidal properties. In this study, we evaluated the effect of bovine LF on the protein composition of <em>M. haemolytica</em> OMVs. A total of 766 proteins were identified, including cytoplasmic, outer membrane (OM), and cell envelope proteins, most of which are related to metabolic processes. The presence of LF in the culture medium led to differential regulation of 162 proteins, affecting pathways such as general metabolism, aminoacyl-tRNA biosynthesis, RNA degradation, and biosynthesis of secondary metabolites. Notably, a decrease was observed in several lipoproteins (Lpps), OM proteins (OMPs)—including the LF-binding protein OmpA—and two components of the Tol–Pal system associated with pathogenicity. In addition, LF treatment was associated with a tendency to increase both the size and number of OMVs. These observations provide valuable insights into the use of LF-modified OMVs as a foundation for alternative prophylactic or therapeutic options against PM.</div></div><div><h3>Significance</h3><div>This work focuses on the analysis of protein cargo in <em>Mannheimia haemolytica</em> A2 OMVs, when bacteria grow in the presence of bovine lactoferrin (LF). This glycoprotein from the innate immune system caused stress in the bacteria which led to more OMVs released and differences in protein cargo present in OMVs, including decrease of several proteins related to pathogenicity. The results in vitro suggest that treatment with LF could reduce the damage caused by OMVs to their host cells, and being LF an alternative for the prevention and treatment of pneumonic mannheimiosis.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"322 ","pages":"Article 105550"},"PeriodicalIF":2.8,"publicationDate":"2025-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145424591","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":"Advancing post-genomics research in Mexico: Opportunities and strategies for the next decade","authors":"Robert Winkler ,&nbsp;Aldo Moreno-Ulloa","doi":"10.1016/j.jprot.2025.105551","DOIUrl":"10.1016/j.jprot.2025.105551","url":null,"abstract":"<div><div>The Mexican Proteomics Society (SMP) is the oldest proteomics organization in Latin America (LATAM), dedicated to advancing research in proteomics, metabolomics, and mass spectrometry (MS). As a non-profit, SMP organizes symposia and academic events, promoting collaboration and education. Since its founding in 2005, SMP has held biennial meetings and workshops in proteomics and metabolomics, invited international experts and fostered a multidisciplinary community. In this perspective manuscript, we provide a brief overview of the SMP, highlighting positive and negative aspects related to the economy, funding, infrastructure, technology and innovation, agriculture, health, and other fields. We also offer an overview of the MS equipment—critical infrastructure for omics research development—available within research centers and universities nationwide. Moreover, we present statistics on the contributions of Mexican-affiliated researchers to published research articles across various omics disciplines comparing Mexico's output to that of other LATAM countries. We believe that with this perspective the community will capture the role of the SMP in the development of omics research in Mexico, what is the country's contribution in the field, what are the country needs and how the new SMP board will contribute to advancing the field.</div></div><div><h3>Significance</h3><div>Omics-based societies play a crucial role in education, scientific development, and fostering multi-institutional collaborations. Although there are various societies worldwide focused on mass spectrometry-based omics, such as proteomics and metabolomics, their presence and impact on academia can be difficult to track. Therefore, in this manuscript, we provide a brief overview of the Mexican Proteomics Society (SMP), highlighting its contributions to omics research, the opportunities envisioned by the 2025-elected SMP leadership to advance omics science nationwide, and insights into the strategic directions for the coming decade.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"322 ","pages":"Article 105551"},"PeriodicalIF":2.8,"publicationDate":"2025-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145364006","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":"Comparative performance of Scribe and database search engines in metaproteomic profiling of a ground-truth microbiome dataset","authors":"Andrew T. Rajczewski ,&nbsp;Subina Mehta ,&nbsp;Reid Wagner ,&nbsp;Wassim Gabriel ,&nbsp;James Johnson ,&nbsp;Katherine Do ,&nbsp;Simina Vintila ,&nbsp;Mathias Wilhelm ,&nbsp;Manuel Kleiner ,&nbsp;Brian C. Searle ,&nbsp;Timothy J. Griffin ,&nbsp;Pratik D. Jagtap","doi":"10.1016/j.jprot.2025.105549","DOIUrl":"10.1016/j.jprot.2025.105549","url":null,"abstract":"<div><div>Mass spectrometry-based metaproteomics, the identification and quantification of thousands of proteins expressed by complex microbial communities, has become pivotal for unraveling functional interactions within microbiomes. However, metaproteomics data analysis encounters many challenges, including the search of tandem mass spectra against a protein sequence database using proteomics database search algorithms. We used a ground-truth dataset to assess a spectral library searching method against established database searching approaches. Mass spectrometry data collected by data-dependent acquisition (DDA-MS) was analyzed using database searching approaches (MaxQuant and FragPipe), as well as using Scribe with Prosit predicted spectral libraries. We used FASTA databases that included protein sequences from microbial species present in the ground-truth dataset along with background protein sequences, to estimate error rates and assess the effects on detection, peptide-spectral match quality, and quantification. Using the Scribe search engine resulted in more proteins detected at a 1 % false discovery rate (FDR) compared to MaxQuant or FragPipe, while FragPipe detected more peptides verified by PepQuery. Scribe was able to detect more low-abundance proteins in the microbiome dataset and was more accurate in quantifying the microbial community composition. This research provides insights and guidance for metaproteomics researchers aiming to optimize results in their analysis of DDA-MS data.</div></div><div><h3>Significance of the study</h3><div>Metaproteomics requires a balance between high numbers of peptide and protein identification and confidence in the accuracy of the identifications made. We demonstrate the utility of the Scribe search engine for metaproteomics applications, as it was found to detect low-abundance proteins with accurate quantitation than other DDA-MS search engines. This tool has great utility for both novel metaproteomics studies as well as hypothesis-generating experiments using previously acquired open source proteomics raw data.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"322 ","pages":"Article 105549"},"PeriodicalIF":2.8,"publicationDate":"2025-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145355158","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":"Proteomic exploration of the recently Re-classified forest cobra Naja species and the potential cytotoxic activity in cancer cell lines","authors":"Phetolo Motsa ,&nbsp;Beric Muller ,&nbsp;Lesetja R. Motadi ,&nbsp;Benedict C. Offor ,&nbsp;Lizelle A. Piater","doi":"10.1016/j.jprot.2025.105548","DOIUrl":"10.1016/j.jprot.2025.105548","url":null,"abstract":"<div><div>The forest cobra (<em>Naja melanoleuca</em>) species represents one of the most widespread medically important elapid snakes across Africa. The immense tissue-damaging effect of cobra venoms is attributed to the cytotoxins (CTX), which predominate in virtually all cobra venoms. In this study a bottom-up venomic approach was followed for deciphering the composition of the <em>N. melanoleuca</em>, <em>N. subfulva</em>, and <em>N. savannula</em> venom proteomes. The results revealed complex venoms that constituted predominantly of proteins belonging to the three-finger toxins (3FTxs) followed by phospholipase A₂ (PLA₂s) and snake venom metalloproteinases (SVMPs). The cytotoxicity and selectivity of the crude venoms and fractions were evaluated against cancer and normal cell lines. The crude <em>N. melanoleuca</em> venom sample demonstrated weak/low cytotoxic activity across the different cell lines as corroborated by SI values of less than 2, thus highlighting its limited application against these cancer cells, while the <em>N. subfulva</em> venom demonstrated its highest cytotoxic activity against the HeLa cancer cell line with a moderate selectivity index of 2.04. It is crucial to emphasize that these findings are still in the preliminary stages, primarily based on in vitro studies, and there remains a significant gap to bridge before any therapeutic applications can be considered.</div></div><div><h3>Significance</h3><div><em>Biological significance:</em> African forest cobra venom is a rich source of bioactive compounds such as cytotoxins, which cause tissue necrosis and descending paralysis. However, the venom has also been identified as a potential source of therapeutic agents, including anticancer agents. In this study, we evaluate the anticancer effects of the <em>N. melanoleuca</em>, <em>N. subfulva</em>, and <em>N. savannula</em> venoms and their fractions against the selected cell lines. The 3FTxs and PLA₂s, which are the most abundant protein families in the venoms, are predominantly responsible for the cytotoxic effects. In conclusion, this research study highlights the important role of forest cobra venoms as potential resources that researchers can further exploit to investigate the molecules responsible for the anticancer effect and investigate their mechanisms of action.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"322 ","pages":"Article 105548"},"PeriodicalIF":2.8,"publicationDate":"2025-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145337249","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":"Hypersensitivity and L-Asparaginase: A structural analysis of the interaction with HLA-DRB1*07:01","authors":"Jhenifer Yonara de Lima ,&nbsp;Emanuella de Castro Andreassa ,&nbsp;Marlon Dias Mariano Santos ,&nbsp;Paulo Costa Carvalho ,&nbsp;Tatiana de Arruda Campos Brasil de Souza","doi":"10.1016/j.jprot.2025.105538","DOIUrl":"10.1016/j.jprot.2025.105538","url":null,"abstract":"<div><div>L-Asparaginase derived from <em>Escherichia coli</em> (EcA) is extensively employed in the treatment of acute lymphoblastic leukemia (ALL); however, immunogenic responses frequently result in therapeutic failure. This study aims to identify residues involved in EcA immunogenicity and to propose structural modifications that may reduce antigenic potential or facilitate the development of targeted therapeutic strategies to inhibit such interactions. To achieve this, we investigate the sites of interaction of HLA-DRB1*07:01 allele and EcA by crosslinking mass spectrometry (XL-MS) using recombinantly expressed proteins. Circular dichroism (CD) spectroscopy confirmed the proper folding of the expressed enzymes, ensuring their structural integrity. Additionally, XL-MS allowed us to experimentally determine the structure of HLA-DRB1*07:01, which was previously unknown. Structural analysis and sequence alignment revealed immunogenic epitopes on the enzyme surface, particularly near the active site and at K288, a highly reactive residue. Our findings highlight key immunogenic sites on EcA, particularly residues 53–58, 283–289, and K288, which represent promising targets for reducing immunogenicity while preserving enzymatic function. These proposed modifications should be experimentally validated to ensure enzyme activity is maintained while effectively mitigating immune responses.</div></div><div><h3>Significance</h3><div>Hypersensitivity to <em>Escherichia coli</em>-derived L-asparaginase (EcA) remains one of the main challenges in the treatment of acute lymphoblastic leukemia (ALL), often forcing patients to interrupt a therapy that could be lifesaving. In this study, we mapped at the structural level how EcA interacts with the HLA-DRB1*07:01 allele, one of the main genetic factors associated with these adverse reactions. By identifying specific regions of the enzyme that trigger the immune response, especially residue K288, we offer a foundation for targeted modifications that may reduce immunogenicity without affecting enzymatic activity. These findings provide a concrete path toward developing safer EcA variants and bring us closer to more effective and personalized treatments.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105538"},"PeriodicalIF":2.8,"publicationDate":"2025-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145329551","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhancing glycopeptide annotation and glycan localization using electron activated dissociation through a multiplexed parallel reaction monitoring design of experiments","authors":"Hiba Salim ,&nbsp;Ruben Almey ,&nbsp;Laura Pont ,&nbsp;Fernando Benavente ,&nbsp;Maarten Dhaenens ,&nbsp;Estela Giménez","doi":"10.1016/j.jprot.2025.105546","DOIUrl":"10.1016/j.jprot.2025.105546","url":null,"abstract":"<div><div>We present an optimized electron activated dissociation (EAD) methodology, based on hot electron capture dissociation, for liquid chromatography-tandem mass spectrometry characterization of N- and O-glycopeptides, using recombinant human erythropoietin as a model glycoprotein. Applying a full factorial design of experiments (DoE) approach, we first optimized LC-MS parameters (i.e., ion spray voltage, ion source temperature, and active gradient time) to enhance glycopeptide ionization efficiency while reducing in-source fragmentation. A second DoE was then applied to fine-tune EAD-specific parameters. Multiplexed parallel reaction monitoring was performed to efficiently and comprehensively optimize the electron beam current, reaction time, and electron kinetic energy of the EAD set-up. Finally, the optimized EAD parameters, initially determined using one glycoform per glycopeptide, were successfully applied in data-dependent acquisition mode to detect the overall glycoform composition of each studied glycopeptide. Byonic, Fragpipe and Mascot softwares, and several peak picking softwares were used to evaluate the potential of our optimized EAD set-up, and compare with collision induced dissociation (CID). The results confirmed that EAD improved confidence in glycan localization, while CID enabled the identification of a greater number of glycoforms but with less confident glycan assignments.</div></div><div><h3>Significance</h3><div>The current manuscript introduces a novel and efficient optimization strategy for electron activated dissociation (EAD) parameters, based on hot electron capture dissociation, using a synergistic combination of design of experiments (DoE) and multiplexed parallel reaction monitoring (PRM) on the ZenoTOF 7600 instrument. The PRM-DoE approach enables rapid and systematic optimization of LC-MS conditions and simultaneous evaluation of key EAD parameters across target glycopeptide glycoforms in a drastically reduced number of experimental runs, significantly saving experimental time and resources. This approach provides the glycoproteomics field with a comprehensive method for achieving improved glycan site localization confidence over conventional collision induced dissociation, as demonstrated by applying a data-dependent acquisition method. The proposed strategy advances the analytical toolkit for LC-MS/MS glycoprotein analysis and potentially other post-translational modifications.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"322 ","pages":"Article 105546"},"PeriodicalIF":2.8,"publicationDate":"2025-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145313131","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}