Journal of proteomics最新文献

{"title":"The dynamics of ubiquitination and its role within the proteome of extracellular vesicles","authors":"Orlando Morales-Tarré ,&nbsp;Xitlally Popa-Navarro ,&nbsp;Alberto Paradela ,&nbsp;Magdalena Hernández-Ortiz ,&nbsp;Oscar Arrieta ,&nbsp;Fernando Corrales ,&nbsp;Sergio Encarnación-Guevara","doi":"10.1016/j.jprot.2025.105566","DOIUrl":"10.1016/j.jprot.2025.105566","url":null,"abstract":"<div><div>Ubiquitination is a multifaceted post-translational modification that plays a crucial role in regulating the degradation of unnecessary cellular proteins and is involved in various cellular processes, including protein export via extracellular vesicles. We investigate how alterations in the intracellular levels of ubiquitinated proteins affect vesicle protein content in BEAS-2B cells. We increased the intracellular levels of ubiquitinated proteins by inhibiting proteasomal degradation with MG-132 and by blocking deubiquitinating enzymes using PR-619. Using centrifugation and ultracentrifugation, were isolate various vesicle types, specifically the largest vesicles (enriched in plasma membrane-derived microvesicles) and the smallest vesicles (enriched in endosomal exosomes). High-resolution mass spectrometry-based proteomics was utilized to quantify their protein content. The content of extracellular vesicles changed in response to both treatments, reflecting cellular changes and the export of stress signals. The increase in intracellular levels of ubiquitinated proteins induced metabolic stress in the cells, generally leading to a reduction in protein translation, an enhanced response to oxidative stress, changes in membrane transport, and alterations in cell-microenvironment interactions. The modifications observed in the vesicular proteome suggest that ubiquitination plays a significant role in regulating protein export. This regulation can be mastered for diagnostic purposes and for describing cells and tissues through liquid biopsies.</div></div><div><h3>Significance</h3><div>Ubiquitination is one of the most abundant post-translational modifications in cells, and its role, beyond marking proteins for degradation, is not fully understood. Characterizing the effect of this modification on protein export to extracellular vesicles can shed light on how a cell selects its contents to influence its microenvironment, send signals to distant tissues, or interact with the immune system. This is particularly relevant in the context of pathologies such as cancer, which hijacks the cellular vesicle-producing machinery and adapts it to its needs to influence the remodeling of its surroundings. Understanding how a cell regulates the specific contents of its vesicles may point the way toward the development of treatments or superior diagnostic and classification tools.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105566"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145530566","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":"2026-01-20","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":"A fast TMT-based proteomic workflow reveals neural enrichment in neurospheres of hiPSC-derived neural stem cells","authors":"Pedro de Lima Muniz ,&nbsp;Michele Rodrigues Martins ,&nbsp;Livia Goto-Silva ,&nbsp;Leticia Rocha Quintino Souza ,&nbsp;Wassali Valadares ,&nbsp;Fábio César Sousa Nogueira ,&nbsp;Stevens Rehen ,&nbsp;Guillaume Nugue ,&nbsp;Magno Junqueira","doi":"10.1016/j.jprot.2025.105568","DOIUrl":"10.1016/j.jprot.2025.105568","url":null,"abstract":"<div><div>Three-dimensional (3D) neural spheroids, or neurospheres, generated from human induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs) more accurately recapitulate the microenvironmental cues of neural tissue compared to traditional two-dimensional (2D) monolayers. However, comparative omics-based characterizations of these models remain limited. Here, we present a streamlined and scalable TMT-based quantitative proteomics workflow to contrast the proteomic landscapes of hiPSC-derived NSCs cultured in 2D monolayers <em>versus</em> 3D neurospheres. A total of 1576 proteins were identified in an unfractionated LC-MS/MS of 68 min, with 542 showing significant differential abundance between groups. Neurospheres exhibited enrichment in neural-related pathways, such as synaptic signaling, neurotrophin signaling, cytoskeletal organization and vesicle trafficking, while monolayers enriched for multipotency features, such as general metabolic activity. Cell-type enrichment analyses confirmed increased neuronal identity in neurospheres, including elevated levels of markers associated with neuronal maturation. Our results demonstrate that 3D culture of NSCs induces a proteomic shift toward a more mature neural phenotype, which was validated by immunofluorescence microscopy. This rapid, multiplexed proteomic approach enables high-content molecular profiling suitable for drug screening and personalized medicine applications.</div></div><div><h3>Significance</h3><div>The present work contributes to the molecular and biological understanding of iPSC-derived neurospheres by exploring the proteome of this model. Neurospheres are a culture model of great potential and applicability in neurobiology research and personalized medicine, which still lacks a robust omic characterization. By studying neurospheres, we also work with a 3D culture model generated <em>in vitro</em>, avoiding the use of primary neural cells culture and animal models. Our fast method is relevant to single-cell proteomics, personalized medicine and screening assays.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105568"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145549911","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":"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":"2026-01-20","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":"Decoding spliceosome inhibition: Isobaric tag-based proteomic profiling of pladienolide B treated human cell lines","authors":"Xcanda Ixchel Herrera Lopez,&nbsp;Karina Martinez-Perez,&nbsp;Sanjukta Guha Thakurta,&nbsp;Benjamin P. Levi,&nbsp;Joao A. Paulo","doi":"10.1016/j.jprot.2025.105565","DOIUrl":"10.1016/j.jprot.2025.105565","url":null,"abstract":"<div><div>Pladienolide B (Pla-B) is a potent splicing modulator that has shown promise in cancer treatment, but its cellular effects remain incompletely understood. We investigated the dose-associated effect of Pla-B on human cell lines using isobaric tag-based quantitative proteomics and phosphoproteomics techniques. We quantified over 10,000 proteins and 19,000 phosphorylation events in SH-SY5Y cells, revealing dose-associated changes in protein abundance and phosphorylation status. Low Pla-B concentrations induced significant alterations in nuclear proteins, specifically those involved in transcription and cell division. Higher concentrations led to more extensive proteome remodeling, affecting chromatin-associated proteins and transcription. Phosphoproteome analysis uncovered alterations in the phosphorylation states of proteins including the splicing factor subunit SF3B, suggesting complex regulation of signaling pathways. Our findings reveal the detailed proteomic landscape of Pla-B's effects, offering insights into its role in the global proteome, which may guide future therapeutic applications and rational drug design.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"323 ","pages":"Article 105565"},"PeriodicalIF":2.8,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145505200","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":"2026-01-06","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":"Interspecific and intraspecific variability in venom composition of Naja naja and Naja kaouthia (Reptilia: Elapidae) populations from different habitats in Bangladesh","authors":"Mohammad Abdul Wahed Chowdhury ,&nbsp;Johannes Müller ,&nbsp;Ibrahim Khalil Al Haidar ,&nbsp;Md. Mizanur Rahman ,&nbsp;Mohammed Noman ,&nbsp;Aniruddha Ghose ,&nbsp;Abdullah Abu Sayeed ,&nbsp;Robed Amin ,&nbsp;Libia Sanz ,&nbsp;Mohammad Abul Faiz ,&nbsp;Ulrich Kuch ,&nbsp;Juan J. Calvete","doi":"10.1016/j.jprot.2025.105544","DOIUrl":"10.1016/j.jprot.2025.105544","url":null,"abstract":"&lt;div&gt;&lt;div&gt;The spectacled cobra (&lt;em&gt;Naja naja&lt;/em&gt;) and monocled cobra (&lt;em&gt;Naja kaouthia&lt;/em&gt;), widespread venomous snakes in South and Southeast Asia, occur in diverse habitats and cause neurotoxic envenoming. Despite reported venom variability of these two cobras across their range, no comparative study has been conducted from the interconnected but distinct habitats of Bangladesh. Using venomics and antivenomics, we analysed 26 individual venom samples of &lt;em&gt;N. kaouthia&lt;/em&gt; and 17 of &lt;em&gt;N. naja&lt;/em&gt; from Bangladesh across age groups and locations, respectively. Significant interspecific and intraspecific venom variability was observed, with geographically connected populations showing minimal divergence, while isolated populations (separated by river barriers or distinct ecosystems) exhibited pronounced compositional differences. Ontogenetic differences in venom composition between adult &lt;em&gt;N. kaouthia&lt;/em&gt; and their juvenile offspring were detected. Commercially available Incepta polyvalent antivenom, produced against India's “Big Four” (including southern Indian &lt;em&gt;N. naja&lt;/em&gt;), demonstrated poor efficacy against Bangladeshi cobra venoms. Collectively, our analyses demonstrate the existence of multi-dimensional variation in cobra venoms of Bangladesh that is influenced by biotic and abiotic factors. We emphasize the urgent need for region-specific antivenoms incorporating venom from ecologically distinct populations and age groups of both species across South Asia to improve snakebite treatment efficacy as well pre-clinical assessments to address biogeographic and ontogenetic venom diversity.&lt;/div&gt;&lt;/div&gt;&lt;div&gt;&lt;h3&gt;Significance&lt;/h3&gt;&lt;div&gt;Snakebite envenoming is a major neglected tropical disease and a leading occupational health hazard especially for rural populations in many low-and middle-income countries. As differences in snake venom composition between and within species can greatly affect the clinical course of envenoming and the efficacy of treatment, detailed knowledge of this variability is highly important for public health planning and the development of better antidotes. In Bangladesh, the monocled cobra (&lt;em&gt;Naja kaouthia&lt;/em&gt;) and the spectacled cobra (&lt;em&gt;Naja naja&lt;/em&gt;) belong to the medically most important and most widely distributed common snake species, but data on the variability of their venoms in this country has been limited and its relation to climatic and other environmental factors remained unexplored. Here we report on the analysis of 43 individual venom samples from 26 &lt;em&gt;N. kaouthia&lt;/em&gt; and 17 &lt;em&gt;N. naja&lt;/em&gt; from different age groups and geographical localities in Bangladesh, using venomics and antivenomics methods. Our findings show that the venoms of these cobras are highly diverse qualitatively and quantitatively, with significant inter- and intraspecific, geographic and ontogenetic variability and differences in their reactivity with a commercial antivenom. The observed geographical variability appear","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"322 ","pages":"Article 105544"},"PeriodicalIF":2.8,"publicationDate":"2026-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145238932","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":"Structural dynamics of the TPS/TPP complex in Saccharomyces cerevisiae: Insights from cross-linking mass spectrometry and computational modeling","authors":"Renata M. Santos ,&nbsp;Viviane A. Bastos ,&nbsp;Felipe F.M. Bagatelli ,&nbsp;Richard H. Valente ,&nbsp;Louise U. Kurt ,&nbsp;Diogo B. Lima ,&nbsp;Carla C. Oliveira ,&nbsp;Fabio C.S. Nogueira ,&nbsp;Elis C.A. Eleutherio ,&nbsp;Paulo C. Carvalho ,&nbsp;Francisco Gomes-Neto","doi":"10.1016/j.jprot.2025.105535","DOIUrl":"10.1016/j.jprot.2025.105535","url":null,"abstract":"<div><div>Trehalose, a ubiquitous disaccharide, plays a vital role in cell viability, including pathogenic fungi, making its synthetic pathway a key target for antifungal drug design. The structural details of the trehalose-phosphate synthase (TPS)/trehalose-phosphate phosphatase (TPP) complex, which is responsible for synthesizing trehalose, have remained elusive. Information on the structure and topology of the TPS/TPP complex remains scarce, significantly limiting the mechanistic understanding of trehalose synthesis. This study presents the first overview of the interactions within the <em>Saccharomyces cerevisiae</em> TPS/TPP complex following a 40 °C heat shock, analyzed by cross-linking mass spectrometry (XL-MS) and computational modeling. Our cross-linking data corroborate the UniProt-available AlphaFold models for isolated subunits. Intrinsically disordered regions are suggested for the regulatory subunits, while cross-linking analysis highlights the disordered N-terminus of Tsl1 as an important region in assembling the TPS/TPP complex. Finally, the phosphorylation prediction indicates that Tps3-disordered regions at the N-terminus and the phosphatase-like domains are preferentially phosphorylated, triggering the inhibition of Tps2 activity and halting T6P accumulation. These insights enhance our understanding of the structural dynamics and flexibility of the TPS/TPP complex, opening new avenues for potential therapeutic applications with diminished or no toxicity to humans.</div></div><div><h3>Significance</h3><div>This research contributes to a comprehensive structural understanding of the TPS/TPP complex in <em>Saccharomyces cerevisiae</em>, a key enzymatic system involved in trehalose synthesis. Trehalose is essential in cellular viability and stress adaptation, particularly in pathogenic fungi, making its biosynthetic pathway a promising target for antifungal drug development. By integrating cross-linking mass spectrometry (XL-MS) and computational modeling, this study uncovers critical interactions and dynamic features of the TPS/TPP complex, including the involvement of intrinsically disordered regions in its regulatory subunits possibly contributing to complex assembly and regulation under heat shock conditions. Furthermore, the phosphorylation predictions shed light on how disordered regions on Tps3 would participate in modulating Tps2 activity to regulate trehalose-phosphate levels, offering insights into the complex's functional dynamics. This work fills a crucial knowledge gap in understanding trehalose biosynthesis and paves the way for novel antifungal strategies with reduced toxicity to human cells.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"322 ","pages":"Article 105535"},"PeriodicalIF":2.8,"publicationDate":"2026-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145131242","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":"2026-01-06","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}

{"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":"2026-01-06","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}