Journal Of Proteomics[JOURNAL]
Sun
200 papers
Skin mucus and blood plasma as non-lethal sources of malnutrition protein biomarkers in meagre (Argyrosomus regius).
Oliveira J, Raposo de Magalhães C, Schrama D … +6 more , Rodrigues PM, Barata M, Soares F, Pousão-Ferreira P, Oliva-Teles A, Couto A
J Proteomics · 2025 May · PMID 40089056 · Publisher ↗
Developing dietary formulations for aquaculture that meet nutritional requirements is essential to production, as nutrition is key for fish growth and health. However, novel dietary formulations may induce malnutrition,...
Developing dietary formulations for aquaculture that meet nutritional requirements is essential to production, as nutrition is key for fish growth and health. However, novel dietary formulations may induce malnutrition, which is complex to evaluate and often requires animal sacrifice. Therefore, finding reliable non-lethal biomarkers to diagnose malnutrition in fish is important. This study aimed to obtain vital information on potential non-lethal biomarkers from blood plasma and skin mucus to assess the fish's nutritional status using meagre (Argyrosomus regius) juveniles. For that purpose, a nutritional challenge was performed with fish fed a fish meal (FM) and fish-oil (FO) based control diet (55.1 % FM; 11 % FO, CTRL), a challenging diet (15 % FM; 7 % FO, CD), and a highly challenging diet (5 % FM; 5 % FO, ED), which, despite being nutritionally complete, may pose digestive and physiological challenges to carnivorous species. Diets significantly affected blood parameters, except for leukocyte counts, peroxidase activity, and immunoglobulin levels. Overall, blood parameters showed potential as non-lethal biomarkers to accurately identify signs of malnutrition. Meagre's plasma and skin mucus proteomes provided crucial information on the species' reaction to malnutrition, and 29 proteins connected to various physiological functions such as metabolism, development and immunity showed potential as non-lethal biomarkers. SIGNIFICANCE: The significance of this study lies in the establishment of potential non-lethal biomarkers for diagnosing malnutrition in fish. The results demonstrate that immunological, haematological, and biochemical parameters measured in fish blood can reveal signs of nutritional deficiencies. The findings further highlight that the proteomes of plasma and skin mucus provide valuable information about the fish's nutritional status. Notably, 29 proteins identified in this study, associated with various physiological functions, exhibit biomarker potential and warrant consideration in future research in the field of aquaculture nutrition. Moreover, the research provides critical insights into the proteome of meagre (Argyrosomus regius), enhancing our understanding of the species and contributing to the future improvement of its aquaculture production.
The dawn of the revolution that will allow us to precisely describe how microbiomes function.
Armengaud J
J Proteomics · 2025 May · PMID 40081757 · Publisher ↗
The community of microorganisms inhabiting a specific environment, such as the human gut - including bacteria, fungi, archaea, viruses, protozoa, and others - is known as the microbiota. A holobiont, in turn, refers to a...
The community of microorganisms inhabiting a specific environment, such as the human gut - including bacteria, fungi, archaea, viruses, protozoa, and others - is known as the microbiota. A holobiont, in turn, refers to an integrated ecological unit where microbial communities function and interact with their host, thus is a more integrative concept. To understand the processes involved, the diversity of microorganisms present must be identified and their molecular components quantified, especially proteins. Indeed, proteins - through their roles as catalytic units, structural components, and signaling molecules - are the main drivers of biological processes. Metagenomics has significantly expanded what we know about the genetic material present in microbiota, revealing their functional potential; metabolomics delivers an overall snapshot of the metabolites produced by the community. But metaproteomics offers a complementary approach to explore microbiome and holobiont functionality by focusing on the active proteins and functional pathways from each taxon. Significant recent advances in high-resolution tandem mass spectrometry have greatly expanded the catalog of peptide sequences accessible in each sample, creating the conditions for unprecedented taxonomical profiling, while also providing more accurate biomass quantification, more detailed protein characterization, and a greater capacity to monitor abundance and distinguish host biomarkers. By integrating artificial intelligence into the metaproteomics pipeline, extended datasets can now be efficiently mined to gain a more comprehensive functional view of complex biological systems, paving the way for next-generation metaproteomics. In this perspective, I discuss the transformative potential of this methodology. We are on the cusp of a remarkable omic revolution that promises to uncover the intricate workings of microbiomes by producing a vast array of new knowledge with multiple applications. SIGNIFICANCE: Metaproteomics provides a powerful lens to investigate microbiome and holobiont functionality by identifying and quantifying active proteins and functional pathways within each taxon. Recent breakthroughs in high-resolution tandem mass spectrometry have dramatically expanded the repertoire of peptide sequences detectable per sample. This progress enables unprecedented taxonomic resolution for microbial identification, more precise biomass quantification, comprehensive protein characterization, abundance monitoring, and the unique identification of host biomarkers. In this commentary, I delve into the distinctive features that make metaproteomics a transformative tool. I discuss the recent advancements in tandem mass spectrometry and argue that the primary challenge in analyzing complex samples is shifting from data acquisition to data interpretation. With the integration of artificial intelligence, I believe next-generation metaproteomics is poised to become the next Big Thing in microbiome research, unlocking profound insights into microbial functionality and ecosystem dynamics.
Proteomics reveals genetic mechanisms of cold resistance in Hezuo pig liver tissue.
Zhang Y, Gao X, Liu C … +4 more , Yang Q, Huang X, Li Y, Gun S
J Proteomics · 2025 May · PMID 40064419 · Publisher ↗
Cold stress poses a significant challenge to pig farming in northern China, leading to reduced productivity and, in severe cases, even mortality. However, the mechanisms underlying cold resistance in pigs are not well un...
Cold stress poses a significant challenge to pig farming in northern China, leading to reduced productivity and, in severe cases, even mortality. However, the mechanisms underlying cold resistance in pigs are not well understood. To explore the genetic mechanism of cold resistance in pigs under low-temperature conditions, the cold-tolerant Hezuo pig was selected as a model. DIA proteomics analysis was performed on liver tissues from Hezuo pigs after 24 h of exposure to low-temperature treatments. The results showed that approximately 149 differential abundance proteins (DAPs) were detected (95 up-regulated and 54 down-regulated). GO analysis showed that these DAPs were mainly associated with lipid metabolism, vesicle fusion, and membrane function. KEGG analysis showed that these DAPs were primarily enriched in lipid metabolism-related pathways such as cholesterol metabolism and vitamin digestion and absorption. Comprehensive analysis identified APOA4, APOA2, SREBF2, ATP23, STX2, USO1, ETFA, RAB11FIP1, ETNPPL, and SGMS1 as potential key proteins involved in cold resistance mechanisms. The mRNA expression of the genes for two key candidate proteins (APOA4 and SREBF2), which are involved in lipid metabolism, was analyzed using qRT-PCR, revealing a significant up-regulation after low-temperature treatment. These findings provide significant insights into the mechanisms of cold resistance in animals and may serve as candidate markers for further studies on cold tolerance. SIGNIFICANCE: Cold resistance is one of the key traits in pigs and involves multiple complex coordinated regulatory mechanisms. However, its genetic mechanisms are not completely understood. In this study, a DIA proteomics approach was used to identify proteins and pathways associated with cold resistance in the liver of low-temperature-treated Hezuo pigs. These findings offer novel candidate proteins and key pathways for investigating the molecular mechanisms of cold resistance in Hezuo pigs, providing a base for further elucidating the mechanisms of cold tolerance in pigs.
Comparative proteomic profiling of glioblastoma and healthy brain cell-derived extracellular vesicles reveals enrichment of cancer-associated proteins.
Hutson HK, Qin G, Cai C … +1 more , Nestorova GG
J Proteomics · 2025 May · PMID 40058457 · Publisher ↗
Extracellular vesicles (EVs)-mediated cellular communication plays a role in cancer development and progression. This study focuses on identifying glioblastoma-specific EV protein markers through a comparative mass spect...
Extracellular vesicles (EVs)-mediated cellular communication plays a role in cancer development and progression. This study focuses on identifying glioblastoma-specific EV protein markers through a comparative mass spectrometry bottom-up proteomic analysis of the LN-229 cell line and human neurons, astrocytes, and endothelial brain cells (HEBCs) using timsTOF Pro 2 instrument. The statistically significant upregulated proteins with fold change greater than 2 in the glioblastoma-derived EVs were clustered based on physical and functional interactions using the STRING database and analyzed using Gene Ontology enrichment. LN229-derived EVs contained an average of 2635 proteins, while human astrocytes, neurons, and HEBC encapsulated 2647, 716, and 2285 proteins, respectively. NanoParticle Tracking Analysis indicated that glioblastoma-derived EVs exhibited greater size variability compared to EVs from healthy cells. Statistical analysis identified 25 statistically significant proteins with increased levels in LN229 EVs relative to at least two healthy cell lines suggesting their potential as glioblastoma markers. Functional clustering using the STRING database and GO analysis indicated involvement in epigenetic regulation, metastasis, angiogenesis, and protein folding. Post-translational modification analysis identified a subset of 17 proteins unique to the cancer-derived EVs involved in chromatin regulation, extracellular matrix remodeling, and basement membrane organization pathways, highlighting their role in tumor progression.
Characterization of substrate distribution and functional implication of lysine acylations in Staphylococcus aureus.
Bian Y, Hu Z, Wang R … +7 more , Xie S, Sun Y, Liu T, Ma S, Liu B, Tan M, Xu JY
J Proteomics · 2025 May · PMID 40057026 · Publisher ↗
Staphylococcus aureus (S. aureus) is a major pathogen whose post-translational modifications (PTMs) regulate key biological processes that exert a substantial impact on protein function within this pathogen. In this stud...
Staphylococcus aureus (S. aureus) is a major pathogen whose post-translational modifications (PTMs) regulate key biological processes that exert a substantial impact on protein function within this pathogen. In this study, we comprehensively analyzed the overall patterns of three lysine acylation in S. aureus including acetylation, succinylation, and malonylation. Using mass spectrometry, we identified 1249 acetylated, 871 succinylated, and 67 malonylated sites. Bioinformatic analysis furtherly revealed that both lysine acetylation and succinylation exhibited a preferential association with glutamate residues near the modified lysine positions. Pathway enrichment showed that modified substrates were associated with ribosomes and metabolic functions. Additional functional exploration showed that lysine succinylation significantly regulates the enzymatic activity of Glutamyl-tRNA amidotransferase and Carbamoyl phosphate synthase. In conclusion, our study enhanced the comprehension of lysine succinylation in S. aureus and highlights potential targets related to its pathogenicity at the post-translational modification level. SIGNIFICANCE NEW: Lysine acylations play important roles in regulating bacterial survival and pathogenicity in Staphylococcus aureus. However, comprehensive and systematic investigations of the lysine acylomes in S. aureus remain insufficient. In this study, we conducted a comprehensive analysis of three lysine acylation modifications in Staphylococcus aureus subspecies aureus ATCC 25923 using mass spectrometry-based proteomic techniques. The objective was to investigate the potential impact of these modifications on protein function. Our bioinformatics analysis identified a significant correlation between lysine acylations and both ribosomal and metabolic pathways. Through additional experimental validation, we have substantiated that lysine succinylation plays a significant regulatory role in the activities of Glutamyl-tRNA amidinotransferase and Carbamoyl phosphate synthetase, consequently exerting a profound impact on cellular energy metabolism and protein synthesis in S. aureus. Collectively, our study underscores the pivotal role of lysine acylation modifications in S. aureus in modulating enzyme function, thereby offering valuable insights into the biology of S. aureus and informing potential therapeutic strategies.
Revisiting toxins with transcriptomics-informed proteomics of venom glands and crude venom from Centruroides bicolor from Panama.
Samudio O, Hernández-Ortiz M, Clement H … +5 more , Encarnación-Guevara S, Cleghorn J, Acosta H, Corzo G, Salazar MH
J Proteomics · 2025 May · PMID 40057025 · Publisher ↗
The sting of the scorpion Centruroides bicolor causes a large morbidity in Panama. To characterize its venom, transcriptomic and proteomic analyses of the venom glands and the crude venom were performed. These two approa...
The sting of the scorpion Centruroides bicolor causes a large morbidity in Panama. To characterize its venom, transcriptomic and proteomic analyses of the venom glands and the crude venom were performed. These two approaches utilized high-throughput sequencing to enhance the likelihood of detecting a wide range of venom proteins correlated with the venom proteome. After RNA venom gland extraction, a cDNA library was constructed and sequenced by RNA-seq. Also, the crude venom was digested using trypsin and chymotrypsin, and the resulting peptides were analyzed using a nano-LC-MS/MS. Notably, transcriptomic and proteomic venom approaches identified a hyaluronidase, alpha- and beta-neurotoxins that affect Na channels, CRISP proteins, metalloproteinases, transferrin, monooxygenase alpha-peptidyl-glycine, serine proteases, alpha pancreatic amylase, lysozyme, neurotoxins targeting K channels, neprilysin, scorpine, angiotensin-converting enzyme, insulin-like growth factor-binding domain proteins, nucleobindin-like proteins, and uncharacterized proteins. Interestingly, some of the venom proteins such as nucleobindin and angiotensin-converting enzymes have been not reported in the proteome, their predicted presence has only been previously derived from the genomic sequence of Centruroides sculpturatus and C. vittatus. These newly identified components enhance the understanding of the venomous nature of C. bicolor. SIGNIFICANCE: The proteins and peptides found in Centruroides bicolor venom by transcriptomic and proteomic analyses were assessed according to the protein and toxin databases available on public domains. Notably, some of the venom proteins such as nucleobindin and angiotensin-converting enzymes have been not reported in the proteome, their predicted presence has only been previously derived from the genomic sequence of Centruroides sculpturatus and C. vittatus. Moreover, enzymatic assays, including hyaluronidase, phospholipase A2, and proteolytic activity were conducted to confirm the presence or absence of those enzymes. Interestingly, neurotoxins from C. limbatus, a related species in the region, were found in the proteome but no mRNAs were identified in the transcriptome. These newly identified components enhance the understanding of the venomous nature of Centruroides bicolor.
Multi-organ proteome reveals different nursing ability between two honeybee srocks.
Wang R, Li J, Meng L
J Proteomics · 2025 May · PMID 40037490 · Publisher ↗
High royal jelly production is an adaptive reproductive investment syndrome in honey bees that enhances their nursing ability to queen bee larvae. However, the biological basis of this reproduction investment at the mult...
High royal jelly production is an adaptive reproductive investment syndrome in honey bees that enhances their nursing ability to queen bee larvae. However, the biological basis of this reproduction investment at the multi-organ level remains elusive. In this study, proteome across 11 organs of two bee stocks: high royal jelly production bees (RJBs) and Italian bees (ITBs) was compared. Our analysis revealed significant differences in protein expression profiles in brain, fat body, mandibular gland, and Malpighian tubule, highlighting their crucial roles in regulating royal jelly secretion in RJBs. The increased energy turnover, protein synthesis, and lipid synthesis observed in RJBs compared to ITBs highlight their enhanced metabolic activity, which is essential for the robust secretion of royal jelly in RJBs. The elevated abundance of major royal jelly proteins (MRJPs), hexamerins, and vitellogenin suggests their critical contributions to the nutritional and material requirement necessary for royal jelly secretion. Furthermore, the high level of vitellogenin and juvenile hormone esterase may suppress juvenile hormones, which contribute to a strong royal jelly secretion and sensitivity of RJBs to larval pheromones relative to ITBs. This comprehensive dataset contributes to a better understanding of nursing behavior and reproductive investment in honey bees. Significiance. The royal jelly secretion syndrome is a colony level social trait dominated by the intricate interplay of multiple organs. However, previous studies have primarily focused on individual organs. In this study, the proteome of 11 organs was compared between high royal jelly production bees (RJBs) and Italian bees (ITBs) to provide knowledge on how multiple organs cooperate to boost the elevated royal jelly production by RJBs. Nutrition supply was sufficient at multiple organs of RJBs when compared to ITBs, indicating that nutrition plays an essential role in boosting energy metabolism, protein and lipid synthesis, and directly contributes to the amount of royal jelly secretion. The high level of secretion of storage proteins, such as MRJPs, hex, and vitellogenin, provides sufficient nutrition and material for royal jelly secretion. Moreover, the higher levels of vitellogenin and juvenile hormone esterase may suppress juvenile hormone synthesis, and contributing to stronger sense of RJBs to larval pheromone relative to ITBs. This suggests that nutrition can influence the hormone levels and sensory abilities of RJBs nurse bees to promote their royal jelly secretion ability. The reported data provide insights into the systematic regulation strategy of honeybee nursing behavior and reproductive investment.
Integrative transcriptomic, proteomic, biochemical and neutralization studies on the venom of Micrurus ephippifer.
Zarzosa V, Neri-Castro E, Lomonte B … +13 more , Fernández J, Rodríguez-Barrera G, Rodríguez-López B, Rodríguez-Solís AM, Olvera-Rodríguez A, Bénard-Valle M, Saviola A, García-Vázquez UO, Fernández-Badillo L, Morales-Capellán N, Borja M, Zamudio F, Alagón A
J Proteomics · 2025 May · PMID 40023277 · Publisher ↗
Species of the genus Micrurus belong to the family Elapidae and possess venoms of significant clinical importance. This study presents an analysis of the venom composition of Micrurus ephippifer, employing transcriptomic...
Species of the genus Micrurus belong to the family Elapidae and possess venoms of significant clinical importance. This study presents an analysis of the venom composition of Micrurus ephippifer, employing transcriptomic and proteomic methodologies. A total of 2885 venom gland transcripts were assembled, of which 42 were identified as toxins. Transcripts for three-finger toxins (3FTx) were the most abundant (80.7 %), followed by PLA transcripts (16.3 %). Tryptic peptide sequences obtained through bottom-up shotgun MS/MS venom analysis were assigned to 46 distinct proteins in the SwissProt/UniProt database, of which 23 belong to the 3FTx family. Peptide spectral matching against the venom gland transcriptome database identified 24 proteins, 12 of which correspond to 3FTx, and three belong to PLA. Venom decomplexation by RP-HPLC followed by N-terminal amino acid sequencing of fractions allowed an estimation of the relative abundance of protein families, indicating that 3FTx comprise over 50 % of the venom. The identified toxic fractions displayed distinct lethality profiles in mice, with certain combinations exhibiting enhanced toxicity, very similar to what has been reported with Brownitoxin-I, with only the PLA sequence showing similarity. Our results emphasize the importance of integrating transcriptomic and proteomic approaches to understand venom diversity and its implications for antivenom development. SIGNIFICANCE: Mexico ranks first in the Americas in snake venom diversity. Paradoxically, very little is known about the composition of coral snake venoms, and Micrurus ephippifer is a clear example of this gap, as nothing was known about its venom composition. This type of study provides valuable information that helps fill these knowledge gaps. This study presents the second report of coral snake venoms containing a complex of phospholipase A and a three-finger toxin, offering important data that, with further research, will contribute to understanding venom evolution and evaluating the efficacy of antivenoms.
Proteomics and phosphoproteomics analysis of acute pancreatitis alleviated by forsythoside B.
Sun L, Li H, Zhang H … +3 more , Guo Y, Wang C, Chen S
J Proteomics · 2025 May · PMID 40015372 · Publisher ↗
Acute pancreatitis (AP) is a common acute abdominal condition in clinical practice, associated with high morbidity and mortality rates. Forsythia constitutes a component of traditional Chinese medicinal decoctions used f...
Acute pancreatitis (AP) is a common acute abdominal condition in clinical practice, associated with high morbidity and mortality rates. Forsythia constitutes a component of traditional Chinese medicinal decoctions used for clinical AP treatment; however, the efficacy of its active monomer in treating AP has yet to be completely substantiated. Here, we engineered an AP cell and mouse model by administering a combination of caerulein and LPS. In vitro experiments utilizing AR42J cells demonstrated that forsythoside B (FST·B) was the most effective monomer in mitigating cellular inflammation. Subsequently, a comprehensive evaluation of FST·B concentrations and efficacy was performed in animal models. Next Mass spectrometry analysis of pancreatic from AP mice treated with 50 mg/kg FST·B was conducted to elucidate its primary regulatory molecular signaling and key targets. FST·B effectively mitigated pathological damage in mice with acute pancreatitis, leading to a reduction in the expression of inflammatory cytokines in both pancreatic tissue and serum. Proteomics and phosphoproteomic profiles revealed that FST·B significantly enhanced the level of oxidative phosphorylation and spliceosome pathway in the AP mice. This research provides initial evidence of the regulatory molecular signals and targets of FST·B in AP, laying a potential foundation for its clinical use in treating AP. SIGNIFICANCE: Acute pancreatitis (AP) is a common acute abdominal condition in clinical practice, associated with high morbidity and mortality rates, and the global incidence of AP has increased by approximately 25 % over the past 15 years. Despite the complexity of AP's causes and the high susceptibility of proteins to degradation during lesions, systems biology studies, such as proteomics, have been limited in investigating the molecular mechanisms involved in its pharmacological treatment. Forsythoside B, a phenylethanol glycoside isolated from the air-dried fruit of forsythia, is a traditional oriental anti-inflammatory drug commonly used in clinical practice. We demonstrated in the AP mouse model that forsythoside B can alleviate pancreatic inflammatory damage in vivo. To elucidate the molecular mechanisms underlying the anti-inflammatory effect of forsythoside B, a comprehensive proteomic and phosphoproteomic analysis was conducted on AP mice models prior to and subsequent to forsythoside B intervention. Finally, 1640 significantly differentially expressed proteins, 1448 significantly differentially expressed phosphoproteins corresponding to 2496 significantly differentially expressed phosphosites were identified. Functional analysis revealed that forsythoside B significantly enhanced the level of oxidative phosphorylation in the AP mice in proteomic profiles, and the spliceosome pathway at the phosphorylation level was significantly affected by forsythoside B. This research provides initial evidence of the regulatory molecular signals and targets of forsythoside B in AP, laying a potential foundation for its clinical use in treating AP.
Trypanosoma cruzi cell cycle progression exhibits minimal variation in histone PTMs with unique histone H4 acetylation pattern.
Menezes APJ, Silber AM, Elias MC … +1 more , da Cunha JPC
J Proteomics · 2025 May · PMID 40010635 · Publisher ↗
Histones are crucial proteins in eukaryotic cells that undergo extensive posttranslational modifications (PTMs) such as methylation, acetylation, and phosphorylation, which are associated to chromatin structure, gene exp...
Histones are crucial proteins in eukaryotic cells that undergo extensive posttranslational modifications (PTMs) such as methylation, acetylation, and phosphorylation, which are associated to chromatin structure, gene expression, DNA damage/repair and cell cycle. In Trypanosoma cruzi, the primary sequence of histones differs from that of other eukaryotes. Despite this, they display a vast range of PTMs, though their modulation throughout the cell cycle remains largely unexplored. In this study, we investigated the dynamic modulation of histone PTMs across G1/S, S, and G2/M phases of T. cruzi cell cycle using hydroxyurea- synchronized parasites. We applied a workflow that included histone derivatization, trypsin digestion followed by a high-resolution mass spectrometry and data independent analysis. Quantitative analysis of 141 histone peptide isoforms revealed that there are only minor variations in histone PTM levels throughout the cell cycle. The H3K76 trimethylation remained predominant throughout all phases, with an increase in monomethylation during G2/M. Additionally, hyperacetylation of the N-terminal region of histone H4 was observed, particularly at lysine residues 2, 5, and 10, suggesting their importance in cell cycle progression. Striking, acetylation of histone H4 at K2 and K5 increases during the S-phase, mirroring the H4K5acK12ac pattern observed in mammals, which are related to histone nuclear import and chromatin deposition. Overall, the results suggest that the T. cruzi cell cycle maintains stable global levels of histone PTMs, relying on variations in only a few specific PTMs. Further investigations are warranted to elucidate the functional significance of these PTMs and their impact on cell cycle regulation and chromatin dynamics in T. cruzi. SIGNIFICANCE: Histone posttranslational modifications (PTMs) are key regulators of chromatin architecture and cellular processes such as gene expression and cell cycle control. In Trypanosoma cruzi, the etiological agent of Chagas disease, histones have a distinct primary structure compared to other eukaryotes, yet they display a wide variety of PTMs. This study provides a comprehensive analysis of histone PTM dynamics across the G1/S, S, and G2/M phases of the T. cruzi cell cycle, revealing that global histone PTM levels remain largely stable, with variations in a few specific marks. Notably, the study highlights the increased acetylation of histone H4 at lysines 2 and 5 during the S-phase, contrasting with the well-conserved acetylation at lysines 5 and 12 observed in mammals involved in nuclear import and chromatin assembly. These findings underscore the evolutionary divergence and functional specificity of histone modifications and provide a foundation for further investigations into their roles in parasite biology, with potential implications for understanding chromatin dynamics and identifying novel therapeutic targets.
Aberrant oxidative modifications of neutrophil myeloperoxidase in anti-neutrophil cytoplasmic antibody-associated vasculitis.
Sato M, Nagai K, Sato T … +15 more , Yoshimoto R, Shibano Y, Shibahara M, Satokawa H, Anzai M, Uchida T, Tsutiya A, Takakuwa Y, Omoteyama K, Arito M, Suematsu N, Ooka S, Kawahata K, Kato T, Kurokawa MS
J Proteomics · 2025 May · PMID 39993524 · Publisher ↗
Anti-neutrophil cytoplasmic antibodies directed to myeloperoxidase (MPO-ANCA) are key molecules in the pathogenesis of ANCA-associated vasculitis (AAV), however, the mechanisms of autoantibody production have not been el...
Anti-neutrophil cytoplasmic antibodies directed to myeloperoxidase (MPO-ANCA) are key molecules in the pathogenesis of ANCA-associated vasculitis (AAV), however, the mechanisms of autoantibody production have not been elucidated. We hypothesized that an aberrant PTM occurs in the MPO of MPO-ANCA-positive AAV (MPO-AAV), which induces immune responses to self MPO. To test this, we purified MPO proteins from neutrophils of 8 patients with MPO-AAV and 8 healthy subjects, digested them with trypsin, and comprehensively quantified PTMs of the MPO peptides using the sequential window acquisition of all theoretical fragment ion spectra (SWATH) method of LC-MS. Among the 1034 detected MPO peptides, 38 peptides were increased in the patients with MPO-AAV relative to the healthy subjects, whereas 10 peptides were decreased in the patients (p < 0.05). Interestingly, oxidative modifications were found in 11 of the 38 increased peptides (1.14- to 3.29-fold), but not in the decreased peptides. These included oxidation of Met577, Phe686, Met688 and Met719, dioxidation of Met409, Phe605, Trp679 and Met719, and kynurenylation of Trp255. Conversely, glycosylation was detected in 4 of the 10 decreased peptides (-1.32- to -2.32-fold), but not in the increased peptides. They were O-type glycans at Ser357 and Ser731, and N-type glycans at Asn355 and Asn729. In animal experiments, immunization of mice with in vitro oxidized or unoxidized mouse MPO (mMPO) showed that not only anti-oxidized mMPO antibodies but also anti-unoxidized mMPO antibodies were preferentially produced in the oxidized mMPO-immunized mice relative to the unoxidized mMPO-immunized mice (anti-oxidized mMPO antibodies, 6/8 vs 1/9, p < 0.05; anti-unoxidized mMPO antibodies, 4/8 vs 0/9, p < 0.05). Our results suggest that the increased oxidative modifications of MPO in MPO-AAV may break immune tolerance and trigger the MPO-ANCA production. SIGNIFICANCE: AAV is a systemic and refractory disease that causes life-threatening multi-organ involvement such as necrotizing glomerulonephritis and lung hemorrhage. MPO-ANCA is an autoantibody that plays a key role in the pathogenesis of AAV. Therefore, elucidation of the mechanism of MPO-ANCA production is crucial to overcoming this disease. In this study, we applied a SWATH-MS analysis to the detection of aberrant PTMs, and found increased oxidative modifications of neutrophil MPO in patients with MPO-AAV for the first time. Immunization of in vitro oxidized MPO induced autoantibodies to the intact unoxidized MPO, suggesting that the increased oxidative modifications of MPO may break the immune tolerance in MPO-AAV. This study suggests a novel trigger mechanism for MPO-ANCA production.
Expanded characterization and localization of male seminal fluid proteins within the female reproductive tract of the dengue vector mosquito Aedes aegypti.
Villa-Arias SV, Mendivil-de la Ossa JA, Avila FW … +2 more , Dorus S, Alfonso-Parra C
J Proteomics · 2025 May · PMID 39984034 · Publisher ↗
Aedes aegypti mosquitoes transmit numerous viruses that impact human health. Contemporary biological control programs aim to reduce Aedes fertility despite our limited understanding of interactions between the sexes requ...
Aedes aegypti mosquitoes transmit numerous viruses that impact human health. Contemporary biological control programs aim to reduce Aedes fertility despite our limited understanding of interactions between the sexes required for reproduction. During mating, males transfer seminal fluid proteins (SFPs) to females which alter their post-mating behavior, physiology and gene regulation, but the contribution of individual SFPs to fertility remains uncharacterized. In Drosophila, a small subset of SFPs localize to the sperm storage organs and oviducts or enter the hemolymph which suggests their participation in specific post-mating processes. We used mass spectrometry-based proteomics in conjunction with whole animal heavy labelling to expand the characterization of the Ae. aegypti ejaculate and identify SFPs that leave the site of insemination and localize to other female tissues. We identified 1031 ejaculate proteins, including a suite of novel SFPs. The expanded ejaculate proteome shows low conservation amongst SFPs when compared to insect model Drosophila, consistent with rapid evolutionary turnover at the genetic and proteomic levels. Further, we identify 25 SFPs that localize to the spermathecae, oviducts, and/or enter the hemolymph. This study expands our knowledge of the Ae. aegypti seminal fluid proteome and identifies candidate SFPs that may have tissue-specific, postcopulatory roles which support fertility. SIGNIFICANCE: Male-derived seminal fluid proteins (SFPs), transferred to females along with sperm during mating, are essential for the fertility of a mating pair. SFPs in aggregate induce several physiological and behavioral changes in mated females. Studies in the insect model Drosophila have shown that individual SFPs often participate in specific post-mating processes. In the dengue vector mosquito Aedes aegypti, 177 high confidence SFPs have been identified, but the contribution of individual SFPs in female fertility has yet not been characterized. In Drosophila, a small subset of SFPs leave the site of insemination and localize to the oviduct and sperm storage organs of the female reproductive tract or are transported to the female hemolymph, with patterns of SFP localization suggesting participation in a specific post-mating process. We used MS/MS proteomic characterization coupled with whole animal heavy labeling to expand characterization of the Ae. aegypti ejaculate proteome, increasing the number of known ejaculate proteins to 1378, including identification of 40 novel SFPs. Further, we identified 25 SFPs that leave the site of insemination and localize to the oviducts and/or spermathecae or enter the hemolymph, which can now be assessed for potential tissue-specific functions in female fertility.
Proteomic and metabolomic analyses reveal the antibacterial mechanism of Cannabidiol against gram-positive bacteria.
Zeng H, Wang X, Tang J … +5 more , Liu P, Zhang S, Chu H, Chen B, Ma M
J Proteomics · 2025 May · PMID 39978755 · Publisher ↗
Cannabidiol (CBD), the primary non-psychoactive cannabinoid isolated from cannabis, exhibits promising antibacterial effects. However, the antibacterial mechanism of CBD remains poorly understood. In this study, the mech...
Cannabidiol (CBD), the primary non-psychoactive cannabinoid isolated from cannabis, exhibits promising antibacterial effects. However, the antibacterial mechanism of CBD remains poorly understood. In this study, the mechanism was investigated using bacterial inhibition assays, label-free proteomics, and untargeted metabolomics, with Bacillus licheniformis (B. licheniformis), Staphylococcus aureus (S. aureus), and Enterococcus faecium (E. faecium) selected as representative Gram-positive bacteria. The results revealed that CBD caused significant damage to bacterial cell walls and membranes, leading to notable changes in proteomic and metabolic profiles. Specifically, 437, 120, and 195 proteins, as well as 52, 153, and 94 metabolites, were differentially expressed in B. licheniformis, S. aureus, and E. faecium, respectively. The antimicrobial mechanism of CBD shares similarities with previously known antibacterial agents, such as penicillin and cephalosporins, particularly in affecting the bacterial cell wall, but differs in its detailed mode of action. CBD disrupted the biosynthesis of primary and secondary metabolites and altered bacterial metabolism, contributing to its antibacterial activity. This study provides valuable insights into the antibacterial mechanism of CBD, supporting its potential development as an antibiotic alternative and its application in food safety. SIGNIFICANCE: It is crucial to find alternatives to antibiotics to mitigate the impact of pathogenic bacteria on food safety and reduce the use of antibiotics. CBD is the primary non-psychoactive cannabinoid derived from cannabis, and it has shown promising antibacterial effects. However, the antimicrobial mechanisms of CBD have not been well elucidated. This study provides a deep understanding of the antibacterial mechanism from the cellular to molecular level, which will contribute to the development of CBD as a novel antibacterial agent.
Comparative proteomics and phosphoproteomics analysis reveals differential sperm motility in Mediterranean buffalo semen.
Xue Q, Ren X, Xu T … +6 more , Yang T, Sun L, Luo X, Huang S, Shi D, Li X
J Proteomics · 2025 May · PMID 39961484 · Publisher ↗
High motility spermatozoa are good for cryopreservation and artificial insemination (AI) of mammalian semen. In this study, normal motility (NM) and low motility (LM) Mediterranean buffalo spermatozoa were compared using...
High motility spermatozoa are good for cryopreservation and artificial insemination (AI) of mammalian semen. In this study, normal motility (NM) and low motility (LM) Mediterranean buffalo spermatozoa were compared using quantitative proteomics and phosphoproteomics techniques to screen for important proteins and phosphorylated proteins related to the motility of spermatozoa and to identify candidate protein molecular markers related to the quality of Mediterranean buffalo semen. Proteomics results identified 2550 proteins, with 119 proteins upregulated and 146 proteins downregulated in the LM spermatozoa versus the NM spermatozoa. The differentially abundant proteins were mainly involved in carbohydrate metabolism, glycolysis/gluconeogenesis, and tricarboxylic acid cycles. The phosphoproteomics analysis revealed 412 proteins, 1228 phosphorylated peptides, and 1465 phosphorylation modification sites. Compared to the NM group, 119 peptides were downregulated in the LM group, corresponding to 98 proteins, and 84 phosphorylated peptides were upregulated in the white matter, corresponding to 61 proteins. Differentially phosphorylated proteins were primarily involved in spermatogenesis, flagellate sperm motility, and glycolysis/gluconeogenesis. The combined proteomics and phosphoproteomics results identified the common proteins HMGB4, POC1B, PKM, LDHA, TBC1D21, and CBY2, whose main roles were related to spermatogenesis, sperm flagellar structure, and energy metabolism, which can be used as potential markers of Mediterranean buffalo sperm quality.
Higher mitochondrial protein-Succinylation detected in lung tissues of idiopathic pulmonary fibrosis patients.
Zhao Y, Hou W, Yang L … +4 more , Chen K, Lang Q, Sun W, Gao L
J Proteomics · 2025 Apr · PMID 39938635 · Publisher ↗
A new pathogenic role for mitochondrial dysfunction has been associated with the development of idiopathic pulmonary fibrosis (IPF). Lysine succinylation (Ksucc) is involved in many energy metabolism pathways in mitochon...
A new pathogenic role for mitochondrial dysfunction has been associated with the development of idiopathic pulmonary fibrosis (IPF). Lysine succinylation (Ksucc) is involved in many energy metabolism pathways in mitochondria, making Ksucc highly valuable for studying IPF. We used liquid chromatography with tandem mass spectrometry (LC-MS/MS) to perform the first global profiling of Ksucc in fibrotic lung tissues from IPF patients, providing a proof of concept for the alteration of Ksucc in IPF and highlighting its potential as a therapeutic target. Selected candidate proteins were further verified by targeted proteomics using parallel reaction monitoring (PRM). In this study, we identified 1964 Ksucc sites on 628 modified proteins, with675 of these Ksucc sites on 124 modified proteins closely related to mitochondrial metabolism. 117 succinylated proteins were associated with energy metabolism in mitochondria by comparing these proteins with those previously reported in normal lung tissues. The Ksucc levels in KYAT3, HSD17B8, GRHPR, and IDH2 were different between control and IPF groups by Using PRM. This study provides insight into Ksucc profile alterations in IPF pathogenesis and Ksucc sites in proteins associated with mitochondrial energy metabolism can also serve as candidate molecules for future mechanism exploration and drug target selection in IPF.
TMT-label comparative proteomics reveals the vernalization mechanism in Wucai (Brassica campestris L.).
Liu X, Liao N, Tang X … +6 more , Wang K, Wang W, Khan A, Wang C, Yuan L, Chen G
J Proteomics · 2025 Apr · PMID 39922279 · Publisher ↗
To investigate the molecular basis of vernalization in Wucai [Brassica campestris L. (Syn. Brassica rapa L.) ssp. chinensis var. rosularis Tsen], we performed differential proteomic analysis using a tandem mass tags (TMT...
To investigate the molecular basis of vernalization in Wucai [Brassica campestris L. (Syn. Brassica rapa L.) ssp. chinensis var. rosularis Tsen], we performed differential proteomic analysis using a tandem mass tags (TMT)-based approach. Proteins from shoot apices subjected to 0, 15, and 30 days of vernalization (V0, V15, and V30) were analyzed to identify differentially abundant proteins (DAPs). A total of 8066 proteins were obtained, and 507 shared DAPs were involved in both initiation and progression of vernalization. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations revealed functional enrichment in cellular processes, metabolic pathways, and translation-related activities, including photosynthesis, glucosinolate biosynthesis, and flavonoid biosynthesis. Proteomic data showed reduced abundance of photosynthesis-related proteins and upregulation of flavonoid biosynthesis during vernalization. Transcriptional validation of 24 proteins across metabolic and regulatory pathways corroborated proteomic findings, with notable peaks in genes associated with flavonoid biosynthesis at 15 days of vernalization, such as VESR1,CH13, CHS1, FHT, and FLS1. The functions of these genes in vernalization will be further analyzed. SIGNIFICANCE: Wucai is prone to premature bolting and flowering under cold conditions, as vernalization plays a key role in controlling flowering time in Chinese cabbage crops. However, the proteomic basis of vernalization remains poorly understood. In this study, TMT-based proteomic analysis identified DAPs associated with vernalization. Pathway enrichment analysis highlighted key DAPs and their roles in significantly enriched pathways relevant to vernalization. Notably, genes in the flavonoid biosynthesis pathway genes, including VESR1, CH13, CHS1, FHT, and FLS1, respond to vernalization. These findings offer novel insights into the molecular mechanisms underlying flowering time regulation in Wucai.
Quantitative phosphoproteomic reveals that the induction of competence modulates protein phosphorylation in Streptococcus pneumonaie.
Lavergne JP, Page A, Polard P … +2 more , Campo N, Grangeasse C
J Proteomics · 2025 May · PMID 39921128 · Publisher ↗
Competence in the pathogenic bacterium Streptococcus pneumoniae (S. pneumoniae) is a developmental genetic program that is key for natural genetic transformation and consequently bacterial horizontal gene transfer. Phosp...
Competence in the pathogenic bacterium Streptococcus pneumoniae (S. pneumoniae) is a developmental genetic program that is key for natural genetic transformation and consequently bacterial horizontal gene transfer. Phosphoproteomic studies have revealed that protein phosphorylation on serine, threonine and tyrosine residues is a widespread regulatory post-translational modification in bacteria. In this study, we performed quantitative proteomic and phosphoproteomic analyses on S. pneumoniae as a function of competence induction. To calculate peptide abundance ratios between non-competent and competent samples we used dimethyl-tag labeling. Titanium dioxide (TiO2) beads were used for phosphopeptide enrichment and samples were analysed by LC-MS/MS. Our proteome analysis covers approximatively 63 % of the total bacterial protein content, identifying 82 proteins with significantly different abundance ratios, including some not previously linked to competence. 248 phosphopeptides were identified including 47 having different abundance ratios. Notably, the proteins with a change in phosphorylation in competent cells are different from the proteins with a change in expression, highlighting different pathways induced by competence and regulated by phosphorylation. This is the first report that phosphorylation of some proteins is regulated during competence in Streptococcus pneumoniae, a key pathway for the bacteria to evade vaccines or acquire antibiotic resistance. SIGNIFICANCE: S. pneumoniae is a prominent model for the study of competence that governs the development of natural genetic transformation. The latter largely accounts for the spread of antibiotic resistance and vaccine evasion in pneumococcal isolates. Many proteins specifically expressed during competence have been identified and extensively studied. However, the potential contribution of post-translational modifications, and notably phosphorylation, during the development of competence has never been investigated. In this study, we used a quantitative phosphoproteomic approach to determine both the protein expression and the protein phosphorylation patterns. Comparison of these patterns allows to highlight a series of proteins that are differentially phosphorylated in the two conditions. This result opens new avenues to decipher new regulatory pathways induced by competence and that are potentially key for natural genetic transformation. Interfering with theses regulatory pathways could represent a promising strategy to combat antibiotic resistance in the future.
P.1 and P.2 SARS-CoV-2 Brazilian variants activate the unfolded protein response with a time and pathway specificity.
de Morais Gomes V, Santos DM, Macedo-da-Silva J … +17 more , Lazari LC, Machado RRG, Dos Santos AF, Araujo DB, Coutinho JVP, Arini GS, Angeli CB, de Souza EE, Marques RF, Boscardin SB, Wrenger C, Marinho CRF, Oliveira DBL, Durigon EL, Labriola L, Rosa-Fernandes L, Palmisano G
J Proteomics · 2025 May · PMID 39909104 · Publisher ↗
COVID-19 is a human respiratory syndrome caused by the infection of the SARS-CoV-2 virus that has a high rate of infection and mortality. Viruses modulate the host machinery by altering cellular mechanisms that favor the...
COVID-19 is a human respiratory syndrome caused by the infection of the SARS-CoV-2 virus that has a high rate of infection and mortality. Viruses modulate the host machinery by altering cellular mechanisms that favor their replication. One of the mechanisms that viruses exploit is the protein folding and processing of post-translational modifications that occur in the endoplasmic reticulum (ER). When ER function is impaired, there is an accumulation of misfolded proteins leading to endoplasmic reticulum stress (ER stress). To maintain homeostasis, cells trigger an adaptive signaling mechanism called the Unfolded Protein Response (UPR) which helps cells deal with stress, but under severe conditions, can activate the apoptotic cell death mechanism. This study elucidated an activation of a diversity of molecular mechanisms by Brazilian variants of SARS-CoV-2 by a time-resolved and large-scale characterization of SARS-CoV-2-infected cells proteomics and immunoblotting. Furthermore, it was shown that pharmacological UPR modulation could reduce viral release by counteracting the different viral activations of its cellular response. Analysis of human clinical specimens and disease outcomes focusing on ER stress reinforces the importance of UPR modulation as a host regulatory mechanism during viral infection and could point to novel therapeutic targets. SIGNIFICANCE: Since the emergence of SARS-CoV-2 and the consequent COVID-19 pandemic, the rapid emergence of variants of this new coronavirus has been a cause for concern since many of them have significantly higher rates of transmissibility and virulence, being called Variants of Concern (VOC). In this work, we studied the VOCs Gamma (P.1) and Zeta (P.2), also known as Brazilian variants. Constant evidence has reported that there are particularities related to each variant of SARS-CoV-2, with different rates of transmissibility, replication and modulation of host biological processes being observed, in addition to the mutations present in the variants. For this reason, this work focused on infections caused by the Brazilian variants of SARS-CoV-2 in different cell lines, in which we were able to observe that the infections caused by the variants induced endoplasmic reticulum stress in the infected cells and activated the UPR pathways, presenting specific modulations of each variant in this pathway. Furthermore, transcriptome analysis of patients revealed a correlation between ER-related genes and COVID-19 progression. Finally, we observed that the use of UPR modulators in host cells decreased viral release of all variants without affecting cell viability. The data presented in this work complement the observations of other studies that aim to understand the pathogenicity of SARS-CoV-2 VOCs and possible new therapeutic strategies, mainly targeting biological processes related to the endoplasmic reticulum.
Antibody-free LC-HRMS/MS method for simultaneous quantification of NGAL, CRP and SAA in serum from sepsis patients.
Derbez-Morin M, Delatour V, Fenaille F … +4 more , Perrot C, Dupuy AM, Boeuf A, Becher F
J Proteomics · 2025 Apr · PMID 39900142 · Publisher ↗
Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS/MS) is a valuable alternative to ligand-binding assay, enabling specific and accurate quantification of protein biomarkers. We developed a rob...
Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS/MS) is a valuable alternative to ligand-binding assay, enabling specific and accurate quantification of protein biomarkers. We developed a robust antibody-free LC-HRMS/MS method for the multiplex quantification of three sepsis biomarkers in serum: NGAL, CRP and SAA. The method was thoroughly optimized from sample preparation to LC-HRMS/MS analysis, alongside the calibration. Specifically, a modified trichloroacetic acid/isopropanol protein precipitation procedure combined with an optimized Parallel Reaction Monitoring acquisition allowed the quantification of the low abundant NGAL at ng/mL levels. While reference material and reference measurement procedure were available for CRP, no such standards existed for NGAL and SAA. Well-characterized peptide calibrators traceable to the international system of units were developed for NGAL and SAA. The method demonstrated suitable trueness and precision for the quantification of NGAL, CRP, and SAA with coefficients of variation (CV%) ranging from 1.6 % to 22.4 % and bias between -12.6 % and +18.0 %. Successful application to pooled serum samples illustrated the method's effectiveness. Our results pave the way toward the development of reference systems for additional sepsis biomarkers.
Shotgun and targeted proteomics of Mycolicibacterium smegmatis highlight the role of arginine phosphorylation in the functional adaptation to its environment.
Xu D, Shi J, Jiang S … +8 more , Meng S, Cheng Z, Wu W, Chang L, Xie Y, Gao Y, Xue Y, Zhang Y
J Proteomics · 2025 Apr · PMID 39884554 · Publisher ↗
Although the phosphorylation of serine (S), threonine (T), and tyrosine (Y) is well-established, arginine phosphorylation (pR) has recently garnered significant attention due to its crucial role in bacteria pathogenicity...
Although the phosphorylation of serine (S), threonine (T), and tyrosine (Y) is well-established, arginine phosphorylation (pR) has recently garnered significant attention due to its crucial role in bacteria pathogenicity and stress response. Mycolicibacterium smegmatis, a nonpathogenic surrogate of Mycobacterium tuberculosis, serves as a model for studying mycobacterial pathogenesis. A recent proteomics study identified six pR proteins in M. smegmatis. To gain a more comprehensive understanding, we performed pR profiling using mass spectrometry in combination with two distinct phosphopeptide enrichment strategies: titanium-immobilized metal ion affinity chromatography (Ti-IMAC) and Fe-NTA cartridge purification. This approach led to the identification of 1192 shared pR peptides with 1553 pR sites in M. smegmatis following both competitive and non-competitive scoring assessments for pR and pS/T/Y. Further stringent filtering through manual verification resulted in 58 high-confident pR sites across 57 proteins. These confirmed pR-proteins are functionally related, particularly in DNA binding and ATP binding. Alterations in the modification of three pR sites during the logarithmic and stationary phases at the phosphorylation level, but not at the total cell protein level, further suggest the role of pR in the bacterium's functional adaptation to its environment. SIGNIFICANCE: Our findings reveal that pR proteins are prevalent and play roles in DNA-binding and ATP-binding activities, providing insights into the broader biological functions of pR peptides in other genetically diverse species. The reliable identification of bacterial pR events in M. smegmatis not only propels the study of pR within the realm of proteomics but also paves the way for exploring its detailed function in bacteria.
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