Journal Of Proteomics[JOURNAL]
Sun
200 papers
Proteomic landscape of crotonylation and acetylation in pig testes across male reproduction.
Wang L, Ao H, Niu X … +4 more , Zhai L, Liu J, Wang C, Xing K
J Proteomics · 2026 May · PMID 41724375 · Publisher ↗
Lysine crotonylation (Kcr) and acetylation (Kac) are evolutionarily conserved post-translational modifications that alter protein's charge, polarity, and structure. To explore the potential roles and critical modified pr...
Lysine crotonylation (Kcr) and acetylation (Kac) are evolutionarily conserved post-translational modifications that alter protein's charge, polarity, and structure. To explore the potential roles and critical modified proteins in male reproduction, we employed the affinity enrichment of pan-anti-Kcr and pan-anti-Kac antibodies with high-resolution LC-MS/MS from testes tissues of six boars (three at 5 months and three at 24 months of age), respectively. As a result, a large number of Kcr and Kac proteins were identified in testicular tissue between two groups. Differential analysis further revealed that Kcr proteins were significantly enriched to Gene Ontology biological process terms concerning reproductive system development, response to steroid hormone and response to hydroxy isoflavone, whereas Kac proteins primarily regulated mitochondria-localized proteins involved in testicular metabolism. RPS5, RPS6, CYP17A1, CYP11A1, and CYP19A1, which may act as important regulators of testicular development through crotonylation modification. In addition, we observed indications of crosstalk between the two modifications, involving shared enzymes (including HDAC1-3), overlapping substrates, and CoA donor metabolism. This study performed a large-scale analysis of Kcr and Kac proteins in porcine testis, providing a post-translational modification perspective to testis development. SIGNIFICANCE: The testis is a key organ determining male reproductive capacity, and pigs of different ages exhibit distinct reproductive performance. Most previous studies have focused on transcriptomic and proteomic differences in testes across developmental stages, while post-translational modifications (PTMs) remain poorly explored. Here, using affinity enrichment with pan-anti-Kcr and pan-anti-Kac antibodies combined with high-resolution LC-MS/MS, we profiled lysine crotonylation (Kcr) and acetylation (Kac) in pig testes. Bioinformatic analyses identified CYP17A1, CYP19A1, CYP11A1, RPS5, and RPS6 as potential regulators of testicular maturation via crotonylation, while acetylation was found to regulate metabolic activity by modifying mitochondrial proteins. Furthermore, crosstalk between crotonylation and acetylation at the metabolic level was revealed. These findings advance our understanding of PTMs in porcine testis development and provide novel data resources for future studies.
First insight into the regulatory role of the glycoprotein hormones GPA2 and GPB5 in the small-spotted catshark (Scyliorhinus canicula L.) during spermiogenesis.
Jeanne F, Pilet S, Bernay B … +1 more , Sourdaine P
J Proteomics · 2026 May · PMID 41724374 · Publisher ↗
Glycoprotein hormones alpha 2 (GPA2) and beta 5 (GPB5) are considered to be ancestral members of the glycoprotein hormone (GPH) family. Despite their potential roles in regulating thyroxine production, the immune respons...
Glycoprotein hormones alpha 2 (GPA2) and beta 5 (GPB5) are considered to be ancestral members of the glycoprotein hormone (GPH) family. Despite their potential roles in regulating thyroxine production, the immune response, and ovarian function, their roles in the testis remains largely unknown. To further explore this regulation, catshark testicular explants containing late spermatid stages were treated in vitro with recombinant GPA2, GPB5 and single-chain GPB5#GPA2. The comparative proteomic analysis of the treated explants revealed that ScGPB5 and ScGPB5#ScGPA2 were the most effective, modulating abundance of 449 and 525 proteins, respectively. In contrast, ScGPA2 only modulated 212 proteins. Among the differentially abundant proteins (DAPs), 103 showed substantial abundance changes in response to at least one treatment, with mean Logratios of 2.5 and - 2.5. Manual functional annotation linked these proteins to spermiogenesis and immunity. Key proteins and candidates involved in spermiogenesis were highlighted, including SPINK2, which is involved in acrosome protection, and LRRC69, which is related to human spermatogenic failure. These results reinforce the hypothesis of a GPA2/GPB5 paracrine regulation of catshark spermiogenesis. They also underscore the necessity of functional and comparative studies to elucidate the physiological functions of GPA2/GPB5 across a broad spectrum of taxa, from non-vertebrates to mammals. SIGNIFICANCE: To our knowledge, this study is the first to report the effects of stimulation with recombinant ScGPA2, ScGPB5, and single-chain ScGPB5#ScGPA2 on vertebrate testicular tissue in vitro. A large-scale, untargeted proteomic analysis revealed for the first time that all three-ScGPA2, ScGPB5, and single-chain ScGPB5#ScGPA2-can modulate the abundance of 103 of the 7619 identified proteins, including proteins related to spermiogenesis and immunity, as well as others that are less well characterized. This report reinforces the importance of functionally characterizing GPA2 and GPB5 by showing the first results toward the characterization of their spermatogenesis related function in a specie of evolutive interest, the small-spotted catshark. In the future, a complete characterization of GPA2 and GPB5 will further our understanding of the functional evolution of glycoprotein hormones-a keystone group of hormones for vertebrate physiology and critical targets for fertility modulation in both livestock and humans.
The acetylation status and metabolic characterization of the HBV-induced macrophages.
Sun Z, Wu S, Luo Y … +3 more , Su T, Xiong Y, Bei J
J Proteomics · 2026 May · PMID 41692303 · Publisher ↗
Hepatitis B virus (HBV) infection drives macrophages toward an M2-polarized phenotype, contributing to immune evasion and viral persistence. However, the underlying mechanisms involving post-translational modifications a...
Hepatitis B virus (HBV) infection drives macrophages toward an M2-polarized phenotype, contributing to immune evasion and viral persistence. However, the underlying mechanisms involving post-translational modifications and metabolic reprogramming remain poorly understood. This study integrates multi-omics and functional analyses to characterize lysine acetylation and lipid metabolic alterations in HBV-induced macrophages. In vitro and in vivo models confirmed HBV-promoted M2 polarization, marked by elevated CD206/CD163 expression and anti-inflammatory cytokine secretion. Acetylomic profiling identified 450 modified proteins and 432 quantifiable sites, with significant upregulation of peptides associated with chromatin remodeling and metabolic regulation. Lipidomic analysis revealed extensive reprogramming, including downregulation of phosphatidylcholines, phosphatidylinositols, and oxidized lipids, and upregulation of specific sphingolipids and triacylglycerols. Functional enrichment linked acetylated proteins to lipid metabolic processes and oxidative stress response. These findings suggest that HBV remodels macrophage acetylation and lipid metabolism, which may contribute to the development of an immunosuppressive microenvironment, providing new insights into potential therapeutic strategies targeting acetylation or lipid pathways in chronic HBV infection. SIGNIFICANCE: This study systematically characterizes lysine acetylation profiles and lipid metabolic reprogramming in HBV-induced macrophages using multi-omics and functional assays. It identifies HBV-driven acetylation changes in 450 proteins, which target chromatin remodeling and metabolic regulation, as well as distinct lipid alterations including reduced lipid storage and modified glycerophospholipids and sphingolipids. The study reveals that crosstalk between acetylation and lipid metabolism fosters an immunosuppressive microenvironment that supports HBV persistence. These findings fill critical gaps in understanding the mechanisms of HBV-induced macrophage polarization and provide novel therapeutic targets for chronic HBV infection.
Corrigendum to 'DSSBU: A novel mass spectrometry-cleavable analogue of the BS cross-linker' [Journal of Proteomics 310 (2025) 105330].
Swati B, Jakub S, Aleš M … +2 more , Petra J, Martin H
J Proteomics · 2026 Apr · PMID 41688246 · Publisher ↗
Abstract loading — click title to view on PubMed.
Novel high-resolution ion mobility mass spectrometry for site-specific quantification of the sirtuin-5 regulated kidney succinylome.
Schilling B, Rorrer L, Royer L … +6 more , Silva-Barbosa A, Pfister K, Goetzman E, Sims-Lucas S, DeBord D, Bons J
J Proteomics · 2026 May · PMID 41687715 · Publisher ↗
Protein post-translational modifications (PTMs) dynamically regulate essential biological and cellular processes. Lysine succinylation changes the amino acid charge, potentially affecting protein structures and functions...
Protein post-translational modifications (PTMs) dynamically regulate essential biological and cellular processes. Lysine succinylation changes the amino acid charge, potentially affecting protein structures and functions, and dysregulation of protein succinylation may lead to metabolic disorders. Proteome-wide succinylation quantification using proteomic tools remains challenging, especially due to the low abundance of succinylated peptides and the frequent presence of isomeric PTM forms. Ion mobility spectrometry workflows that can differentiate peptidoforms with different PTM distributions represent a powerful strategy to alleviate these challenges. Recently, a new Parallel Accumulation with Mobility Aligned Fragmentation (PAMAF™) operating mode for high-resolution ion mobility-mass spectrometry (HRIM-MS) analysis based on the structures for lossless ion manipulation (SLIM) technology was introduced. Here, we first assessed the performance of PAMAF mode for protein succinylation analysis using synthetic succinylated peptides, demonstrating residue-level differentiation of co-eluting isomers and isobars and precise PTM site localization. We leveraged this novel approach to investigate succinylome remodeling in kidney tissues from wild-type and Sirtuin-5 (Sirt5) knock-out mice, a NAD-dependent lysine de-succinylase. PAMAF acquisitions yielded ∼1000 confidently identified and accurately quantified succinylated peptides and sites from mouse kidney. Sirt5 regulated succinylation of mitochondrial proteins involved in metabolic processes, including fatty acid oxidation, the tricarboxylic acid cycle, and propionate metabolism. SIGNIFICANCE: Understanding the dynamic remodeling of the protein post-translational modification landscape is critical to gain insights into the underlying molecular mechanisms of biological systems. Lysine succinylation is a recently discovered reversible post-translational modification (PTM), that regulates various biological processes and associates with diverse diseases. However, this PTM is poorly characterized, partly due to analytical barriers. Here, we present a novel mass spectrometry (MS) methodology leveraging high-resolution ion mobility (HRIM) spectrometry and Parallel Accumulation with Mobility Aided Fragmentation (PAMAF) technology to profile and quantify succinylated peptides. The unique combination of liquid chromatography, ion mobility in a very long ion path (13 m), and alternate acquisition of MS and MS/MS spectra for all ions entering the mass spectrometer provided comprehensive profiling and accurate quantification of succinylated peptides in complex matrices. This technology enabled confident resolution of succinylated isomeric peptides, that could not be differentiated without high-resolution ion mobility separation and subsequent MS/MS PTM site identification. We investigated the kidney succinylome of Sirtuin-5 (desuccinylase) knockout mice compared to wild-type mice, with over 1000 succinylated peptides identified and quantified. We analyzed the hypersuccinylation of proteins upon Sirtuin-5 deletion, especially of mitochondrial proteins involved in diverse metabolic processes.
Ablation of ACSS2 drives alteration to cardiac and hepatic proteomic landscapes in a tissue- and sex-specific manner.
Winters AM, Wohlfahrt J, Wolf T … +5 more , Sarkar A, Patel SJ, Guergues J, Burkhardt BR, Stevens SM
J Proteomics · 2026 May · PMID 41679503 · Full text
ACSS2 catalyzes the conversion of acetate into acetyl-CoA, linking nutrient availability to cellular processes such as lipid biosynthesis, energy production, and epigenetic regulation. Although ACSS2 has been studied und...
ACSS2 catalyzes the conversion of acetate into acetyl-CoA, linking nutrient availability to cellular processes such as lipid biosynthesis, energy production, and epigenetic regulation. Although ACSS2 has been studied under metabolically stressful conditions, its basal sex- and tissue-specific functions remain poorly defined. Here, we employed comprehensive proteomic characterization of the impact of global ACSS2 ablation in the liver and heart of adult male and female mice. Over 6000 proteins were identified per tissue, providing deep proteomic coverage. Despite the liver exhibiting greater baseline abundance of ACSS2, the most extensive remodeling occurred in the heart. Both tissues displayed marked sex differences, with males showing greater overall proteomic shifts, and minimal overlap in differentially abundant proteins occurring between males and females. Shared alterations across tissues converged on metabolic and immune regulation, whereas sex-specific changes implicated distinct structural and signaling pathways. Comparatively modest hepatic changes may reflect compensatory processes in the liver, in contrast to the strong inhibitory remodeling observed in the heart. These findings reveal a previously unrecognized degree of tissue- and sex-specificity in regulation by ACSS2, while also highlighting the importance of including female mice in proteomic studies, as male only approaches may overlook key sex-dependent adaptations.
Molecular diversity and antimicrobial properties of extracts from different organs of Tunisian extremophilic plants.
Ben Brahim R, Voisin SN, Fouzai K … +2 more , Bulet P, Regaya I
J Proteomics · 2026 May · PMID 41679502 · Publisher ↗
Plants are a rich source of bioactive molecules, including anti-infective compounds. Antimicrobial peptides have been found in seeds, leaves, roots, stems, and flowers. In a previous study, we reported antimicrobial acti...
Plants are a rich source of bioactive molecules, including anti-infective compounds. Antimicrobial peptides have been found in seeds, leaves, roots, stems, and flowers. In a previous study, we reported antimicrobial activity in extracts from the leaves and roots of Astragalus armatus and Anthyllis sericea. In this study, we characterized the molecular diversity of several plant extracts using MALDI mass spectrometry and off-gel bottom-up proteomics approaches. In addition, the biological properties of the acidic extracts of the roots, leaves, and seeds of A. armatus, A. sericea, Genista microcephala, and Rhanterium suaveolens, 4 extremophilic plants from southern Tunisia, were evaluated. Their MALDI profiles showed substantial variability between plant species and organs. For the 4 plants, the seed profiles showed a higher molecular content compared to the leaf and root profiles. Furthermore, most of the masses determined for the different organs of the 4 plants were between 1 kDa and 3 kDa. This MALDI profiling revealed a large variety of molecules (401 in A. sericea, 564 in A. armatus, 398 in G. microcephala, and 300 in R. suaveolens) that remain to be further explored for their antimicrobial potential. The resulting extracts were tested against Gram-positive and Gram-negative bacteria, the filamentous fungus Aspergillus fumigatus, and the yeast Candida albicans, and showed variable antimicrobial activity. LC-MS analysis enabled the identification of 89 proteins in the plants, and the analysis of a fraction of A. artmatus roots, which successfully inhibited A. fumigatus, led to the identification and partial characterization of 3 proteins: Endochitinase 2, Thaumatin-Like protein and Peroxidase. SIGNIFICANCE: Antimicrobial resistance is one of the most pressing health challenges currently. In this context, extremophile plants represent an insufficiently explored source of bioactive molecules with potential therapeutic value. By applying proteomic approaches (MALDI-MS and LC-MS), our study provides new insights into the molecular diversity of acidic extracts from 4 Tunisian extremophile species and highlights their antimicrobial activities against resistant pathogenic bacteria and fungi. The importance of this work resides in its dual contribution: (i) expanding proteomic knowledge of Tunisian extremophile plants, which remain still poorly characterized, and (ii) opening new perspectives for the discovery of plant peptides and proteins as promising alternatives to conventional antimicrobials. This integrative approach bridges the gap between molecular characterization and functional validation, thereby contributing to the ongoing search for innovative strategies to combat multidrug resistance.
Proteomics-driven innovations in plant-based foods: Current advances, emerging technologies, and future perspectives.
Boukid F
J Proteomics · 2026 May · PMID 41672347 · Publisher ↗
Despite rapid market growth, plant-based foods such as meat analogs, plant-based milk, yogurt alternatives, and fermented products still fall short of matching the sensory, structural, and nutritional qualities of animal...
Despite rapid market growth, plant-based foods such as meat analogs, plant-based milk, yogurt alternatives, and fermented products still fall short of matching the sensory, structural, and nutritional qualities of animal-based counterparts, primarily due to simple ingredient substitution that fails to reproduce the molecular structure, interactions, and functional properties required for optimal texture, flavor, and nutritional performance. Proteomics, using advanced mass spectrometry (MS) and label-free quantification methods, provides an approach to analyze plant protein composition, structure, interactions, and modifications, enabling targeted functional improvements. This review describes how proteomic workflows inform formulation across three areas. First, protein compositional and structural characterization employs techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) and differential scanning calorimetry (DSC) coupled with MS to map protein composition and structural behavior, supporting decisions on protein sources, fractionation, and purification. Second, indirect proteomic methods coupled with other non-proteomic complementary tools are used to determine structure-function relationships induced by processing to examine processing-induced crosslinking, enzymatic modifications, and lipid-protein interactions that influence texture. Third, targeted MS methods, including selected reaction monitoring (SRM) and parallel reaction monitoring (PRM), are applied to profile off-flavor compounds and identify protein modification sites relevant to sensory and nutritional properties. By integrating proteomic data with processing strategies, this review outlines how proteomics can be used to examine key functional attributes related to texture, flavor, and nutritional quality in plant-based foods. SIGNIFICANCE: This review highlights the pivotal role of proteomics in advancing next-generation plant-based foods. Proteomic analysis enables an in-depth understanding of plant protein structure, composition, interactions, and bioactivity, providing critical insights for the development of functionally enhanced and consumer-acceptable alternatives. By integrating proteomics with AI, machine learning, multi-omics approaches, and cutting-edge analytical tools such as spatial proteomics and mass spectrometry imaging, the review demonstrates how protein functionality, flavor, texture, nutrition, and allergenicity can be optimized. Furthermore, it emphasizes the potential of proteomics to accelerate innovation in personalized nutrition, support sustainable and circular food systems, improve food safety, and reduce waste by valorizing plant-based by-products. This work serves as a roadmap for researchers and industry stakeholders seeking to leverage proteomics to design novel, high-quality, and sustainable plant-based protein products.
Comparative proteomics of amaranth and quinoa seeds reveals species-specific solubility traits of 11S globulins after Osborne and polarity-based extractions.
Bojórquez-Velázquez E, Vidal-Limón AM, Zamora-Briseño JA … +4 more , Llamas-García ML, Barrera-Pacheco A, Espitia-Rangel E, Barba de la Rosa AP
J Proteomics · 2026 May · PMID 41653972 · Publisher ↗
Amaranth and quinoa are nutritious grains rich in essential amino acids, vitamins, and phytochemicals. The use of these emergent functional foods is still limited and their proteins are poorly characterized. Here, we com...
Amaranth and quinoa are nutritious grains rich in essential amino acids, vitamins, and phytochemicals. The use of these emergent functional foods is still limited and their proteins are poorly characterized. Here, we compared amaranth and quinoa seeds by profiling their proteins using Osborne and polarity-based extraction methods and evaluating their relative protein content. The Osborne fractions and the two fractions generated by the polarity-based method (hydrophilic and hydrophobic) were quantified and analyzed by 1D-SDS-PAGE in the absence and presence of a reducing agent, as well as by diagonal electrophoresis. In addition, hydrophilic and hydrophobic proteins were analyzed by 2-DE, and the representative spots for each species were identified by LC-MS/MS. Both methods yield similar total protein amounts. The electrophoretic profiles showed differentiated patterns between the two seeds. All the extracts reflect the formation of high-molecular-mass aggregates because of interchain disulfide bonds. Intrachain disulfide bonds were also detected in 2S albumins. A differential behavior in the solubility of 11S globulins was observed across both species, and molecular modelling and molecular dynamics simulations were performed to explain this phenomenon. This study provides valuable insights into the structural differences between amaranth and quinoa proteins, which could help inform decisions about potential food applications. SIGNIFICANCE: This work addresses two main topics: the implementation of alternative methods for characterizing plant proteins and the detailed comparison of the protein profiles of amaranth and quinoa seeds using different electrophoretic approaches. The polarity-based method we propose represents an alternative to reduce sample handling and the number of extracts required for proteome characterization without sacrificing the protein yield. This study generated relevant information on the storage proteins of the two seeds analyzed, primarily 2S albumins, prolamins, and 11S globulins, to inform decision-making on their application in food technology.
Prospective serial proteomic analysis uncovers mechanistic pathways of chemotherapy resistance in advanced non-small cell lung cancer.
Kuo WK, Chu HY, Ko YK … +1 more , Hsu PH
J Proteomics · 2026 Apr · PMID 41653971 · Publisher ↗
Predicting chemotherapy response in advanced non-small cell lung cancer (NSCLC) remains a clinical challenge, as baseline profiles often fail to capture dynamic molecular adaptations under treatment. This prospective stu...
Predicting chemotherapy response in advanced non-small cell lung cancer (NSCLC) remains a clinical challenge, as baseline profiles often fail to capture dynamic molecular adaptations under treatment. This prospective study employed serial plasma proteomics to identify mechanistic pathways associated with chemotherapy resistance in 44 patients with stage IV NSCLC receiving platinum-based doublet chemotherapy. By analyzing blood samples collected immediately before the first and second cycles using liquid chromatography-tandem mass spectrometry, we demonstrated that a ratio-based proteomic model (early-treatment/pre-treatment) yielded superior separation between controlled and uncontrolled disease (UCD) compared to baseline-only assessment. Among 159 quantified proteins, 13 showed significant differential abundance, with UCD patients exhibiting marked upregulation of tetranectin, coagulation factor XIII A chain, and complement factor H-related protein 2. Ingenuity Pathway Analysis revealed that therapeutic resistance was characterized by three dominant axes: the activation of complement-coagulation-acute-phase signaling, the induction of lipid-nuclear receptor activity (LXR/RXR and DHCR24 signaling), and the relative attenuation of immune-regulatory pathways such as IL-12 signaling. These findings highlight the potential of serial proteomic profiling to uncover treatment-induced molecular adaptations, providing insights for therapeutic monitoring and hypothesis generation in precision oncology. SIGNIFICANCE: This study demonstrates the added value of prospective serial plasma proteomic profiling, compared with baseline-only approaches, for capturing early treatment-associated molecular adaptations in advanced non-small cell lung cancer (NSCLC) receiving chemotherapy. By quantifying proteomic changes between pre-treatment and early-treatment time points, we identified coordinated alterations involving the complement-coagulation-acute-phase axis and lipid-nuclear receptor signaling programs, including LXR/RXR and DHCR24, alongside relative attenuation of immune-regulatory pathways. Rather than reflecting isolated protein effects, these findings highlight interconnected host-tumor response programs that emerge under therapeutic pressure and may contribute to early adaptive resistance. Importantly, this work moves beyond static baseline markers by emphasizing dynamic, pathway-level changes and provides a hypothesis-generating framework for longitudinal therapeutic monitoring. Candidate proteins such as tetranectin and coagulation factor XIII A chain are proposed as molecular features associated with treatment response, warranting further validation in larger, prospective cohorts before translational application.
Proteomic profile of the extracellular matrix following cerebral ischemia-reperfusion injury identified HCK as a target for ischemic stroke therapy.
Li X, Mao Z, Chao P … +2 more , Liao Y, Li Y
J Proteomics · 2026 Apr · PMID 41628829 · Publisher ↗
Ischemic stroke is a detrimental central nervous system (CNS) disorder with high morbidity and disability rates, caused by local cerebral ischemia. Extracellular matrix (ECM) is a complex network structure secreted by ce...
Ischemic stroke is a detrimental central nervous system (CNS) disorder with high morbidity and disability rates, caused by local cerebral ischemia. Extracellular matrix (ECM) is a complex network structure secreted by cells and located in the intercellular compartment, which is significant changed following ischemic stroke. Here, we quantified the proteomic profile of the ECM of brain from young (2-month-old) and aged (18-month-old) mice underwent cerebral ischemia/reperfusion (I/R) at 24 h and 60 d post I/R. The proteomics results indicated that proteins associated with the tricarboxylic acid (TCA) cycle and neutrophil extracellular trap (NET) formation were significantly up-regulated in the brain ECM from mice underwent cerebral I/R during acute stage, while those associated with synaptic vesicle cycle were significantly down-regulated in all stage post cerebral I/R. Differently, the brain ECM from aged mice underwent I/R expressed higher levels of lysosomal proteins and lower levels of autophagy and synaptic vesicle cycle associated proteins than the brain ECM from young mice underwent I/R. Furtherly, the proteomics identified that Hematopoietic Cell Kinase (HCK) is a regulator for NET formation. Inhibition of HCK could down-regulate LPS-induced phosphorylation of ERK1/2 and IKKα/β, as well as blocking the LPS plus nigericin induced activation of NLRP3 inflammasome and NET-like trap formation in vitro. In addition, inhibition of HCK significantly ameliorated cerebral I/R-induced brain injury and NET formation in vivo, suggesting HCK is a therapeutic target for ischemic stroke treatment. SIGNIFICANCE: Our results systemically analyzed the protein profiles of ECM in the brain post- acute and chronic ischemic stroke, and identified upregulation of NET-associated proteins was a common feature of the cerebral ECM during the acute phase, while down-regulation of synaptic vesicle cycle associated proteins was a common character of the brain ECM in all stage. What's more, HCK was identified as a regulator for NET formation, inhibition of HCK could block the formation of NET by inhibiting the activation of ERK1/2, IKKα/β and NLRP3 inflammasome, suggesting HCK is a therapeutic target for ischemic stroke treatment.
Proteomics analysis of brain tissues of cerebral ischemic rat treated with Xingnaojing injection and its brain component muscone.
Gao C, Hao H, Chen X … +9 more , Mao N, Zhang X, Zhao J, Li Y, Deng N, Jia P, Zheng X, Liao S, Bian Y
J Proteomics · 2026 Apr · PMID 41611168 · Publisher ↗
Cerebral ischemia-reperfusion injury is categorized as "stroke" in traditional Chinese medicine. For thousands of years, traditional Chinese medicine has accumulated rich experience in the treatment of stroke and other d...
Cerebral ischemia-reperfusion injury is categorized as "stroke" in traditional Chinese medicine. For thousands of years, traditional Chinese medicine has accumulated rich experience in the treatment of stroke and other diseases, and with remarkable curative effects. Currently, Xingnaojing injection and its component musk are commonly used in the treatment of acute stroke, and muscone is the main active ingredient of musk. In this study, a rat model of transient middle cerebral artery occlusion was established, and the neuroprotective effects of Xingnaojing and muscone on transient middle cerebral artery occlusion rats were validated by Zea-Longa neurological function score, behavioral test and 2,3,5-triphenyltetrazolium chloride staining. Quantitative proteomics analysis was then performed on the brain tissues from different groups to investigate the mechanisms by which Xingnaojing and muscone act on cerebral ischemia-reperfusion injury. Our data indicate that Xingnaojing and muscone significantly affect proteins related to oxidative phosphorylation in CIRI rats, highlighting mitochondrial energy metabolism as a potentially important pathway contributing to their neuroprotective effects. Furthermore, the limited proteolysis-coupled mass spectrometry, target-responsive accessibility profiling, and lysine reactivity profiling methods were used to identify the direct protein targets of muscone in rat brain tissue lysate. A total of 36 potential target proteins were commonly identified by all the three methods. Bioinformatics analysis suggested that muscone was more significantly enriched in glycolysis/gluconeogenesis related pathways and closely associated with oxidative phosphorylation. Finally, the glycolytic key enzyme phosphoglycerate kinase 1, one of the binding proteins with muscone, was selected and verified by drug affinity responsive target stability. The molecular docking and dynamics simulation analysis further confirmed the interaction of glycolytic key enzyme phosphoglycerate kinase 1 and muscone. This study provides evidences for the clinical application and mechanisms of Xingnaojing and muscone in treating cerebral ischemia-reperfusion injury, and identifies candidate protein targets of muscone.
Label-free proteomics revealed drought stress tolerance mechanisms in the sugar beet monosomic addition line M14.
Guo X, Qiu W, Zhu J … +2 more , He Y, Yu B
J Proteomics · 2026 May · PMID 41581655 · Publisher ↗
Sugar beet M14 line is a diploid cultivated sugar beet (Beta vulgaris L.) that carries a monosomic addition of chromosome 9 from the wild white-flowered beet (B. corolliflora Zoss.), developed through distant hybridizati...
Sugar beet M14 line is a diploid cultivated sugar beet (Beta vulgaris L.) that carries a monosomic addition of chromosome 9 from the wild white-flowered beet (B. corolliflora Zoss.), developed through distant hybridization. It exhibits enhanced salt and drought tolerance compared to the diploid cultivated beets. In this study, the M14 line exhibited superior water retention capacity under dehydration conditions compared with five major diploid cultivated varieties grown in northern China. Through integrated analysis of phenotype, photosynthetic parameters, physiological and biochemical indicators, and the expression of key drought-responsive genes, 3 days and 5 days of 20% PEG-6000 treatment were identified as two critical time points for the drought stress response of the M14 line. Through label-free quantitative proteomics, 903 and 526 DAPs were identified at 3 and 5 days, respectively. PPI network analysis further revealed key protein interaction modules in the M14 line under drought stress. Furthermore, qRT-PCR analysis of 12 key DAP-encoding genes revealed that their transcript levels generally corresponded to the protein expression trends. This study helped to produce molecular network maps of drought tolerance in the M14 line, uncovering the mechanisms underlying its drought tolerance. SIGNIFICANCE: The drought tolerance of the sugar beet M14 line and ive major diploid sugar beet varieties cultivated in northern China was evaluated, revealing that the M14 line showed the strongest drought resistance. This study uncovered the dynamic regulatory network responsible for drought tolerance in the M14 line at the proteomic level, highlighting the main response pathways and key functional proteins at 3 and 5 days after stress exposure. These results not only deepen our understanding of the molecular mechanisms behind the sugar beet drought tolerance but also identify important candidate proteins and key regulatory modules for molecular breeding drought-tolerant varieties.
The untapped potential of metaproteomics in microbial systems ecology.
Hu J, Zheng Y, Li L
J Proteomics · 2026 Apr · PMID 41577126 · Publisher ↗
As a direct readout of protein presence and abundance in complex microbiomes, metaproteomics has become central to uncovering functionality across diverse microbial ecosystems. Recent collaborative efforts within the res...
As a direct readout of protein presence and abundance in complex microbiomes, metaproteomics has become central to uncovering functionality across diverse microbial ecosystems. Recent collaborative efforts within the research community have driven methodological advances, computational innovations, and diverse applications. With the advancing maturity of metaproteomics techniques, we highlight the Proteome-level Microbial Systems Ecology (ProMiSE) framework as a complementary framework for the next phase, providing a conceptual lens to interpret metaproteomics data with a bottom-up perspective that extends beyond functional profile and taxonomic composition to the quantitative assessment of system properties, processes, and dynamics. Building on existing metrics such as β-diversity and functional redundancy, future work can further develop quantitative approaches for resilience, exergy, and functional energy landscapes, thereby enabling a deeper systems-level understanding of microbiome dynamics and opening new avenues for the precise functional regulation of microbiomes. SIGNIFICANCE: This Perspective reviews the development of metaproteomics-particularly in the Journal of Proteomics-and envisions it as a transformative tool in microbial systems ecology, introducing the ProMiSE framework to integrate protein-resolved data with ecological theory and properties such as redundancy, resilience, and functional energy landscapes for the future of precise microbiome modulation.
The in vitro effect of omentin-1 on the global proteome of granulosa cells from normal weight Large White and fat Meishan pigs.
Pich K, Respekta-Długosz N, Rytelewska E … +5 more , Świderska B, Malinowska A, Smolińska N, Dupont J, Rak A
J Proteomics · 2026 Apr · PMID 41570894 · Publisher ↗
Ovarian granulosa cells (Gc) play a vital role in follicle maturation and successful ovulation. Omentin-1 (ITLN1) is an adipokine involved in energy metabolism and insulin resistance; its expression has been demonstrated...
Ovarian granulosa cells (Gc) play a vital role in follicle maturation and successful ovulation. Omentin-1 (ITLN1) is an adipokine involved in energy metabolism and insulin resistance; its expression has been demonstrated in the ovary and varies depending on the degree of pig fatness. However, its effect on the global proteome of Gc has not been previously investigated. It was hypothesized that ITLN1 affects the abundance of proteins involved in key processes occurring in Gc in pigs. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of Gc identified 208 significantly differentially abundant proteins (DAPs) in the Large White pigs, with 99 proteins upregulated and 109 downregulated. In fatter Meishan pigs, 42 statistically significant DAPs were identified, including 25 upregulated and 17 downregulated proteins. The identified DAPs were associated with the estrogen signaling pathway, cell cycle and DNA replication, protein synthesis, transport and maturation, cytoskeleton dynamics, cell signaling, and hormonal regulation. Notably, the number and identity of DAPs differed markedly between the two breeds, suggesting that ITLN1-mediated effects are modulated by fatness and breed-specific metabolic status. To further illustrate the observed differences, selected proteins were also analyzed using Western blotting and ELISA, which were consistent with the LC-MS/MS findings. The results indicate that ITLN1 has a modulatory influence on the porcine Gc proteome, which is dependent on fat content. This highlights the important role of ITLN1 in regulating ovarian functions. SIGNIFICANCE: This study provides a comprehensive proteomic analysis of porcine granulosa cells (Gc) after treatment with omentin-1 (ITLN1) in pigs with different fat content (Large White < Meishan). The identified differentially abundant proteins (DAPs) are intricately linked to critical biological pathways, including estrogen signaling, cell cycle regulation, DNA replication, protein synthesis and transport, cytoskeleton organization, and hormonal regulation. These findings enhance our understanding of the molecular mechanisms underpinning ovarian follicle development and breed-related reproductive traits in pigs. The insights gained could inform future strategies to improve fertility and reproductive efficiency in swine production, as well as provide a valuable resource for comparative studies on ovarian biology across species.
Time-resolved proteomic and phosphoproteomic profiling of Angiotensin-(1-7) signaling in A549 cells.
Melo-Braga MN, Magalhães GC, Silva FA … +4 more , Kjeldsen F, Larsen MR, Santos RAS, Verano-Braga T
J Proteomics · 2026 Apr · PMID 41570893 · Publisher ↗
Angiotensin-(1-7) [Ang-(1-7)] is a heptapeptide of the renin-angiotensin system (RAS) with antitumoral effects reported in various tumoral cell lines, including the human lung adenocarcinoma A549 lineage. While previous...
Angiotensin-(1-7) [Ang-(1-7)] is a heptapeptide of the renin-angiotensin system (RAS) with antitumoral effects reported in various tumoral cell lines, including the human lung adenocarcinoma A549 lineage. While previous studies have shown that Ang-(1-7) modulates MAPK and PI3K-AKT signaling, the precise molecular mechanisms involved remain incompletely understood. To investigate the signaling events of Ang-(1-7) in lung cancer-derived cells, we employed an integrated proteomic and phosphoproteomic approach in A549 cells. We analyzed early (minutes) and late (hours) molecular responses to Ang-(1-7) treatment. The treatment resulted in time-dependent modulation of multiple signaling pathways, including significant alterations in the MAPK, PI3K-AKT, and mTOR pathways at both the protein and phosphorylation levels. Notably, widespread early dephosphorylation events were observed, similar to the effects seen with other RAS peptides with antitumoral effects. Additionally, Ang-(1-7) promoted a long-lasting nuclear accumulation (up to 24 h) of the transcription factor FOXO1 indicating its activation. FOXO1 is known to regulate genes involved in apoptosis, cell cycle arrest, and oxidative stress, suggesting a role in mediating the peptide's antitumoral effects. The study provides new insights into the molecular basis of Ang-(1-7)'s antitumoral activity in A549 cells and reinforce its therapeutic potential in lung cancer. Raw data are available via ProteomeXchange with identifier PXD066687. SIGNIFICANCE: This study provides the first comprehensive, time-resolved proteomic and phosphoproteomic analysis of Angiotensin-(1-7) signaling in the lung cancer cell line A549. By capturing both early and late molecular events in A549 cells, we reveal that Ang-(1-7) modulates critical pathways involved in tumor progression, including MAPK, PI3K-AKT, and mTOR signaling. Importantly, we demonstrate the nuclear accumulation of FOXO1, a key transcription factor associated with tumor suppression, as part of the Ang-(1-7) response in A549 cells.
Rapid high-throughput antibody analysis using microwave-assisted digestion.
de Toledo TM, Valerio HP, Neto AMM … +4 more , Mule SN, Dos Santos Donado PR, Pascale CBA, Palmisano G
J Proteomics · 2026 Apr · PMID 41570892 · Publisher ↗
Monoclonal antibodies are a class of biotherapeutic proteins that have been developed over the past decade, leading to improved standards of care for the treatment of multiple diseases. Multi-attribute methods have emerg...
Monoclonal antibodies are a class of biotherapeutic proteins that have been developed over the past decade, leading to improved standards of care for the treatment of multiple diseases. Multi-attribute methods have emerged as powerful tools for critical quality attributes (CQAs). They leverage high-resolution accurate mass spectrometry and automated computational pipelines to identify pre-established modifications using DDA. In this study, we describe the development of a mass spectrometry-based workflow capable of processing up to 96 samples simultaneously while monitoring a broad panel of PTMs. We evaluated microwave-assisted digestion under different buffers and pHs, assessing sequence coverage, missed cleavages, and the occurrence of chemical artifacts. Analyses were performed using both DDA and DIA. Raw data were processed in dependent-peptide search(DDA) and PTM-probing search(DIA), enabling PTM discovery without prior knowledge. Our results demonstrate that microwave-assisted digestion, combined with control of temperature and pH, provides a fast and reliable alternative for efficiently digesting biotherapeutic proteins. It achieves high sequence coverage while minimizing artificial PTM formation. We also show that DIA combined with MW digestion improved peptide identification, highlighting its potential for comprehensive characterization of antibodies. Among the tested buffers, sodium acetate under MW conditions was the most effective in reducing deamidation and oxidation levels. SIGNIFICANCE: This study presents a detailed and optimized protocol for microwave-assisted (MW) protein digestion, enabling simultaneous reduction and alkylation for antibody samples. The method is rapid and minimizes chemical artifacts typically introduced during sample preparation. By combining MW-assisted digestion with both data-dependent (DDA) and data-independent acquisition (DIA), we performed a comprehensive and unbiased multi-attribute analysis (MAM). Notably, the use of DIA alongside MW digestion allowed for higher reproducibility and more complete peptide and post-translational modification (PTM) detection compared to DDA alone. Compared to conventional overnight digestion, MW-assisted digestion significantly reduced deamidation levels, with evident influences of buffer composition and pH on PTM identification. Although the levels of protein oxidation persisted, indicating that further optimization is necessary, this approach substantially decreased other artifacts, particularly deamidation, highlighting its potential as a fast, reliable, and highly informative strategy for antibody characterization.
Sputum proteomics and phosphoproteomics for improving chronic obstructive pulmonary disease knowledge.
Long F, Zeng X, Wang F … +14 more , Wang X, Shi W, Lan Y, Cheng J, Zhu C, Yang Y, Xiao J, Hu L, Tan L, Yang Y, Chen R, Liang Z, Peng T, Lu S
J Proteomics · 2026 Apr · PMID 41565155 · Publisher ↗
Analysis of proteins and other molecular components in induced sputum provides critical insights for the diagnosis, pathological assessment, and therapeutic monitoring of respiratory diseases. In this study, we collected...
Analysis of proteins and other molecular components in induced sputum provides critical insights for the diagnosis, pathological assessment, and therapeutic monitoring of respiratory diseases. In this study, we collected three distinct types of induced sputum samples from patients with chronic obstructive pulmonary disease (COPD) and subjected them to proteomic and phosphoproteomic analysis using three different enzymatic digestion methods. We found that raw sputum samples yielded a higher number of uniquely identified proteins and phosphoproteins (1313 proteins and 1603 phosphorylation sites, corresponding to 782 phosphoproteins) and provided a more comprehensive characterization of COPD pathology. Furthermore, compared to in-gel digestion and in-solution digestion, the filter-aided sample preparation method increased protein identification by approximately 30% and yielded the highest number of unique protein identifications. Our study is the first to demonstrate that raw induced sputum can serve as a viable alternative source for liquid biopsy in respiratory diseases. We have also established the first methodological framework and dataset for proteomic and phosphoproteomic analysis of raw induced sputum, generating a preliminary map of the COPD sputum proteome and phosphoproteome. This novel proteomic and phosphoproteomic approach has untangled biologically relevant pathways in respiratory physiology, highlighting potential avenues for future research. SIGNIFICANCE: In this study, we aimed to investigate the feasibility of establishing and evaluating proteomic research methods using sputum samples from patients with chronic obstructive pulmonary disease (COPD). The ultimate goal was to develop analytical approaches suitable for sputum proteomics and phosphoproteomics and to preliminarily map the sputum proteome and phosphoproteome in COPD. It was found that raw sputum samples more comprehensively reflect the disease characteristics of COPD and are therefore more suitable for proteomic and phosphoproteomic studies of COPD. Among three mainstream enzymatic digestion methods, the Filter-Aided Sample Preparation (FASP) method demonstrated superior identification rates and was deemed most suitable for processing raw sputum samples. Furthermore, this study reports for the first time a draft map of the proteome and phosphoproteome of COPD sputum. This research provides valuable insights into sputum proteomic analysis and offers a useful resource for the study of respiratory diseases.
Barley protein: From agricultural staple to sustainable protein solution.
Boukid F
J Proteomics · 2026 Apr · PMID 41554437 · Publisher ↗
Barley protein is a multifunctional, sustainable plant-based ingredient with potential in food, nutraceutical, and industrial applications. This review synthesizes current knowledge on barley protein, emphasizing how pro...
Barley protein is a multifunctional, sustainable plant-based ingredient with potential in food, nutraceutical, and industrial applications. This review synthesizes current knowledge on barley protein, emphasizing how proteomics and processing methods influence its composition, digestibility, and functional properties. Proteomic analyses reveal the distribution of major protein fractions, albumins, globulins, hordeins, and glutelins and their bioactive peptides, which exhibit antioxidant, antihypertensive, antidiabetic, and appetite-regulating activities. Protein concentrates and isolates offer improved digestibility and functional quality, though lysine remains limiting. Advanced techniques, including enzymatic hydrolysis, ultrasound-assisted extraction, and post-processing modifications, are evaluated for their impact on protein structure and functionality. Barley protein's potential applications in novel foods, micro- and nano-encapsulation, and targeted bioactive delivery are highlighted. By integrating proteomics insights with nutritional and technological perspectives, this work underscores the role of barley proteins in sustainable food systems. SIGNIFICANCE: This review synthesizes current knowledge on barley protein composition, emphasizing insights gained from proteomic analyses. By characterizing protein fractions, bioactive peptides, and allergenic determinants, proteomics enables a deeper understanding of barley's functional, nutritional, and health-related properties. The work highlights how extraction and processing influence protein quality and bioactivity, informing strategies for the development of novel plant-based foods. These insights provide a foundation for future research and industrial applications, advancing barley as a sustainable and functional protein source in human nutrition.
Two-dimensional electrophoresis-based proteomics reveals soybean seed hypocotyl proteoforms.
Tan JZ, Kagawa H, Kizawa K … +1 more , Hirano H
J Proteomics · 2026 Apr · PMID 41554436 · Publisher ↗
Shotgun proteomics is widely used for comprehensive profiling of protein expression. However, this approach has inherent limitations in resolving "proteoforms". Two-dimensional gel electrophoresis (2DE) has the potential...
Shotgun proteomics is widely used for comprehensive profiling of protein expression. However, this approach has inherent limitations in resolving "proteoforms". Two-dimensional gel electrophoresis (2DE) has the potential to separate and visualize proteoforms effectively. To evaluate its utility, proteins extracted from soybean seed hypocotyls were analyzed by 2DE/LC-MS/MS. As a result, a total of 693 proteins were separated by 2DE, of which 302 were identified by LC-MS/MS. The dynamic range of protein abundance was approximately 10 to 10. This analysis revealed that the hypocotyls contain numerous proteoforms of proteins essential for seed physiology, including late embryogenesis abundant protein, glycinin, β-conglycinin, trypsin inhibitor, sucrose-binding protein, and glyceraldehyde-3-phosphate dehydrogenase. Furthermore, 2DE revealed that the expression of proteoforms of the major seed storage proteins, glycinin A2 and A3 subunits, and β-conglycinin β subunit in hypocotyls differed from that in cotyledons, suggesting distinct functional roles beyond nutrient storage during germination. Overall, the results demonstrate that 2DE is a valuable complementary technique to shotgun proteomics, providing proteoform-specific information that cannot be resolved by shotgun analysis alone. SIGNIFICANCE: Shotgun proteomics is widely used for comprehensive profiling of protein expression. However, this approach has inherent limitations when analyzing "proteoforms". In the present study, we analyzed soybean seed hypocotyls using two-dimensional gel electrophoresis (2DE)-LC-MS/MS, demonstrating that 2DE is useful for effective proteoform analysis. 2DE provides proteoform-specific information that cannot be obtained from shotgun proteomics alone, making it a useful complementary method.
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