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The field of proteomics has rapidly evolved over the last five years enabled by rapid advances in instrumentation and computation. At the same time, the proteomics community is also growing. This is reflected by the increasing participation in international conferences such as those organized by the European Proteomics Association and the Human Proteome Organization. These events provide early-career researchers with unique opportunities to exchange ideas, develop collaborations, and build networks that support professional development. One such network is the Young Proteomics Investigators Club, a European initiative supported by European Proteomics Association and led by early-career researchers. In this Community-Driven project, we investigate recent trends in proteomics by screening conference abstracts and evaluating the session attendance at Human Proteome Organization Congresses and European Proteomics Association conferences. Based on these analyses, we identified five areas that, from our perspective, are shaping the current trends in proteomics: clinical proteomics, proteomics of post-translational modifications, single-cell proteomics, systems biology and multi-omics, and computational proteomics. For each area, we highlight both unique challenges and identify a common theme: a shift from exploratory studies with manageable sample numbers towards large screenings and cohorts and the generation of big data, which often comes with the lack of computational support, organizational networks, and infrastructure. In this light, we describe the unique challenges and opportunities faced by early-career researchers. We point to actionable directions for enabling reproducible and transparent proteomics as well as community-driven projects and initiatives, which are often providing training and support. SIGNIFICANCE: In this perspective, the Young Proteomics Investigators Club (YPIC) discusses advances in analytical developments and computational approaches in proteomics research. Based on empirical analysis of recent European Proteomics Association conference and Human Proteome Organization congresses contributions, we identify clinical, single-cell, post-translational and systems-level proteomics as the research areas that have gained most momentum in the last three to five years. What makes this work distinctive is that it is written by and for early-career researchers, thereby uniquely identifying where momentum, challenges, and unmet needs converge for the newest generation of proteomics researchers. Rather than cataloguing advances, we examine the widening gap between what modern proteomics can generate and what individual researchers can realistically process, validate, and interpret. We describe specific structural barriers including access to high performance computing, limited formal training in scalable data analysis, the need for unified benchmarking standards and navigating clinical collaboration frameworks. We then highlight opportunities for the field, such as community-curated benchmarks, interdisciplinary mentorship models, and shared computational infrastructure. By making these challenges explicit from an early-career researchers standpoint, we aim to inform how training, funding, and community initiatives can be shaped to support the next generation of proteomics researchers.
The Mexican Proteomics Society (MPS), founded in 2005, is the earliest proteomics society in Latin America. It is a non-profit organization comprising academics and professionals committed to advancing research in proteomics, metabolomics, and mass spectrometry. To fulfill its mission, MPS organizes symposia and academic events that foster collaboration and knowledge exchange among researchers and professionals interested in these fields. MPS actively promotes human resource training and scientific outreach. Mexican researchers are engaged in international initiatives such as HUPO and consortia, including pi-HuB and the Chromosome-Centric Human Proteome Project, underscoring the country's growing role in global proteomics. The biennial MPS symposium has become a solid academic forum in proteomics and metabolomics, where international experts share their latest findings, and technology developers present advances in analytical instrumentation and software. The topics covered at the symposium held in Oaxaca, Mexico, on November 9-13, 2024, were as diverse as our country's biodiversity and as broadly applicable as mass spectrometry. These include human health (e.g., cancer, chronic degenerative diseases, allergies), plants, foodomics, lipidomics, venomics, microbial proteomics, computational omics, and analytical methods development. This Special Issue, which features research presented at the symposium, reflects the diversity of our community and highlights the value of scientific collaboration. SIGNIFICANCE: For the past 20 years, the Mexican Proteomics Society (MPS) has advanced proteomics and metabolomics in México. The society hosts a trusted biennial event where global experts and vendors share the latest breakthroughs in mass spectrometry, metabolomics, and proteomics. Through hands-on workshops, the MPS trains students and professionals in essential protein and metabolite analysis and the use of open-access bioinformatics tools. The society has also hosted prestigious global events, including the 2022 HUPO World Congress. The topics represented in the articles of this special issue reflect the diversity of our community and highlight the value of scientific collaboration.
Environmental proteomics has emerged as a powerful approach for elucidating the molecular mechanisms underlying pollutant-induced biological effects. Although this field has developed rapidly, the systematic review of recent proteomics applications in environmental pollution research remains limited. This review explored the emerging roles of toxicoproteomics in biomarker discovery and mechanistic elucidation, as well as ecotoxicoproteomics in ecological risk assessment and bioremediation strategies. Here, we review the field, highlighting recent trends such as the integration of proteomics with genomics, transcriptomics, and metabolomics to provide a comprehensive view of biological responses to environmental stressors. We further discuss the growing application of artificial intelligence in improving proteomics data interpretation and accelerating biomarker discovery. In addition, recent technological advances in environmental proteomics are highlighted, including next-generation tissue microarray proteomics, nanoscale proteomics, single-cell proteomics, and spatial proteomics. Despite its potential, proteomics faces challenges, such as high operational costs, computational complexity in analysis, and technical limitations in low-abundance protein detection. We propose that the convergence of proteomics with artificial intelligence and multi-omics approaches offers promising solutions to these challenges, enhancing the practical application of proteomics in environmental monitoring and risk assessment.
Control of the stomatal aperture is multifaceted, involving a complex interplay of environmental cues and intracellular signaling pathways. It is well established that changes in ion gradients drive water movement into and out of the guard cell, thereby altering cell volume and modulating the opening or closing of the stomatal pore. These rapid responses are often regulated by phosphorylation cascades to efficiently transmit environmental status and either reduce water loss or enhance carbon assimilation. The role of endomembrane trafficking networks in stomatal dynamics is not well characterized. Here, we investigated the regulation of stomatal opening and closing by generating a proteome and phosphoproteome of guard cell-enriched tissue. This deep proteome captured a protein profile that was similar to previously characterized guard cell proteomes. The guard cell-enriched tissue with closed stomata showed greater levels of phosphorylation of proteins related to endomembrane trafficking and vacuoles when compared to both whole leaf tissue with closed stomata and guard cell-enriched tissue with open stomata. These results support the hypothesis that phosphorylation of endomembrane proteins may contribute to the regulation of stomatal movements. SIGNIFICANCE: Stomatal movements are tightly regulated and are critical for gas exchange and water retention in plants. Stomatal movements are regulated by light, temperature, CO2, humidity and hormones, and are driven by changes in cellular turgor and metabolism. Stomatal opening is the result of a complex signaling pathway involving the activity of blue light photoreceptors, plasma membrane H+ ATPases, and inward-rectifying K+ channels. Stomatal closing is induced by the stress hormone ABA, and involves the activation of specific anion channels that lead to K+ efflux. Phosphorylation cascades are key signaling cues for this pathway, and many kinases and phosphatases have been implicated in regulating stomatal movements. This work describes the first phosphoproteome of guard cell-enriched tissue. Isolation of intact guard cells, followed by treatment with opening or closing conditions, allowed the isolation of proteins in their open or closed-responsive state. Quantitative phosphoproteomics enabled quantitative detection of changes in phosphorylated peptide abundance between open and closed states. GO enrichment analysis supports the hypothesis that phosphorylation cascades induced by stomatal closing may target endomembrane proteins, which could be important for the endomembrane remodeling that follows decreases in cellular volume. Overall, this dataset serves as a key resource for hypothesis generation regarding plant stomatal function.
Proteomic analysis revealed that Bacillus subtilis exhibits markedly different physiological adaptations under pellicle biofilm and swarming growth conditions, and that these lifestyles strongly influence the bacterial response to CeO₂ nanoparticles. In pellicle biofilms, proteins involved in respiration, amino acid acquisition, Mn/Fe uptake, and SUF-mediated iron‑sulfur cluster synthesis were upregulated, together with oxidative stress defense systems, indicating adaptation to microaerobic and heterogeneous biofilm conditions. In contrast, swarming cells displayed increased sporulation-associated processes and a stronger stringent response. Exposure to CeO₂ nanoparticles induced a pronounced response, particularly under swarming conditions, where central carbon metabolism enzymes were strongly repressed and stringent response pathways were reinforced. In biofilms, CeO₂ effects were more moderate, with limited metabolic perturbation and a slight stimulation of biofilm formation. The contrasting responses between lifestyles appear primarily linked to differences in metabolic state, oxidative stress physiology, and nanoparticle accessibility within the biofilm matrix rather than to direct nanoparticle toxicity alone. Overall, these findings demonstrate that nanoceria impacts B. subtilis physiology in a growth mode-dependent manner and highlight the importance of considering bacterial lifestyle when evaluating nanoparticle toxicity. SIGNIFICANCE: This research provides a biologically understanding of how Bacillus subtilis, an important bacterium for soil, plant, and animal health, adapts to environmental stress under more physiologically realistic growth conditions. Using shotgun proteomics, we demonstrated that pellicle biofilm and swarming lifestyles are associated with profoundly distinct physiological states, notably in metabolism, oxidative stress management, metal homeostasis, and developmental regulation. Extending this approach to exposure to cerium oxide nanoparticles (CeO₂ NPs), a widely distributed environmental nanomaterial, we showed that bacterial responses are strongly dependent on growth mode. Swarming cells exhibited pronounced metabolic repression and activation of stringent response pathways, whereas biofilms displayed more limited perturbations together with slight stimulation of biofilm formation. These contrasting responses appear to result primarily from lifestyle-dependent differences in metabolic activity, oxidative stress physiology, and nanoparticle accessibility within the biofilm matrix rather than from direct nanoparticle toxicity alone. Since regulatory toxicology frameworks such as REACH often overlook subtle physiological adaptations, this study highlights the importance of considering microbial lifestyle and physiological context when assessing the ecological risks of emerging chemicals and nanomaterials.
Tear fluid is a promising minimally invasive source of biomarkers for ocular surface diseases (OSDs). However, variability in sampling and pre-analytical processing remains a major limitation for reproducibility and cross-study comparability. Here, we quantitatively evaluated two commonly used tear collection methods, microcapillary tubes (CT) and Schirmer strips (SCH), and assessed the effect of elution buffer composition on protein recovery from SCH. In Study I, tears were collected from the same healthy donors using CT and SCH. In Study II, SCH from additional donors were longitudinally split and eluted with phosphate-buffered saline (PBS) or a denaturing cell lysis buffer (CLB; 7 M urea, 2 M thiourea, 4% CHAPS), enabling paired within-strip comparisons. Samples were analyzed by Evosep-timsTOF Pro DIA mass spectrometry and quantified using library-free DIA-NN. Overall, 3749 proteins were identified. SCH markedly increased proteome coverage compared with CT, reflecting recovery of both soluble tear proteins and ocular surface-derived cellular material. Functional annotation showed enrichment of intracellular compartments in SCH samples, particularly cytoplasmic, nuclear, and cytoskeleton-related proteins. For SCH extraction, CLB modestly increased unique protein identifications compared with PBS while preserving high quantitative concordance. These findings show that pre-analytical choices strongly influence tear proteome depth and abundance profiles. SIGNIFICANCE: Tear proteomics is increasingly recognized as a valuable platform for biomarker discovery in ocular, neurological, and systemic diseases. However, substantial methodological heterogeneity in tear collection and pre-analytical processing remains a major source of variability, limiting reproducibility and cross-study comparability. By directly comparing microcapillary tube and Schirmer strip sampling within the same donors, and by evaluating mild versus denaturing elution conditions from Schirmer strips using a within-strip paired design, this study provides quantitative evidence that pre-analytical choices systematically shape both proteome depth and quantitative abundance profiles. Schirmer strip sampling markedly expands detectable proteome coverage, partly through co-recovery of ocular surface-derived cellular material, whereas denaturing extraction enhances the recovery of protein subsets insufficiently solubilized under mild conditions while preserving high quantitative concordance. Collectively, these findings demonstrate that sampling and extraction strategies are not neutral technical variables but key determinants of the biological space interrogated by tear proteomics. The data presented here inform rational study design, improve inter-laboratory comparability, and support the development of harmonized standard operating procedures for robust tear-based proteomic biomarker discovery and translational applications.
SIRT1 (sirtuin 1), a nicotinamide adenine dinucleotide-dependent protein deacetylase, regulates cardiovascular inflammation by modulating cellular stress, inhibiting NLRP3 (NLR family, pyrin domain-containing 3) activation, and promoting the clearance of damaged mitochondria. However, its precise role in the pathogenesis of Kawasaki disease, a pediatric systemic vasculitis that causes coronary artery aneurysms in children, remains unclear. Using the Lactobacillus casei cell wall extract (LCWE) murine model of Kawasaki disease, we evaluated the severity of vasculitis in mice supplemented with nicotinamide adenine dinucleotide precursors, as well as transgenic mice overexpressing SIRT1, and mice with specific deletion of Sirt1 in vascular smooth muscle cells and myeloid cells. Proteomics and echocardiographic analyses were conducted on wild-type and SIRT1-overexpressing mice. We performed immunofluorescent staining, flow cytometry, and Western blot analyses of cardiovascular tissues, heart and abdominal aorta, to assess immune cell infiltration and the expression of proteins related to the autophagy/mitophagy pathway. Western blot analysis was also performed on primary vascular smooth muscle cells to determine the impact of SIRT1 on autophagic flux. The production of proinflammatory cytokines was measured in bone marrow-derived macrophages and peritoneal lavage of transgenic mice using ELISAs. SIRT1 expression was downregulated in cardiovascular lesions of LCWE-injected mice, which was associated with a significant reduction of circulating levels of nicotinamide. Supplementation of mice with nicotinamide adenine dinucleotide precursors or genetic overexpression of SIRT1 significantly reduced the development of LCWE-induced vasculitis, while the specific deletion of Sirt1 in vascular smooth muscle cells or myeloid cells exacerbated vasculitis. Furthermore, overexpression of SIRT1 restored impaired ejection fraction and reduced immune cell infiltrations in LCWE-induced cardiovascular lesions. Proteomics analysis indicated impaired mitophagy/autophagy and the pathogenic synthetic switch of vascular smooth muscle cells in LCWE-injected mice, which was rescued with SIRT1 overexpression and associated with reduced production of proinflammatory cytokines. This study reveals an impaired nicotinamide adenine dinucleotide-SIRT1 axis in the pathogenesis of LCWE-induced vasculitis and the therapeutic potential of targeting this axis to reduce cardiovascular lesions and inflammation.
Tuberous sclerosis complex (TSC) is a rare disease caused by mutations in TSC1 and TSC2, resulting in activation of mechanistic target of rapamycin complex 1 (mTORC1). Neurological manifestations in TSC patients include epilepsy, autism and intellectual disability. Two types of brain lesions, cortical tubers and subependymal giant cell astrocytomas (SEGAs), cause the majority of neurological manifestations in TSC. We have limited understanding of the molecular changes that occur in tubers and SEGAs and how these contribute to disease pathogenesis. To investigate this, we performed proteomic and phosphoproteomic analysis of TSC patient tuber and SEGA tissue. Tubers showed evidence of alterations in mitochondrial respiration, cytoskeleton organisation and neuronal function. However, we were unable to detect mTORC1 activation in tubers, likely due to the small number of cells with complete inactivation of TSC1 or TSC2. By contrast, SEGAs showed evidence of strong mTORC1 activation and large-scale changes in the proteome and phosphoproteome. SEGAs exhibited increased expression of ribosomal proteins and activation of a neuroinflammatory response. Phosphoproteomics identified 6060 phosphosites within 2154 proteins increased in SEGAs. Phosphorylation of multiple proteins involved in RNA-metabolism, including mRNA splicing, was increased in SEGAs. Consistent with this, we found evidence of extensive alterations in mRNA transcript splicing in SEGA tissue that is shared with a wide range of cancers. These data greatly expand the repertoire of known mTORC1 target proteins in the human brain and reveal that large-scale mis-regulation of mRNA splicing may promote the formation of SEGAs in TSC.
Dynamic modulation of α5-GABAAR expression and synaptic distribution plays a pivotal role in neuronal homeostatic plasticity, critically influencing memory processes. This study aims to investigate the spatiotemporal dynamics of α5-GABAAR in hippocampal subregions (CA1, CA3, and DG) and their behavioral correlation in mice following sevoflurane exposure across eight timepoints. Eight-week-old female C57BL/6 mice were exposed to 3% sevoflurane for 1 h and they were subjected to trace fear conditioning followed by sevoflurane. Hippocampal tissues were harvested for proteomic analysis and immunofluorescence staining to quantify the expression of α5-GABAAR and P-gephyrin. Three-dimensional spatial colocalization of α5-GABAAR and gephyrin was reconstructed in IMARIS software. By integrating trace fear conditioning with molecular profiling, we identified 2 days postexposure (Sev2d) as the critical phase for sevoflurane-induced memory impairment and 6 days postexposure (Sev6d) as the recovery phase. The time-dependent biphasic pattern of α5-GABAAR regulation was demonstrated by proteomics, immunofluorescence, and 3D imaging: (1) At Sev2d, α5-GABAAR expression and postsynaptic clustering were significantly elevated, which coincided with peak cognitive deficits; (2) by Sev6d, both receptor density and synaptic localization normalized to baseline level, paralleling memory restoration. These findings indicate that changes in the expression and distribution of α5-GABAAR are correlated with sevoflurane-induced memory impairment and recovery, providing potential insights into sevoflurane-induced memory fluctuation.
Medication-related osteonecrosis of the jaw (MRONJ) is a complex condition associated with the use of antiresorptive drugs, such as bisphosphonates and denosumab. The condition is characterized by the presence of exposed bone in the maxillofacial region that fails to heal. MRONJ remains highly intractable, as its pathogenic mechanisms are not yet fully understood. It is therefore essential to elucidate the molecular mechanisms underlying the disease. MiRNA expression analysis and proteomic studies were performed on a selected cohort of patients with MRONJ on jawbone tissue, using qRT-PCR and 2D electrophoresis followed by mass spectrometry. MiRNAs and proteomics data validation was carried out by Western blot analysis of differentially expressed proteins highlighted by a proteome study and predicted targets of differentially expressed miRNAs. Nineteen miRNAs were overexpressed and two downregulated in jawbone tissue from all MRONJ patients. Notably, five of these dysregulated miRNAs are involved in the regulation of angiogenesis and desmosome functions, suggesting a potential link to the molecular alterations observed at the protein level. Proteomic analysis revealed decreased concentrations of the pigment epithelium-derived factor, and of desmoglein-1, a desmosomal cadherin. Validation analysis confirmed the dysregulation of pathways involved in bone remodeling and necroptosis. The pathophysiology of MRONJ arises from a complex interplay of factors, including impaired bone remodeling, affected angiogenesis, and altered cell adhesion and differentiation mechanisms, ultimately leading to necroptosis. Through proteomic analysis and validation of miRNA expression, our study proposes specific molecular alteration in MRONJ-compromised bone tissue, involving desmosomal component imbalance and angiogenesis inhibition.
ALG13 mutations cause congenital disorders of glycosylation and neurodevelopmental deficits, but how asparagine-linked glycosylation 13 (ALG13) deficiency impairs brain development remains unclear. This study aimed to elucidate the underlying mechanisms in Alg13 knockout (ALG13KO) mice. We first confirmed neurodevelopmental delays and abnormal cortical neuron distribution in ALG13KO mice. Quantitative proteomic analysis of the postnatal day 7 cerebral cortex revealed widespread protein abundance changes. Subsequent bioinformatic and protein-protein interaction net-work analyses pinpointed the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) pathway. Pathway as a central hub. Parallel reaction monitoring validated the downregulation of key upstream regulators Laminin γ-1 (LAMC1), Focal Adhesion Kinase (FAK), and Integrin α6 (ITGA6). Western blot confirmed the inhibition of PI3K/AKT/mTOR phosphorylation. Our findings demonstrate that ALG13 deficiency disrupts cortical development, likely via suppression of the PI3K/AKT/mTOR pathway through the LAMC1-ITGA6-FAK axis. This study reveals a critical, early-developmental suppression of mTOR signaling, contrasting with its reported hyperactivation in adult epileptic ALG13KO mice, highlighting a stage-dependent role. SIGNIFICANCE: This study provides the first proteomic evidence of early postnatal suppression of the PI3K/AKT/mTOR pathway in a mouse model of ALG13-congenital disorder of glycosylation (ALG13-CDG). By integrating unbiased quantitative proteomics, targeted validation, and phenotyping, we identify the LAMC1-ITGA6-FAK axis as a novel upstream regulator mediating this suppression, linking a glycosylation defect directly to a key neurodevelopmental signaling hub. Importantly, our finding contrasts with reported mTOR hyperactivation in adult epileptic mice, revealing a critical, previously unrecognized stage-dependent duality of mTOR signaling in ALG13-CDG pathophysiology. This work not only advances the mechanistic understanding of neurodevelopmental deficits in CDG but also showcases the power of a focused, early time-point proteomic strategy to disentangle primary developmental pathophysiology from secondary disease states.
Caregiver roles and responsibilities in chronic kidney disease (CKD) patients have pervasive effects on the physical, social, and emotional well-being of caregivers, yet these issues are often under-prioritized in medical management. This study assessed depression, anxiety, burden, and quality of life (QOL) in caregivers of CKD patients. Caregiving burden, quality of life, depression, and anxiety were assessed using the Zarit Burden Interview (ZBI), SF-36 QOL questionnaire, Beck Anxiety Inventory, and Beck Depression Inventory, respectively, among caregivers of CKD patients on maintenance hemodialysis (HD), peritoneal dialysis (PD), and kidney transplantation (TX). The study groups consisted of 100, 105, and 62 caregivers among PD, HD, and TX patients, respectively. The mean scores of depression, anxiety, and burden in all caregivers were 13.44 (SD 11.57), 15.77 (SD 12.49), and 13.33 (SD 9.51), respectively. More than 40% of caregivers had depression, while 68.5% reported moderate to severe anxiety. Although anxiety, depression, and burden did not differ significantly across the three groups, the physical function scores of caregivers of PD patients were significantly better than those of the other groups (P = .033). Caregivers with higher depression, anxiety, and burden scores had significantly lower scores in both physical (P < .001, P < .001, P = .022) and psychological health (all P < .001) domains of QOL. Sex and education had a strong influence on depression, anxiety, burden, and QOL. Caregivers of CKD patients, especially females with lower education, are at increased risk of psychological burden. Early screening for mental health issues in caregivers, along with supportive interventions, should be incorporated into treatment guidelines for CKD patients.
Human pluripotent stem cells can be differentiated into a variety of different cell types, for instance cardiomyocytes. Especially in the context of future application in regenerative medicine, it is crucial to understand the developmental processes taking place upon differentiation. Moreover, the identification of a panel of cell surface markers suitable for characterization and purification is necessary to ensure quality of human pluripotent stem-cell derived products. In this study, we used quantitative mass spectrometry to characterize proteomic changes in early mesendodermal differentiation. Two human pluripotent stem cell lines, one embryonic (H3) and one induced pluripotent stem cell line (I2), were analyzed under pluripotent conditions and after two days of embryoid body-based differentiation. Functional clustering and enrichment analysis showed down-regulation of proteins associated with pluripotency and the tricarboxylic acid cycle at day two. In contrast, proteins related to the proteasome and annexin family were up-regulated upon differentiation. Among the proteins that were down-regulated upon differentiation in both, H3 and I2, the membrane protein junctional adhesion molecule A (JAM-A) emerged as potentially associated with pluripotency. Flow cytometry and immunocytochemistry further confirmed down-regulation of JAM-A on the cell surface of human induced pluripotent stem cells that were differentiated toward mesendoderm for just two days. STATEMENT OF SIGNIFICANCE: Understanding early molecular changes during human pluripotent stem cell differentiation is essential for stem cell biology and regenerative medicine. This study provides a comparative proteomic analysis of two cell lines during early differentiation and identifies coordinated metabolic and pluripotency-associated changes. The understanding of changes that occur in the early phase of cardiomyocyte differentiation might be helpful to more precisely monitor the differentiation process. Importantly, we identify the membrane protein JAM-A as a robustly down-regulated cell surface protein, highlighting its potential as marker for human pluripotent stem cells. In the future, JAM-A might be used in a panel of cell surface markers for pluripotent stem cells in order to remove pluripotent stem cells from stem cell-derived therapeutic products.
Partial downregulation of pancreatic endoplasmic reticulum kinase (PERK) activity recovered insulin content in human islets exposed to glucolipotoxicity (GLT), resulting in improved insulin secretion and glucose-lowering effects in a mouse model of type 2 diabetes. We conducted this study to elucidate the beta-cell-enhancing mechanisms of PERK attenuation. Pancreatic islets isolated from non-diabetic living donors were divided into three groups: control, GLT mimicking diabetes conditions, and GLT with treatment of a PERK inhibitor (PERKi, GSK2606414) for 24 h. Proteomic analysis was conducted on these samples. Differentially expressed proteins (DEPs) altered by GLT and reversed by PERKi were analyzed using bioinformatics. Validation studies were followed using western blotting, RT-PCR, and immunocytochemistry. Using nine islet samples pooled from seven participants, 161 DEPs were identified among 5513 quantifiable proteins across the three groups. On a subset of 42 proteins that were downregulated by GLT and upregulated by PERKi, GO, and KEGG analyses highlighted nucleocytoplasmic transport (NCT) as a key pathway, involving genes such as XPO4, KPNA4, NUP43, and NUP58. The involvement of NCT, particularly XPO4, was further supported by a replication proteomic analysis using islets from four independent donors. Based on these findings, we examined the NCT of representative β-cell transcription factors, including PDX1 and FOXO1. PERKi significantly increased their nuclear localization under GLT conditions (both p < 0.05), accompanied by heightened expression of their target genes, such as FBXW5. These results suggest that PERKi-mediated modulation of NCT may enhance the functional activity of PDX1 and FOXO1. In conclusion, proteomic analysis revealed that PERKi appears to modulate NCT of human islets under metabolic stress, thereby contributing to the restoration of β-cell function through regulation of relevant transcription factors. These findings suggested a novel mechanism of low-dose PERKi as a therapeutic approach to diabetes, in addition to the canonical unfolded protein response.
Coronary artery disease (CAD) is a leading global cause of mortality, yet the predictive accuracy of conventional risk models is limited. Here, we integrate conventional risk factors, polygenic risk scores, and large-scale proteomics to develop a unified model for enhanced CAD risk prediction. Using data from UK Biobank, participants with plasma proteomics and genetic risk data were included after excluding prevalent CAD. Participants from England were split into training (n=32 330) and internal validation (n=13 857) sets, and Scotland/Wales participants formed an external validation set (n=5775). Incident CAD was ascertained from linked health records. A 202-protein proteomic risk score was derived by least absolute shrinkage and selection operator Cox regression, and CatBoost models were trained using conventional risk factors alone and with incremental addition of polygenic risk scores and protein proteomic risk scores; Shapley Additive Explanations-guided forward selection identified a compact protein panel. Across cohorts, the median age was 58 years and ∼45% were men. Protein proteomic risk score was dose-dependently associated with CAD risk. Compared with conventional risk factors alone, integrating polygenic risk scores and protein proteomic risk scores improved discrimination, with the area under the curve increasing from 0.750 (95% CI, 0.732-0.767) to 0.789 (95% CI, 0.772-0.805) in internal validation and from 0.717 (95% CI, 0.683-0.750) to 0.762 (95% CI, 0.732-0.791) in external validation. A 9-protein panel (GDF15 [growth differentiation factor 15], MMP12 [matrix metalloproteinase 12], NPPB [natriuretic peptide B], PGF [placental growth factor], REN [renin], ADGRG2 [adhesion G-protein coupled receptor], ACE2 [angiotensin-converting enzyme 2], CDCP1 [CUB domain-containing protein 1], CXCL17 [C-X-C motif chemokine ligand 17)]) captured most proteomic predictive information. Our findings demonstrate that integrating conventional risk factors, polygenic risk scores, and proteomic data improves CAD risk prediction. This study highlights the utility of proteomics in precision cardiovascular medicine and simplified risk stratification tools.
The temporal proteomic and phosphoproteomic reprogramming during early M1 macrophage polarization (0-6 h) remains poorly understood. We performed time-resolved proteomic and phosphoproteomic analyses of LPS-stimulated RAW264.7 macrophages at seven time points within 6 h. Time-clustering of differentially expressed molecules revealed two patterns: initial change with partial recovery, and sustained dysregulation. Upregulated proteins and phosphorylation sites were enriched in the Rho GTPase signaling pathway, T-cell receptor signaling pathway, NF-κB cascade, osteoclast differentiation pathway, and antiviral immune pathway. Downregulated pathways were associated with cell cycle regulation, chromatin remodeling, RNA metabolism, and mRNA processing, indicating resource reallocation to prioritize acute inflammatory responses. Kinase-substrate network analysis confirmed the mitogen-activated protein kinase (MAPK), cyclin-dependent kinase (CDK), protein kinase B (AKT), and ribosomal S6 kinase (RSK) families as core upstream phosphorylation regulators. Integrated analysis revealed synergistic and antagonistic relationships between proteomic and phosphoproteomic changes. This study provides a temporal molecular atlas of M1 polarization, delineating inflammatory signaling dynamics and offering a basis for therapeutic target discovery in inflammatory diseases. SIGNIFICANCE: Macrophage M1 polarization is a central event in innate immune defense against pathogenic invasion, yet its dysregulation is a pivotal driver of the onset and progression of a broad spectrum of inflammation-associated disorders, spanning autoimmune diseases, infectious conditions and inflammatory bone diseases, making the dissection of its molecular regulatory mechanisms an urgent research priority in immunology and translational medicine. Dynamic molecular events within 0-6 h after LPS stimulation are critical for initiating and shaping M1 inflammatory activation, yet systematic time-resolved proteomic and phosphoproteomic profiling remains insufficient.In this study, we comprehensively characterized temporal proteome and phosphoproteome changes at seven consecutive time points during macrophage polarization, clarified two distinct dynamic molecular patterns, identified core signaling pathways and key kinase regulators involved in inflammatory reprogramming, and uncovered the leading role of post-translational phosphorylation modifications in initiating polarization. This work delineates the time-series molecular atlas of early macrophage activation, provides novel insights into the temporal regulatory mechanism of inflammatory signaling networks, and lays a solid experimental foundation for exploring new intervention targets and regulatory nodes in clinical translational research.
Polycomb repressive complex 2 (PRC2) and its catalytic subunit EZH2 regulate transcriptional repression and display subcellular organization patterns important for cell function. Here, we present a protocol that applies image-guided optoproteomics to profile the in situ proteome of endogenous EZH2-containing PRC2 bodies in the triple-negative breast cancer cell line BoM-1833. We describe steps for cell culture, EZH2 immunofluorescence, and Microscoop-based photolabeling. We then detail procedures for protein enrichment and mass spectrometry for profiling chromatin-associated signaling. For complete details on the use and execution of this protocol, please refer to Pelzer et al.1.
Lung cancer remains the leading cause of cancer-related mortality worldwide, with major subtypes including lung adenocarcinoma, lung squamous cell carcinoma (LUSC), and small cell lung cancer, yet the causal role of plasma protein-protein ratios in subtype-specific development remains poorly understood. We performed a bidirectional, two-sample Mendelian randomization (MR) analysis to assess the causal effects of plasma protein-protein ratios on lung cancer. Genetic instruments for protein-protein ratios were derived from a large-scale proteomic genome-wide association study, and lung cancer summary statistics were obtained from the FinnGen and Transdisciplinary Research in Cancer of the Lung and International Lung Cancer Consortium databases. Our study identified 17 plasma protein-protein ratios associated with lung cancer across both databases, including 5 for overall lung cancer (LC), 4 for lung adenocarcinoma, 5 for LUSC, and 4 for small cell lung cancer. Notably, a higher melanoma inhibitory activity (MIA)/DAN family BMP antagonist ratio was associated with increased risk for both LC and LUSC. When analyzing the constituent proteins of these ratios individually, only the causal association between MIA and LC remained significant. Subsequent 2-step MR analysis revealed that the MIA/DAN family BMP antagonist ratio mediated the effect of body mass index on LUSC development, with consistent mediation proportions across both databases (13.46%, 95% confidence interval: 3.14%-23.77% in FinnGen; 9.26%, 95% confidence interval: 0.05%-18.47% in Transdisciplinary Research in Cancer of the Lung and International Lung Cancer Consortium). Additional exploratory mediation pathways were identified for smoking initiation and body mass index; however, these findings should be interpreted with caution, given the assumptions underlying 2-step MR and the exploratory nature of the analysis. Multiple sensitivity analyses confirmed the robustness of these findings. This MR study offers novel proteomic insights into lung cancer pathogenesis and identifies candidate protein-protein ratios that warrant further functional investigation to assess their therapeutic potential.
Cellular senescence drives aging and age-related disease through the accumulation of senescent cells and their senescence-associated secretory phenotype (SASP). Emerging evidence suggests intermittent ("hit-and-run") senolytic interventions may improve healthspan by reducing senescent cell accumulation and the SASP. Healthy adults aged 45-79 were recruited for a decentralized, single-arm pilot study (NCT06953518) evaluating 2 days of nutraceutical supplementation (Qualia Senolytic). Fingerstick blood samples and validated quality of life (QoL) questionnaire data were collected on days 0 and 7. Primary outcomes were SASP biomarkers measured by the Olink® Target 48 Cytokine panel, including tumor necrosis factor (TNF), interleukin-1 beta (IL-1β), interleukin-8 (CXCL8), and vascular endothelial growth factor A (VEGFA). Protein data were analyzed using linear mixed models and Wilcoxon signed-rank tests. Seventy-one adults enrolled and 53 (74.6%) provided paired protein samples. No significant changes occurred in primary outcomes. Exploratory unadjusted analyses revealed significant reductions in the established senescence chemokines CXCL9 and CXCL10, as well as CCL8 and CXCL11, and increases in interleukin-17F and oncostatin M. QoL significantly improved without safety concerns, though results are expectation-sensitive. Preliminary findings support the feasibility of this decentralized approach and identify candidate SASP biomarker signals in healthy adults warranting validation in randomized, placebo-controlled trials.
In oncology, biomarkers have various probable applications. They are crucial in cancer detection and are vital in diagnosis, predicting outcomes, and determining the prognosis of various types of cancer. Biomarkers are critical in enhancing treatment outcomes and reducing healthcare costs. Numerous biomolecules can be used as diagnostic, predictive, and prognostic cancer biomarkers. These biomolecules include genetic, epigenetic, transcriptomic, metabolomic, proteomic, cellular, physiological, or imaging-based biomarkers. Cancer cells or surrounding non-cancerous cells produce cancer biomarkers in response to the presence of tumors. It offers a critical insight into molecular and cellular changes associated with malignancy. Once a biomarker is identified, it helps elucidate the molecular pathways linked to the onset and progression of cancer and is used to formulate treatment. Many cancer biomarkers are already in therapeutic practice. One major challenge is the development of early-stage detection and prognostic biomarkers, as most early-stage cancers are often asymptomatic and therefore difficult to detect. This chapter will cover the broad spectrum of the role of cancer biomarkers and their application in the context of cancer detection, diagnosis, and treatment.