J. Proteome Res. [JOURNAL]
Sun
200 papers
Automated Metadata Extraction from mzML Files with RunAssessor.
Andken M, Zheng C, Sun Z … +1 more , Deutsch EW
J Proteome Res · 2026 May · PMID 41988924 · Publisher ↗
The reusability of proteomics data sets depends on the ability to obtain accurate metadata to guide reprocessing pipelines. However, many data sets deposited in public data repositories lack sufficient and reliable annot...
The reusability of proteomics data sets depends on the ability to obtain accurate metadata to guide reprocessing pipelines. However, many data sets deposited in public data repositories lack sufficient and reliable annotation, limiting large-scale reanalyses. To address this challenge, we developed RunAssessor, a tool that systematically extracts and summarizes information directly from mass spectrometry data files prior to peptide identification analysis. RunAssessor extracts and summarizes sample preparation and instrument acquisition parameters directly from the data where possible. Using one complete data set and test files from 18 other data sets as examples, we demonstrate RunAssessor's ability to extract instrument models, isobaric labels, phosphoenrichment, precursor and fragment ion tolerances, along with the dynamic exclusion time used by the instrument. These extracted metadata are stored in a comprehensive output file, and summarized in a standard Sample and Data Relationship Format (SDRF) file, thereby reducing the burden of manual curation and improving the reliability of proteomics data set metadata, facilitating the reuse of public data.
Altered Sphingolipid Metabolism is Associated with Osimertinib Resistance in Nonsmall-Cell Lung Cancer.
Babuta J, Gruevska A, Wickremesinghe C … +10 more , Montoya A, Roumelioti G, Shliaha P, Balcells C, McKinney F, Bray C, Athersuch T, Martin M, Keun H, Hall Z
J Proteome Res · 2026 May · PMID 41988688 · Full text
Nonsmall-cell lung cancer (NSCLC) accounts for more than 80% of lung cancer cases. Epidermal growth factor receptor mutations (EGFRm) occur in 15 and 40% of NSCLC in Western and Asian populations, respectively. Current t...
Nonsmall-cell lung cancer (NSCLC) accounts for more than 80% of lung cancer cases. Epidermal growth factor receptor mutations (EGFRm) occur in 15 and 40% of NSCLC in Western and Asian populations, respectively. Current treatment for advanced NSCLC targets EGFRm with tyrosine kinase inhibitors (TKIs). Osimertinib is a third-generation EGFR-TKI now used as a first-line treatment in advanced/metastatic NSCLC; however, drug resistance frequently develops. Dysregulation of metabolism has been suggested to play a role in the development of drug resistance. Here, we investigated the role of lipid metabolism in the development of osimertinib resistance (OR) using pharmacologically-induced resistant cellular models. We used a multiomics approach, combining lipidomics with proteomics analyses. We found alterations in processes relating to metabolism, such as dysregulated sphingolipid metabolism. In particular, we identified that OR lines reduce free ceramides in favor of complex glycosphingolipids. Mechanistically, this metabolic shift avoids ceramide-mediated apoptosis via caspase-3 activation. Importantly, when we combined osimertinib with D-PDMP, an inhibitor of the key enzyme responsible for the conversion of ceramide to glucosylceramide, we increased the sensitivity to osimertinib. Overall, we have identified the glycosphingolipid metabolic pathway as a potential therapeutic target to reinstate sensitivity to osimertinib in NSCLC.
Longitudinal Plasma Proteomic Profiling Reveals Divergent Immune Responses in Durably Cured and Relapsed Pulmonary Tuberculosis.
Call DH, Day LZ, Phillips PPJ … +4 more , Powell SM, Gritsenko MA, Nahid P, Jacobs JM
J Proteome Res · 2026 May · PMID 41983578 · Publisher ↗
: Although clinical biomarkers of tuberculosis (TB) relapse are well characterized, the biological mechanisms underlying different treatment outcomes remain poorly understood. Elucidating these mechanisms may reveal impr...
: Although clinical biomarkers of tuberculosis (TB) relapse are well characterized, the biological mechanisms underlying different treatment outcomes remain poorly understood. Elucidating these mechanisms may reveal improved biomarkers of long-term treatment outcomes. : We conducted a longitudinal, global proteomic study on 60 participants with active pulmonary TB, half who were durably cured and half who relapsed. Plasma was collected at seven time-points: at treatment initiation (baseline), during therapy, and 52 weeks postbaseline. Samples were analyzed by high-resolution LC-MS/MS. : 2,418 proteins were identified across all samples, with 1,756 being differentially expressed relative to baseline ( < 0.05). 956 proteins were differentially abundant between cured and relapsed participants. Relapsed participants showed heightened humoral immunity throughout treatment, as well as upregulated complement activation and HDL particles. Cured participants exhibited elevated recovery-related pathways by week 4, including downregulated epithelial invasion and upregulated oxygen transport. : Heightened humoral and innate immune responses were associated with relapse, whereas recovery signatures were associated with durable cure. Several proteins showed potential as relapse biomarkers and warrant future validation in independent cohorts. These findings advance our understanding of host responses to treatment and provide a basis for developing blood-based biomarkers to identify patients at increased risk of relapse.
PeptideVisualizer: A Novel Software Solution for PROTOMAP Analysis.
Kolarič M, Ivanovski S, Sever T … +2 more , Turk B, Fonović M
J Proteome Res · 2026 May · PMID 41982141 · Full text
PeptideVisualizer is an open-source software for PROTOMAP analysis, offering an intuitive graphical user interface and command-line compatibility. The software processes mass spectrometry data output from MaxQuant to vis...
PeptideVisualizer is an open-source software for PROTOMAP analysis, offering an intuitive graphical user interface and command-line compatibility. The software processes mass spectrometry data output from MaxQuant to visualize protein migration in polyacrylamide gel electrophoresis and sequence coverage. In addition, it integrates information regarding protein secondary structure and features from the UniProt database to visualize them within comprehensive peptographs. These features allow us to assess the occurrence of a proteolytic event based on sequence context and peptides abundance. The key advantages of PeptideVisualizer include the integration of quantitative information, handling of multiple experiments with biological replicates, and the introduction of a novel mismatch factor─a metric designed to rapidly identify proteolytic events. Herein, the software was validated in an apoptosis-related data set, demonstrating its effectiveness and usefulness in large-scale proteomic data analysis.
Time-Resolved Proteomic Analysis in Zebrafish Using Bioorthogonal Noncanonical Amino Acid Tagging.
Miller SE, Wang TY, Quan B … +4 more , Cammidge T, Chou TF, Prober DA, Tirrell DA
J Proteome Res · 2026 May · PMID 41980224 · Full text
Tracking time-dependent proteomic changes remains challenging. Bioorthogonal noncanonical amino acid tagging (BONCAT) offers a targeted approach for identifying proteins synthesized within defined time windows. Here, we...
Tracking time-dependent proteomic changes remains challenging. Bioorthogonal noncanonical amino acid tagging (BONCAT) offers a targeted approach for identifying proteins synthesized within defined time windows. Here, we describe BONCAT proteomic analysis in larval zebrafish, a model organism widely used for its genetic tractability and utility in developmental biology and neuroscience. We enriched and identified azidohomoalanine-labeled proteins via mass spectrometry after labeling durations as short as 12 h, achieving significant signal above background compared to unlabeled controls. As a proof of concept, we investigated proteomic changes in response to heat shock. BONCAT analysis revealed upregulation of heat shock-induced proteins with greater sensitivity than global proteomics. Gene set enrichment analysis confirmed that heat shock response proteins were significantly enriched in BONCAT samples but not in whole lysates, highlighting BONCAT's ability to detect otherwise-masked transient molecular responses. Beyond the expected changes in synthesis of heat shock proteins, BONCAT identified differentially expressed proteins implicated in stress responses, lipid metabolism, and neural regulation, offering insights into the zebrafish heat shock response. These findings establish BONCAT as a powerful tool for time-resolved proteomic analysis in zebrafish, and for elucidation of the molecular underpinnings of behavior, stress responses, and development in this versatile model organism.
Multiomics Analysis Reveals the Protective Effect of a Novel Bioactive Peptide (BP1) in Cardiomyopathy Using a Zebrafish Model.
Thakur S, Kumari S, Punia A … +3 more , Patial V, Singh D, Kumar R
J Proteome Res · 2026 May · PMID 41980054 · Publisher ↗
Medicinal plants have been used in traditional healthcare systems and drug discovery since ancient times, owing to their storage of natural products, including bioactive metabolites and peptides. Bioactive peptides (BPs)...
Medicinal plants have been used in traditional healthcare systems and drug discovery since ancient times, owing to their storage of natural products, including bioactive metabolites and peptides. Bioactive peptides (BPs) are reported to have diverse therapeutic potential and are used for the treatment/prevention of many lifelong diseases, including cardiovascular. In our previous work, we isolated and characterized a bioactive peptide, ASGLCPEEAVPRR (BP1), from and hypothesized that BP1 possesses angiotensin-converting enzyme (ACE) inhibitory activity, which has a cardioprotective role; however, a system-level understanding of its cardioprotective and metabolic control is needed to validate the hypothesis. Here, we investigated the cardioprotective effect of BP1 in zebrafish larvae and adult models of isoproterenol (ISO)- and doxorubicin (DOX)-induced cardiac damage, respectively. Following the treatment, the cardiac morphology, cardiac functional parameters, transcriptome, and metabolome were studied. Results indicated that treatment with BP1 significantly reduced ISO-induced cardiac dysfunction in zebrafish larvae. Similarly, BP1 pretreatment effectively mitigated the DOX-induced pathological changes in the myocardium of zebrafish. Transcriptomic and weighted gene coexpression network analysis (WGCNA) showed attenuation in the expression of genes associated with protein synthesis, metabolic pathways, signaling pathways, and cardiac muscle contraction, among others, following BP1 treatment in the DOX-induced cardiotoxicity model. Untargeted metabolomics revealed metabolic pathways such as sphingolipid, riboflavin, and glutathione metabolism, among others, involved in attenuating DOX-induced cardiotoxicity by BP1. Our results concluded that BP1 treatment showed cardioprotection in zebrafish targeting multiple pathogenic pathways involved in cardiotoxicity.
Exploring Metabolic Changes in Children with Congenital Hypothyroidism: A Serum Metabolomic Study Combined by Machine Learning.
Guo Y, Li X, Fang K … +6 more , Huang J, Huang R, Lin Y, Feng J, Chen J, Shen G
J Proteome Res · 2026 May · PMID 41973905 · Publisher ↗
Congenital hypothyroidism (CH) is a genetic endocrine disorder that can cause developmental delays if it is untreated. In this study, NMR-based metabolomics was employed to analyze serum samples from CH children and heal...
Congenital hypothyroidism (CH) is a genetic endocrine disorder that can cause developmental delays if it is untreated. In this study, NMR-based metabolomics was employed to analyze serum samples from CH children and healthy controls across different age groups. Multivariate statistical analysis screened for 17, 16, 33, and 21 differential metabolites in the respective age groups and identified seven common metabolites, including lysine, 1-methylhistidine, glycerophosphocholine, phosphocholine, β-glucose, lipids, and creatine. The results indicated that CH children experienced metabolic disturbances in multiple pathways, particularly glycerophospholipid metabolism and glycine, serine, and threonine metabolism. Following recursive feature elimination (RFE) for feature selection, the top five core metabolites were selected to construct an optimized artificial neural network (ANN) model for CH diagnosis, achieving a prediction accuracy of 89.4%. These findings suggest that the identified metabolites can be used as potential diagnostic biomarkers for CH in children. This may help improve the early diagnosis accuracy of CH, serve as a rapid screening tool for newborns, and provide an auxiliary diagnostic method for suspected CH cases to facilitate early clinical intervention.
Proteomic Analysis of Paired FFPE Tissue and Extracellular Vesicles Reveals Proteins Associated with Recurrence in Stage II Colorectal Cancer.
Rejas-González R, Fernández-Aceñero MJ, Tirado-Zambrana PS … +11 more , Sanz-Criado L, Dziaková J, Sanz-López R, Poves C, Feliu J, Heredia-Soto V, Mendiola M, Martínez-Useros J, Peláez-García A, Montero-Calle A, Barderas R
J Proteome Res · 2026 May · PMID 41966223 · Full text
Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide. Stage II CRC poses a clinical challenge due to its heterogeneous outcomes, with 15-20% of patients experiencing recurrence. Current p...
Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide. Stage II CRC poses a clinical challenge due to its heterogeneous outcomes, with 15-20% of patients experiencing recurrence. Current prognostic models based on clinicopathologic features lack sufficient precision, highlighting the need for new molecular markers. In this study, we employed an integrative TMT-based proteomics strategy to identify biomarkers of recurrence in stage II CRC. We analyzed paired formalin-fixed paraffin-embedded (FFPE) tissues and small extracellular vesicles (sEVs) from stage II recurrent and nonrecurrent CRC patients. Validation of proteomics data was performed in silico and by WB, immunohistochemistry, ELISA, and in vitro functional cell-based assays. This multifaceted approach identified several dysregulated proteins associated with CRC recurrence, including MANF, TLN1, TALDO1, and CDCA2, among others. CDCA2 knockdown altered the tumorigenic properties of CRC cells in vitro, correlating findings with its association with prognosis. Conversely, higher plasma levels of MANF were found in nonrecurrent CRC patients, aligning results with its favorable prognosis profile. Collectively, our findings support the value of combining paired FFPE tissue and sEVs proteomics analyses to uncover recurrence-associated biomarkers, offering the potential for risk stratification and management of stage II CRC patients.
Enhanced Proteomics Analysis with a Novel Recombinant Chymotrypsin Analogue Engineered for High Cleavage Specificity.
Adoni KR, Ditcham JE, González Rivera AK … +4 more , Charlton GH, Saveliev SV, Thalassinos K, Zenezini Chiozzi R
J Proteome Res · 2026 May · PMID 41961476 · Full text
Chymotrypsin is widely used in shotgun proteomics, owing to its orthogonal cleavage specificity relative to trypsin, which enhances sequence coverage of hydrophobic protein regions. However, commercial preparations often...
Chymotrypsin is widely used in shotgun proteomics, owing to its orthogonal cleavage specificity relative to trypsin, which enhances sequence coverage of hydrophobic protein regions. However, commercial preparations often display variable cleavage specificity, trypsin contamination, and elevated missed cleavage rates, which can collectively reduce the proteome coverage and data reproducibility. To address these limitations, we present a novel recombinant chymotrypsin () engineered for improved cleavage specificity and robustness in proteomics workflows. Benchmarking against standard bovine chymotrypsin revealed 97% C-terminal cleavages after tyrosine (Y), phenylalanine (F), and leucine (L) for , compared to 72% for the standard enzyme. This enhanced cleavage specificity reduced missed cleavages and increased peptide-spectrum matches across charge states. Across 3,638 identified proteins, yielded 22.2% unique identifications compared with 8.2% for standard chymotrypsin, while maintaining similar peptide length, /, and hydrophobicity distributions. The enzyme remained active in up to 6 M urea and achieved near-maximal proteome coverage within 2 h (only a 2.4% gain after overnight digestion). These results establish as an advanced tool with improved cleavage specificity that reduces analytical complexity and enhances the reliability of proteomic analysis, while expanding chymotryptic digestion to hydrophobic and high-denaturant proteomics applications.
Comparative Proteomics of Adult Honeybee Brains Reveals Differential Lysine Crotonylation Associated with Cognitive Impairment from Prepupal Cold Exposure.
Zhu C, Cao M, Sun J … +6 more , Shang J, Li C, Li H, Xu X, Zhou S, Zhu X
J Proteome Res · 2026 May · PMID 41958437 · Publisher ↗
Honeybee larvae and pupae are extremely stenothermic. Cold exposure during development causes mortality and developmental arrest and impairs adult cognitive functions. Lysine crotonylation (Kcr) regulates neural activity...
Honeybee larvae and pupae are extremely stenothermic. Cold exposure during development causes mortality and developmental arrest and impairs adult cognitive functions. Lysine crotonylation (Kcr) regulates neural activity, yet its role in cold-impaired honeybee cognition is unknown. Adult honeybees with 8 h/12 h/16 h cold exposure during the prepupae stage (T8/T12/T16) were compared to controls with optimal brood-rearing temperature (CK). The learning and memory assay revealed graded levels of cognitive impairment in the adult honeybee cohorts. Western blot analysis revealed that as learning and memory capacity declined, the pan-Kcr levels in adult bee brains also decreased. Further brain LC-MS/MS identified 3017 proteins with 15,233 Kcr sites. Relative to CK, T12 and T16 exhibited 402 differentially modified sites (DMSs) on 304 proteins and 216 DMSs on 325 proteins, respectively. Enrichment analysis of proteins with differentially modified Kcr suggested that Kcr may regulate cognitive function by modulating protein quality control and energy metabolism. Key pathway proteins were validated by parallel reaction monitoring. This first comprehensive profiling of honeybee brain Kcr elucidates its metabolism-linked neural regulation and provides a novel, accessible model for neurological health research.
Integrated Proteomics and Metabolomics Analyses Reveal That Phosphatidylethanolamine Reprograms Macrophage Immunometabolism and Attenuates LPS-Driven Inflammation.
Maurício T, Neves B, Domingues MR … +1 more , Domingues P
J Proteome Res · 2026 May · PMID 41950066 · Full text
Phospholipids are key regulators of immune metabolism, yet their specific influence on macrophage function remains incompletely defined. We investigated how phosphatidylethanolamine (PE) species with distinct acyl chains...
Phospholipids are key regulators of immune metabolism, yet their specific influence on macrophage function remains incompletely defined. We investigated how phosphatidylethanolamine (PE) species with distinct acyl chains (PE18:0/22:6 and PE18:0/20:4) modulate RAW264.7 macrophages under resting and LPS-stimulated conditions using LC-MS/MS-based proteomics and metabolomics, followed by qPCR validation. LPS elicited a robust M1-like phenotype with strong upregulation of Ptgs2, Nos2, Nfkb1, and Nfkb2. PE supplementation alone did not induce a classical pro-inflammatory profile but significantly remodeled protein expression, enhancing antioxidant defenses, including catalase, Hmox1 and Prdx1. In the context of LPS activation, PE selectively attenuated inflammatory signaling by downregulating Nfkb1, Nfkb2, and Ptgs2 while further enhancing proteins linked to oxidative stress response (Prdx1 and Hmox1) and lipid metabolism (CD36 and Abcc1). qPCR corroborated these effects: both PE species reduced LPS-induced and mRNA levels while increasing , , and transcription. Metabolomics converged with these findings, indicating reinforced glutathione metabolism and context-dependent shifts in purine and amino-acid pathways consistent with a restrained inflammatory phenotype. Collectively, native PE species reprogram macrophage immunometabolism, mitigating LPS-driven inflammation while strengthening Nrf2-mediated antioxidant and immune-supportive pathways.
Time-of-Day Differences in the Plasma -Glycome of Normal and Obese Mice and the Effects of Dapagliflozin Administered in the Morning or at Night.
Ozawa Y, Muramoto F, Senda H … +9 more , Kada M, Torii-Goto A, Hanamatsu H, Miura N, Fujita A, Furukawa JI, Yoshioka H, Yoshikawa M, Shinohara Y
J Proteome Res · 2026 May · PMID 41948954 · Publisher ↗
Circadian rhythms regulate a wide range of physiological functions, including metabolism and protein expression. While the influence of circadian timing on plasma glycosylation remains poorly understood, aberrant -glycos...
Circadian rhythms regulate a wide range of physiological functions, including metabolism and protein expression. While the influence of circadian timing on plasma glycosylation remains poorly understood, aberrant -glycosylation is implicated in metabolic diseases. Here, we investigated diurnal variations in the plasma -glycome of mice fed a high-fat diet (HFD), with or without treatment using the sodium-glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin (Dap), administered either in the morning or at night. Using a quantitative glycomic platform based on glycoblotting and MALDI-TOF MS, we identified time-of-day-dependent changes in specific plasma -glycans following HFD feeding. These alterations were more pronounced in plasma collected at night. Moreover, repeated administration of Dap induced glycan profile changes that differed depending on the dosing time. Interestingly, nighttime administration of Dap normalized several HFD-induced glycan alterations to levels closer to those of control mice, despite limited effects on blood glucose or body weight. These results suggest that -glycan profiles may serve as sensitive and potentially early biomarkers of pharmacological effects in this experimental model and support the importance of time-of-day-dependent sampling when evaluating glycosylation-targeted interventions.
Stability-Based Machine Learning Identifies a Minimal Two-Protein Serum Signature for Early Silicosis.
Chu X, Li Y, Wang F … +6 more , Li W, Wang N, Zhou L, Gao Y, Zhou P, Han L
J Proteome Res · 2026 May · PMID 41937675 · Publisher ↗
BACKGROUND: The early diagnosis of silicosis, an irreversible fibrotic lung disease, is challenged by the low sensitivity of current radiological methods in early-stage disease and their susceptibility to interobserver v...
BACKGROUND: The early diagnosis of silicosis, an irreversible fibrotic lung disease, is challenged by the low sensitivity of current radiological methods in early-stage disease and their susceptibility to interobserver variability. Consequently, a pressing need exists for noninvasive, objective biomarkers to facilitate timely detection and intervention. METHODS: We employed a multistage study design comprising a discovery cohort (57 Stage I silicosis patients, 57 matched controls) and an independent, unmatched validation cohort (40 patients, 40 controls). Serum protein profiles were generated using Olink targeted proteomics. We utilized a rigorous, stability-based machine learning framework, which integrated Lasso, Random Forest, and SVM-RFE algorithms over 100 iterations, to perform feature selection and identify a robust biomarker signature from the discovery cohort. Based on the selected features, a logistic regression model was subsequently constructed, and its performance was evaluated using both internal and external validation. RESULTS: Our discovery strategy identified a two-protein signature comprising IL8 and CCL3. This signature demonstrated excellent diagnostic performance in the discovery cohort, achieving a cross-validation AUC of 0.986 (95% CI: 0.975-1.000). Importantly, the model's robustness was confirmed in the heterogeneous validation cohort, where it achieved an outstanding AUC of 0.973 (95% CI: 0.936-1.000), with 95.0% specificity and 77.5% sensitivity. Bioinformatic analysis revealed that decreased serum levels of IL8 and CCL3 were associated with silicosis, providing novel diagnostic biomarkers and highlighting a complex, paradoxical shift in circulating chemokines during early-stage disease.
Discovery and Validation of Lung Cancer Biomarkers Based on Proteomic Analysis of Bronchoalveolar Lavage Fluid from the Self-Controlled Pulmonary Lobes.
Wei S, Chen T, Jin Y … +8 more , Jin X, Zhao Y, Chen X, Cao Q, Li L, Zhou M, Qi R, Cao C
J Proteome Res · 2026 May · PMID 41921188 · Publisher ↗
Distinguishing indeterminate pulmonary nodules remains a significant challenge in the early diagnosis of lung cancer. This study aims to identify lobe-specific proteomic biomarkers to address this issue. Using a data-ind...
Distinguishing indeterminate pulmonary nodules remains a significant challenge in the early diagnosis of lung cancer. This study aims to identify lobe-specific proteomic biomarkers to address this issue. Using a data-independent acquisition (DIA) strategy, we performed proteomic profiling of bronchoalveolar lavage fluid (BALF) obtained from the lesioned lobe of lung cancer patients (LC-L), their paired nonlesioned lobe (LC-N), and the lesioned lobe of patients with benign nodules (BN-L). We quantified 4,305 proteins and identified 134 upregulated and 44 downregulated proteins in LC-L compared to both control groups ( < 0.05; |FC| > 1.2). Pathway analysis revealed significant enrichment of these dysregulated proteins in metabolic pathways and the TCA cycle. Subsequent validation in an independent cohort confirmed that two candidate proteins, SUCLA2 and FKBP9, were significantly upregulated specifically in cancerous lobes. To our knowledge, this is the first report identifying SUCLA2 and FKBP9 as lung cancer-specific biomarkers detectable in BALF using a self-controlled study design. These findings provide crucial insights into the metabolic reprogramming of early stage lung cancer and offer promising novel targets for improving the differential diagnosis of indeterminate pulmonary nodules.
MR-SP: A Microreactor-Based Workflow for Few-Cell Spatial Proteomics on the Legacy Zeiss PALM MicroBeam.
Metzger M, Maldacker M, Hutzenlaub T … +3 more , Paust N, Schilling O, Klatt JN
J Proteome Res · 2026 May · PMID 41914927 · Full text
Laser capture microdissection (LCM) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables spatial proteomics at the few-cell level but is constrained by cumulative losses during specimen capture...
Laser capture microdissection (LCM) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables spatial proteomics at the few-cell level but is constrained by cumulative losses during specimen capture, surface adsorption during processing, and sample transfer prior to LC-MS/MS analysis. The capture-associated losses are particularly relevant for pressure catapulting systems such as the legacy Zeiss PALM MicroBeam, which, despite discontinuation, remains in active use and therefore requires compatible low-loss workflows. We present MR-SP (microreactor-based sample preparation for spatial proteomics), a one-pot workflow integrating reproducible Zeiss LCM-cut specimen capture, processing with minimized adsorptive losses, and pipetting-free transfer with Evotip disposable precolumns. The workflow was evaluated using a formalin-fixed paraffin-embedded (FFPE) murine kidney tissue analyzed by timsTOF flex LC-MS/MS analysis. Across 50,000 μm regions (22 cells), MR-SP modestly improved proteome depth (3381 ± 80 versus 3174 ± 59 proteins). Decreasing sample input further accentuated the advantage of MR-SP in maintaining higher identification rates, highlighting the successful reduction of the adsorptive losses. At 12,500 μm (5-6 cells), identifications increased to 1145 ± 188 versus 302 ± 126. At 3125 μm (1-2 cells), identifications reached 695 ± 112 versus 206 ± 51. MR-SP improves identification depth for few-cell FFPE samples by nearly 3-fold compared to conventional tube-based workflows.
Microglial Adaptations to Chronic Nicotine in the Cerebellum: Proteomic Evidence for Neuroimmune Vulnerability.
Nusir A, Anthony SM, Zhou W … +1 more , Kabbani N
J Proteome Res · 2026 May · PMID 41911483 · Full text
Smoking is a major public health concern with widespread effects on multiple organ systems, including the immune system. Chronic nicotine exposure can alter immune cell function through nicotinic receptors expressed on p...
Smoking is a major public health concern with widespread effects on multiple organ systems, including the immune system. Chronic nicotine exposure can alter immune cell function through nicotinic receptors expressed on peripheral macrophages and microglia in the brain. Recent evidence indicates that the cerebellum is impacted by nicotine, contributing to motor and nonmotor outcomes, during drug use. In this study, we investigated the effect of chronic nicotine on microglia proteomes in the adult mouse cerebellum. Microglia were isolated by fluorescence-activated cell sorting (FACS) based on CD11b CD45 expression from male and female mice ( = 9 per group) exposed to 200 μg/mL nicotine (dissolved in 2% saccharin drinking water) for 30 days. Proteomic analysis was performed using liquid chromatography electrospray ionization (LC-ESI) mass spectrometry (MS) comparing the effect of nicotine relative to vehicle control. Our results reveal a sex-dependent effect of nicotine on microglial proteomes. While both males and females exhibited histone-related genomic responsiveness to nicotine, males demonstrated enrichment in cytoskeletal and metabolic proteins, and females showed complement-protein adaptations. The microglial proteome in male mice displayed nicotine-related adaptations in proteins that can contribute to neurodisorders including Huntington's disease and amyotrophic lateral sclerosis (ALS), of which smoking is a known risk factor. Together, these results highlight an effect of nicotine on the proteome of microglia providing insight into immune pathways that can contribute to smoking-related behavior and disease.
A Sensor-to-Initiation Proteome Architecture Governing Regeneration Commitment in .
Liu S, Takemasa E, Mogi M
J Proteome Res · 2026 May · PMID 41911482 · Publisher ↗
() is well-known for its remarkable adaptability to environmental stress and its capacity for rejuvenation The current study was undertaken to identify an upstream cue that senses stress changes in the external milieu an...
() is well-known for its remarkable adaptability to environmental stress and its capacity for rejuvenation The current study was undertaken to identify an upstream cue that senses stress changes in the external milieu and governs a binary fate decision to maintain dormancy or unlock regeneration. We performed proteome-scale profiling across the cyst and early stolon stages of sp., with an emphasis on extracellular signaling and translational control. Proteome dynamics from the cyst to early stolon stage converge on a coherent "sensor-to initiation" architecture, including a sensor layer (TRP/PIEZO mechanotransducers, purinergic receptors, and integrin/FAK), an initiation layer (mTORC1-eIF4F signaling), and a stress-modulation layer (PERK-ISR signaling). We also nominate three actionable upstream hubs whose changes could be sufficient, in principle, to create a pro-translation state: CUL3-Kelch adaptors, Rag GTPase regulators and FKBP8-linked quality-control nodes. We therefore we propose a compact, testable mechanism for regeneration commitment in which sensor-integrated cues drive a calibrated mTORC1-eIF4F "initiation switch" buffered by a protective ISR. The identification of CUL3-Kelch, Rag GTPases, and FKBP8 as leverage points yields immediate hypotheses for transiently unlocking initiation to hasten repair.
A Framework for Database Search with AI Models in Mass Spectrometry-Based Proteomics.
Kalogeropoulos K, Van Goey J, Jenkins TP … +1 more , Eloff KM
J Proteome Res · 2026 May · PMID 41911267 · Full text
Database search remains the primary strategy for peptide detection in mass spectrometry-based proteomics, but growing data sets and increasingly expansive peptide search spaces now challenge its computational limits. At...
Database search remains the primary strategy for peptide detection in mass spectrometry-based proteomics, but growing data sets and increasingly expansive peptide search spaces now challenge its computational limits. At the same time, machine learning has transformed multiple aspects of spectrum identification and is increasingly applied directly to peptide-spectrum matching. Neural network models have been proposed as core engines for database search, yet the computational complexities of such approaches have not been systematically defined or compared. Here, we present a range of emerging approaches for database search and a theoretical framework for runtime and scaling in spectrum identification, contrasting classical search strategies with emerging neural network-based methods. We analyze asymptotic complexity in the number of spectra and peptide candidates and estimate practical wall time and memory requirements under realistic hardware assumptions. Our framework highlights trade-offs and provides a guide for selecting and developing scalable peptide search strategies in the era of large models and proteomics data sets. We therefore consider whether learned scoring models may progressively replace or augment classical similarity functions at the peptide-spectrum scoring level.
Advancing the Reproducibility and Repeatability of Capillary Zone Electrophoresis-Mass Spectrometry-Based Top-Down Proteomics by an Improved Capillary Coating Procedure.
Yue Y, Fang F, Gao G … +8 more , Sadeghi SA, Colón Rosado JA, Tabatabaeian Nimavard R, Falamarzi Askarani M, Thorp L, Dashtaki MR, Zhu G, Sun L
J Proteome Res · 2026 May · PMID 41907012 · Full text
Capillary zone electrophoresis (CZE)-mass spectrometry (MS) has attracted tremendous attention in top-down proteomics (TDP). However, its reproducibility and long-term repeatability for TDP remain concerns, most likely d...
Capillary zone electrophoresis (CZE)-mass spectrometry (MS) has attracted tremendous attention in top-down proteomics (TDP). However, its reproducibility and long-term repeatability for TDP remain concerns, most likely due to capillary coating. Here, we present an improved procedure for making linear polyacrylamide (LPA) coating, the most widely used coating in CE-MS-based proteomics, to boost the reproducibility and long-term repeatability of CZE-MS-based TDP. We focused on the step of degassing the polymerization solution, a critical step for achieving consistent LPA coating quality. The CZE-MS system using LPA-coated capillaries prepared with the optimal degassing procedure produced excellent reproducibility and repeatability for proteoform analysis. The 210 CZE-MS runs of three protein samples (a standard protein mixture, an cell lysate, and a HeLa cell lysate) across 200 h of instrument time with two MS instruments demonstrated the reliability of our conclusion. The optimal condition produced relative standard deviations (RSDs) of less than 2.6% in the migration time of proteoforms across dozens of CZE-MS runs without migration time alignment. CZE-MS/MS analysis of a HeLa whole cell lysate identified 274 ± 20 proteoforms (RSD ∼ 7%) and 146 ± 10 proteins (RSD ∼ 7%) across 10 successive runs and yielded consistent proteoform intensities. The coating procedure can be easily adopted by other research groups, as evidenced by our 2025 CE-MS summer school data. All results demonstrate that CZE-MS using the optimal LPA-coating procedure is ready for broad adoption to enable reproducible and repeatable measurements of proteoforms in complex samples.
Proteome Profiling Reveals Inflammation and Fibrosis Biomarkers in Urinary Migrasomes of Patients with Diabetic Kidney Disease.
Zhou X, Guo Z, Zhang R … +1 more , Yan R
J Proteome Res · 2026 May · PMID 41906798 · Publisher ↗
Urinary extracellular vesicles are emerging markers reflecting the disease status of diabetic kidney disease (DKD), but their types and functions in DKD disease are not yet fully understood. This study applied Astral-DIA...
Urinary extracellular vesicles are emerging markers reflecting the disease status of diabetic kidney disease (DKD), but their types and functions in DKD disease are not yet fully understood. This study applied Astral-DIA proteome for the in-depth profiling of urinary migrasomes (umig, a novel type of extracellular vesicles) and serum from DKD patients, aiming to identify protein biomarkers indicative of disease status. Proteome analysis revealed that protein-protein interactions were identified among LAMA1, ITGB3, FGG, and FGB in the differentially expressed proteins of serum and umig in the NC group and the DKD group. ITGB3 and FGB were closely related to inflammation and fibrosis pathways. The cellular source of umig may mainly come from podocytes, with some from monocytes/macrophages. NLRC4, ITGB3, and FGB were detected in umig. HK2 cells stimulated by DKD patient-derived umig showed increased expression of TLR4, -NF-κB p65, NLRC4, caspase-1 p20, IL-1β, and TNF-α, suggesting that umig promotes inflammation through the TLR4-NF-κB pathway. In addition, inflammation and fibrosis occurred in the renal tissues of patients with DKD and / mice, accompanied by significantly elevated levels of NLRC4, ITGB3, and FGB. The abundance of NLRC4, ITGB3, and FGB in urinary migrasomes highlights the promise of migrasomes as noninvasive biomarkers for gauging inflammation and fibrosis status in DKD.
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