Proteomics最新文献

{"title":"Integrated Transcriptomic, Proteomic, and Pharmacologic Profiling of 2D and 3D Patient-Derived GCTB Cell Lines Reveals Culture-Dependent Drug Response Determinants.","authors":"Yomogi Shiota, Ikumi Fujita, Sakura Hayashi, Sumio Ohtsuki, Tadashi Kondo","doi":"10.1002/pmic.70136","DOIUrl":"https://doi.org/10.1002/pmic.70136","url":null,"abstract":"<p><p>Giant cell tumor of bone (GCTB) is an intermediate bone neoplasm defined by recurrent H3F3A mutations and limited systemic treatment options beyond denosumab. Patient-derived cancer cell lines (PDCs) offer a scalable platform for mechanistic studies and therapeutic discovery, yet the extent to which culture dimensionality alters baseline molecular states and drug response in GCTB remains unclear. Here, we performed integrated transcriptomic, proteomic, and pharmacologic profiling of thirteen patient-derived GCTB cell lines cultured under two-dimensional (2D) monolayer and three-dimensional (3D) spheroid conditions. RNA sequencing and data-independent acquisition (DIA)-based quantitative proteomics were conducted on paired cultures, and drug sensitivity was assessed using a panel of 221 anticancer agents. 3D culture reproducibly induced compact spheroid formation across all cell lines and was accompanied by broad remodeling of gene expression, protein abundance, and drug-response profiles. Unsupervised analyses consistently demonstrated that samples clustered primarily by culture condition rather than by cell-line identity at both the transcriptome and proteome levels. Although global trends were shared, a substantial fraction of molecules showed RNA-protein discordance, indicating that transcriptomic changes alone do not fully explain culture-dependent functional remodeling. Pathway analyses highlighted enrichment of extracellular matrix-related processes, stress-response programs, and metabolic regulation in 3D cultures, with several features more prominent at the protein level. Functionally, 3D culture generally reduced sensitivity to many agents while preserving compound-dependent vulnerabilities. These results establish culture dimensionality as a key determinant of therapeutic susceptibility in GCTB PDCs and support incorporating proteome-informed 3D models into translational pipelines to prioritize clinically relevant drug candidates and biomarkers.</p>","PeriodicalId":224,"journal":{"name":"Proteomics","volume":" ","pages":"e70136"},"PeriodicalIF":3.9,"publicationDate":"2026-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147831595","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identifying Subcellular Structure Components in Escherichia Coli by Crosslinking and SEC-MS.","authors":"Rachel A Victor, Austin Lipinski, Paul R Langlais, Jacob C Schwartz","doi":"10.1002/pmic.70105","DOIUrl":"10.1002/pmic.70105","url":null,"abstract":"<p><p>Cells are comprised of a broad spectrum of structures that compartmentalize biochemical and signaling mechanisms. These structures can be comprised of many biomolecules, but especially lipids, proteins, and nucleic acids. Techniques are limited to quantify or discover new subcellular structures. We explored whether a proteomics approach using chemical crosslinking followed by size-exclusion chromatography and mass spectrometry (SEC-MS) of whole cell lysates can address this challenge. Formaldehyde crosslinking was used to preserve the weak molecular interactions responsible for many protein and nucleic acid assemblies. In this study, we perform the first formaldehyde crosslinking-assisted SEC-MS in a bacterial system. We demonstrate that when expressed ectopically in E. coli, large structures of a known assembly protein, FUS, can be detected through SEC-MS. We then show that E. coli proteins are enriched in particles of large or medium size due to formaldehyde crosslinking, which is the first analysis by formaldehyde and SEC-MS for a bacterial system. Last, analysis identified previously characterized E. coli protein assemblies and condensates, as well as potentially novel associations of prokaryote metabolism with large subcellular bodies. We propose this unbiased method can be used to stimulate or supplement targeted methods for discovery of new cellular bodies in a wide range of cell types.</p>","PeriodicalId":224,"journal":{"name":"Proteomics","volume":" ","pages":"27-36"},"PeriodicalIF":3.9,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13106914/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146016737","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reduction in Synaptic Vesicle Protein Abundance but Increased Amounts of Nsg2 and Lpcat1 in Cerebral Cortices Without the Endosomal SNARE Proteins Vti1a and Vti1b.","authors":"Julia Gottschalk, Katharina Kotschnew, Julia Hahn, Thomas Patschkowski, Gabriele von Fischer von Mollard","doi":"10.1002/pmic.70117","DOIUrl":"10.1002/pmic.70117","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Absence of the endosomal SNAREs vti1a and vti1b results in perinatal death and severe neuronal phenotypes in mice, while lack of one of these proteins results in minor phenotypes. Proteomic differences were investigated to obtain a deeper insight into processes in which vti1a and vti1b are involved. Here we applied a bottom-up shotgun proteomic approach to investigate the differences in wild-type, double heterozygous (DHET), vti1a&lt;sup&gt;-/-&lt;/sup&gt; vti1b&lt;sup&gt;-/-&lt;/sup&gt; double knockout (DKO), vti1a&lt;sup&gt;-/-&lt;/sup&gt; knockout and vti1b&lt;sup&gt;-/-&lt;/sup&gt; knockout cerebral cortices. Single deletions did not affect protein levels significantly. A total of 1725 proteins were detected of which 69 were less abundant and 191 proteins were more abundant in DKO cortices. Many less abundant proteins belonged to cellular components and reactome pathways synapse, synaptic vesicle cycle, vesicle mediated transport, L1CAM interaction, and cholesterol biosynthesis in pathway enrichment analysis. More abundant proteins were enriched in cellular components and Kyoto Encyclopaedia of Genes and Genomes (KEGG)-pathways such as spliceosome, ribosome, carbon metabolism, and ribonucleoprotein complex. Immunoblotting validated reduced expression levels of the tested synaptic vesicle proteins as well as increased amounts of lysophosphatidylcholine acyltransferase 1 (Lpcat1) and neuron-specific gene 2 (Nsg2), which is involved in postsynaptic AMPA-receptor recycling. These data indicate that the synapse and cell adhesion were strongly affected in DKO brains. STATEMENT OF SIGNIFICANCE OF THE STUDY: Distinct populations of neurons and glia cells are generated and organize into layers during brain development. Neurons develop an elaborate morphology to transmit information via axons and synapses to dendrites in receiving neurons. These neurites form via several specialized pathways of vesicle secretion and endocytosis. Fusion between these membranes requires members of the SNARE protein family. Double knockouts of the endosomal SNAREs vti1a and vti1b (DKO) result in perinatal lethality in mice with massive defects in the brain. In this study we compared the proteome of DKO brain cortices with double heterozygous controls to obtain insights into the molecular alterations and affected pathways. DKO brains contained lower amounts of synaptic proteins and proteins involved in cell adhesion, membrane trafficking and cholesterol biosynthesis. Several proteins of spliceosomes, ribosomes and carbon metabolism were more abundant in DKO brains, which may be a consequence of the reduced amounts of synaptic proteins or a shift in cell populations. Lysophosphatidylcholine acyltransferase 1 (Lpcat1) and neuron-specific gene 2 (Nsg2), which is involved in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) recycling, were confirmed to be more abundant by Western blotting. These data point to defects in trafficking especially in the synapse and in cell adhesion, which is required f","PeriodicalId":224,"journal":{"name":"Proteomics","volume":" ","pages":"86-97"},"PeriodicalIF":3.9,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13106906/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147484016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deep Proteomic Profiles of the Antarctic Diatom Fragilariopsis Cylindrus Under Varying Iron and Manganese Conditions.","authors":"Loay J Jabre, Elden Rowland, J Scott P McCain, Erin M Bertrand","doi":"10.1002/pmic.70109","DOIUrl":"10.1002/pmic.70109","url":null,"abstract":"<p><p>Fragilariopsis cylindrus is a key diatom in the Southern Ocean, where low iron and manganese availability constrain primary production and biogeochemical activity. The molecular mechanisms used by polar diatoms, including F. cylindrus, to cope with trace metal limitations remain largely unexplored. Here we present phenotypic characterizations and proteomic profiles of F. cylindrus grown under controlled iron (low, medium, high) and manganese (low, high) conditions that reflect those observed in the Southern Ocean. Using data-independent acquisition mass spectrometry, we measured over 8000 unique proteins capturing diverse metabolic responses, including those related to photosynthesis, elemental transport, and intracellular trafficking. We confirm consistent expression of canonical iron stress proteins (e.g., phytotransferrin) under low iron, and identify additional candidate biomarkers for iron and manganese stress that could be explored in future laboratory and field experiments. Our data also support the notion that one flavodoxin isoform in F. cylindrus is iron responsive and one is not, and show that PsaE, a protein associated with the iron-rich photosystem-I, is upregulated under low iron. Altogether, this dataset is among the most comprehensive proteomic characterizations of trace metal physiology in polar diatoms to date, providing a foundation for connecting molecular responses to trace metal availability and ocean biogeochemistry.</p>","PeriodicalId":224,"journal":{"name":"Proteomics","volume":" ","pages":"112-119"},"PeriodicalIF":3.9,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13106929/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146176885","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative Proteomics Reveals the Adaptive Mechanisms of Aeromonas hydrophila Under Cobalt Stress.","authors":"Xiaowei Zhang, Chenghao Shen, Zhen Qiu, Linbin Chen, Binghui Zhang, Chunyan Jia, Jinting Guo, Feiliao Lai, Xiangmin Lin","doi":"10.1002/pmic.70106","DOIUrl":"10.1002/pmic.70106","url":null,"abstract":"<p><p>Cobalt is an essential micronutrient but becomes toxic at elevated concentrations, requiring microorganisms to balance acquisition and detoxification. Aeromonas hydrophila, an opportunistic aquatic pathogen, is often encountered in metal-contaminated aquatic environments; however, its adaptive responses to cobalt stress have not been systematically characterized. Here, we applied quantitative proteomics to characterize the global protein response of A. hydrophila under cobalt stress. A total of 2767 proteins were identified, of which 724 were differentially abundant. Enrichment analyses indicated that cobalt exposure was associated with alterations in energy metabolism, oxidative phosphorylation, and ribosome-related pathways. Gene set enrichment analysis suggested an overall upregulation of ribosome-associated functions, accompanied by down regulation of carbon metabolism and the tricarboxylic acid cycle. Protein-protein interaction network mapping identified 15 functional clusters, with core modules linked to oxidative phosphorylation, ABC transport, carbohydrate metabolism, and Fe-S cluster biogenesis. Ten hub proteins associated with respiratory and transport systems were identified based on network topology. Functional validation using seven deletion mutants indicated that genes encoding shikimate kinase, glutaminase, and arsenate reductase contribute to cobalt tolerance. Together, these findings provide a systems-level view of how A. hydrophila adapts to cobalt stress, reveal candidate factors mediating metal resistance, and suggest potential targets for antimicrobial development and bioremediation strategies.</p>","PeriodicalId":224,"journal":{"name":"Proteomics","volume":" ","pages":"37-47"},"PeriodicalIF":3.9,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146008002","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mass Spectrometry Proteomics of the Nanoparticle Corona Is Highly Dependent on Sample Preparation Protocol.","authors":"Asia Saorin, Alberto Martinez-Serra, Michael Henry, Paula Meleady, Marco P Monopoli","doi":"10.1002/pmic.70118","DOIUrl":"10.1002/pmic.70118","url":null,"abstract":"<p><p>It is widely established that the layers of proteins bound to the surface of nanoparticles (NPs), also known as the protein corona, critically influence their behavior in biological environments. Despite numerous proteomics studies performed in different NP-corona systems, the impact of proteomic sample preparation methods on corona profiling remains poorly understood. In this study, we systematically compared five digestion protocols, namely S-Trap^TM, iST^TM, ProteaseMAX^TM, RapiGest^TM, and a routine in-gel digestion protocol commonly used in several laboratories. Protein corona samples were isolated from silica NPs incubated in 80% human plasma in phosphate-buffered saline. Protocol performance was assessed in terms of digestion efficiency, protein and peptide yield, reproducibility, and identification bias. Proteomic characterization revealed marked differences across protocols, highlighting the influence of protocol on protein digestion, recovery, and representation. Our findings emphasize the need for method standardization and tailored protocol selection in corona studies to ensure accurate and reproducible proteomic profiling.</p>","PeriodicalId":224,"journal":{"name":"Proteomics","volume":" ","pages":"98-111"},"PeriodicalIF":3.9,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13106918/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147519502","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Practical Impact of Imputation and Batch-Effect Correction for Proteomics/Peptidomics Differential-Abundance Analysis.","authors":"Charis Gonidaki, Agnieszka Latosinska, Antonia Vlahou, Rafael Stroggilos, Harald Mischak","doi":"10.1002/pmic.70111","DOIUrl":"10.1002/pmic.70111","url":null,"abstract":"<p><p>MS-based proteomics offers powerful opportunities for biomarker discovery; nevertheless, it is associated with technical challenges, including missing values and batch effects. Although imputation and batch-correction methods are well established in proteomics, their impact remains incompletely characterized in large-scale clinical proteomics datasets. Here, we examine the practical impact and interaction of three popular imputation methods (Gaussian, ½ LOD, KNN) in combination with three batch-effect correction approaches (ComBat, ComBat with disease covariate, MNN) on differential abundance analysis in a CE-MS urine peptidomics dataset of 1,050 samples across 13 batches from chronic kidney disease (CKD) patients and controls. Downstream effects were assessed based on peptide validation between discovery and validation sets. Imputation method choice had minimal impact on the final list of disease-associated peptides (DAPs), given the missingness structure and normalization strategy. In contrast, batch-effect correction largely affected the results: MNN and especially unadjusted ComBat removed a large proportion of DAPs ( <math><semantics><mo>∼</mo> <annotation>$sim $</annotation></semantics> </math> 50% and >90%, respectively), whereas inclusion of disease status in the ComBat model largely preserved biological signal. This study highlights how popular preprocessing choices can affect biological signal, showing that imputation and batch-effect correction interact and jointly influence downstream results, underscoring the need for caution when applying batch-effect correction. STATEMENT OF SIGNIFICANCE OF THE STUDY: Finding reliable biomarkers in clinical proteomics requires addressing the technical noise that can hide true biological signals. In this work, we examine the practical impact and interaction of commonly used imputation and batch correction methods on the list of peptides that emerge as differentially abundant. Instead of relying on simulations or small datasets, we examine a large, real-world urine-peptidomics cohort of more than 1,000 samples screened for chronic kidney disease. The results demonstrate that, in datasets such as the one used here, different preprocessing strategies can lead to substantially different outcomes. Imputation and batch-effect correction were found to be interdependent, and batch effect removal can lead to loss of meaningful biological differences, highlighting the importance of applying such corrections with caution.</p>","PeriodicalId":224,"journal":{"name":"Proteomics","volume":" ","pages":"48-58"},"PeriodicalIF":3.9,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13106926/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146217946","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Brownotate, a Comprehensive Solution to Generate Protein Sequence Databases for Any Species.","authors":"Adrien Brown, Alexandre Burel, Sarah Cianférani, Christine Carapito, Fabrice Bertile","doi":"10.1002/pmic.70094","DOIUrl":"10.1002/pmic.70094","url":null,"abstract":"<p><p>Proteomics is strengthening research in biology and the diversification of the model organisms studied is very promising for fully understanding the complexity of biological principles. However, the lack of protein sequence databases for many species is a major bottleneck. Existing computational solutions are usually incomplete and/or only usable by bioinformaticians. We have built an open-source, user-friendly pipeline, called Brownotate, which allows anyone to generate protein sequence databases for any species as long as sequencing information is available. The pipeline can extract already existing protein sequences, but also automatically annotate any genome assembly or assemble and annotate any DNA sequence dataset. By testing the pipeline with numerous sequencing and assembly datasets covering a large part of the phylogenetic tree, we show that Brownotate generates fragmented but good quality assemblies and good quality annotations when compared to reference data. By comparing the use of protein databases generated by Brownotate or downloaded from NCBI to interpret proteomic data, we show very comparable results. The Brownotate pipeline is, therefore, an important new addition to the proteomics toolbox. The pipeline and its web interface are freely available at https://github.com/LSMBO/Brownotate and https://github.com/LSMBO/brownotate-app, respectively. SUMMARY: This study evaluated the performance of a newly developed pipeline, Brownotate, for the assembly and annotation of sequencing data for multiple species, from prokaryotes to eukaryotes. We compared their fragmentation level (assembly) and completeness based on evolutionary expectations of gene content, and we evaluated their overlap. Brownotate generated fragmented, slightly less complete assemblies. However, the overlap of proteins predicted was very good, despite an excess of predicted sequences of small size with Brownotate. In addition, the interpretation of proteomics data downloaded from PRIDE repository for 27 species was found to lead to very similar results regardless of the origin of the protein sequencing database used, whether it was generated by Brownotate or downloaded from NCBI. Brownotate, made available to the community, will, therefore, be a tool of choice to mitigate the lack of an appropriate protein sequence database for many species, and allow proteomists to analyse without delay samples from species for which only sequencing data are available.</p>","PeriodicalId":224,"journal":{"name":"Proteomics","volume":" ","pages":"13-26"},"PeriodicalIF":3.9,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13106930/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145909824","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Imaging Mass Spectrometry-Based Assessment of ER, PR, and HER2 Protein Expression in Breast Cancer.","authors":"Kiran K Mangalaparthi, Daigo Gunji, Olajide E Olaleye, Amy J French, Gunveen S Sachdeva, Shilpa Venkataraman, Neha Joshi, Tianqi Gao, Sumankalai Ramachandran, Julie A Vrana, Kenneth J Rothschild, Mark J Lim, Gargey Yagnik, Saba Yasir, Michael Keeney, Akhilesh Pandey","doi":"10.1002/pmic.70110","DOIUrl":"10.1002/pmic.70110","url":null,"abstract":"<p><p>Imaging mass spectrometry has made significant advancements in recent years owing to its ability to study the spatial localization and abundance of proteins, peptides, lipids, glycans, metabolites, and drugs. In this study, we employed an imaging mass spectrometry-based workflow called MALDI-IHC, which uses antibodies conjugated with photocleavable mass-tags, to investigate the detection of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) along with pan-cytokeratin in formalin-fixed paraffin-embedded (FFPE) sections from cases with breast cancer. The expression of ER, PR, and HER2 was consistent with the clinical diagnosis based on conventional immunohistochemistry, lacking signal in triple-negative cases and concordant signals in the cases that were classified as ER+/PR+/HER2+, ER+/PR+/HER2-, and ER-/PR-/HER2+ groups, respectively. In addition, the potential utility of a seconday antibody-based MALDI-IHC approach for HER2 expression was evaluated. In agreement with immunohistochemistry results, HER2 expression co-localized within tumor regions in a HER2+ breast cancer case, while no expression was observed in a HER2- case. Collectively, these results highlight the potential of imaging mass spectrometry-based MALDI-IHC workflow for multiplexed detection of proteins in clinically relevant tissue sections.</p>","PeriodicalId":224,"journal":{"name":"Proteomics","volume":" ","pages":"6-12"},"PeriodicalIF":3.9,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147288996","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single-Cell Nanodroplet Processing Proteomics Pipeline for Analysis of Human-Derived Microglia.","authors":"Ashley N Ives, James M Fulcher, Alex R Bautista, Reta Birhanu Kitata, Sarah M Williams, David A Bennett, Philip L De Jager, Vladislav A Petyuk","doi":"10.1002/pmic.70112","DOIUrl":"10.1002/pmic.70112","url":null,"abstract":"<p><p>Single-cell omics tools provide unique insights into heterogeneous cell populations and their responses to stimuli. For example, single-cell RNA sequencing has identified several transcriptionally distinct populations of microglia, which are resident immune cells of the central nervous system (CNS) that are responsive to CNS injury, infection, and neurodegeneration. To date, single-cell studies of microglia have focused on RNA-sequencing or cytometry by time of flight (CyTOF), which provide indirect readouts of protein abundance or quantification of a limited number of targets. Herein, we present a workflow based on FACS-assisted isolation, cryopreservation, and nanodroplet-based processing for single-cell mass spectrometry proteomics analysis of the postmortem human brain cortex-derived microglia. From a single microglial cell, 1039 proteins could be identified on average. As a proof-of-principle, we applied single-cell proteomics for exploring the heterogeneity of brain microglia at the cellular level. This pilot proteomics data partially recapitulates the prior microglia subtypes. Specifically, we determined that mitochondrial proteins, in particular members of NADH dehydrogenase (Complex I), cytochrome b-c1 (Complex III), cytochrome c oxidase (Complex IV), F1-ATPase (Complex V), and Na+/K+-ATPase complex, drive variation across microglia. This pipeline offers the potential for identifying functionally and analytically relevant protein targets for microglia in Alzheimer's disease and other neurological disorders. SIGNIFICANCE OF THE STUDY: Microglia are a key brain cell type that may contribute to pathogenesis in neurodegenerative disease. Transcriptomic profiling of microglia from the central nervous system of humans and animal models has identified several subtypes of microglia, and complementary proteomic profiling of microglia is likely to provide functionally and therapeutically relevant targets. Single-cell proteomics studies of human-derived microglia are lacking. This work describes a label-free, single-cell proteomics approach for microglia isolated by fluorescence-activated cell sorting from a human donor that yields comparable numbers of identifications in comparison to prior single-cell RNA sequencing studies of microglia. This approach holds promise for enabling large-scale proteomics-based subtyping of microglia and studying their roles in neurodegenerative diseases.</p>","PeriodicalId":224,"journal":{"name":"Proteomics","volume":" ","pages":"59-68"},"PeriodicalIF":3.9,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13106911/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147300575","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}