Molecular & Cellular Proteomics最新文献

{"title":"Data-Independent Acquisition: A Milestone and Prospect in Clinical Mass Spectrometry-Based Proteomics.","authors":"Klemens Fröhlich, Matthias Fahrner, Eva Brombacher, Adrianna Seredynska, Maximilian Maldacker, Clemens Kreutz, Alexander Schmidt, Oliver Schilling","doi":"10.1016/j.mcpro.2024.100800","DOIUrl":"10.1016/j.mcpro.2024.100800","url":null,"abstract":"<p><p>Data-independent acquisition (DIA) has revolutionized the field of mass spectrometry (MS)-based proteomics over the past few years. DIA stands out for its ability to systematically sample all peptides in a given m/z range, allowing an unbiased acquisition of proteomics data. This greatly mitigates the issue of missing values and significantly enhances quantitative accuracy, precision, and reproducibility compared to many traditional methods. This review focuses on the critical role of DIA analysis software tools, primarily focusing on their capabilities and the challenges they address in proteomic research. Advances in MS technology, such as trapped ion mobility spectrometry, or high field asymmetric waveform ion mobility spectrometry require sophisticated analysis software capable of handling the increased data complexity and exploiting the full potential of DIA. We identify and critically evaluate leading software tools in the DIA landscape, discussing their unique features, and the reliability of their quantitative and qualitative outputs. We present the biological and clinical relevance of DIA-MS and discuss crucial publications that paved the way for in-depth proteomic characterization in patient-derived specimens. Furthermore, we provide a perspective on emerging trends in clinical applications and present upcoming challenges including standardization and certification of MS-based acquisition strategies in molecular diagnostics. While we emphasize the need for continuous development of software tools to keep pace with evolving technologies, we advise researchers against uncritically accepting the results from DIA software tools. Each tool may have its own biases, and some may not be as sensitive or reliable as others. Our overarching recommendation for both researchers and clinicians is to employ multiple DIA analysis tools, utilizing orthogonal analysis approaches to enhance the robustness and reliability of their findings.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100800"},"PeriodicalIF":6.1,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11380018/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141331433","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Chemo-Phosphoproteomic Profiling with ATR Inhibitors Berzosertib and Gartisertib Uncovers New Biomarkers and DNA Damage Response Regulators.","authors":"Rathan Jadav, Florian Weiland, Sylvie M Noordermeer, Thomas Carroll, Yuandi Gao, Jianming Wang, Houjiang Zhou, Frederic Lamoliatte, Rachel Toth, Thomas Macartney, Fiona Brown, C James Hastie, Constance Alabert, Haico van Attikum, Frank Zenke, Jean-Yves Masson, John Rouse","doi":"10.1016/j.mcpro.2024.100802","DOIUrl":"10.1016/j.mcpro.2024.100802","url":null,"abstract":"<p><p>The ATR kinase protects cells against DNA damage and replication stress and represents a promising anti-cancer drug target. The ATR inhibitors (ATRi) berzosertib and gartisertib are both in clinical trials for the treatment of advanced solid tumors as monotherapy or in combination with genotoxic agents. We carried out quantitative phospho-proteomic screening for ATR biomarkers that are highly sensitive to berzosertib and gartisertib, using an optimized mass spectrometry pipeline. Screening identified a range of novel ATR-dependent phosphorylation events, which were grouped into three broad classes: (i) targets whose phosphorylation is highly sensitive to ATRi and which could be the next generation of ATR biomarkers; (ii) proteins with known genome maintenance roles not previously known to be regulated by ATR; (iii) novel targets whose cellular roles are unclear. Class iii targets represent candidate DNA damage response proteins and, with this in mind, proteins in this class were subjected to secondary screening for recruitment to DNA damage sites. We show that one of the proteins recruited, SCAF1, interacts with RNAPII in a phospho-dependent manner and recruitment requires PARP activity and interaction with RNAPII. We also show that SCAF1 deficiency partly rescues RAD51 loading in cells lacking the BRCA1 tumor suppressor. Taken together these data reveal potential new ATR biomarkers and new genome maintenance factors.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100802"},"PeriodicalIF":6.1,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11338954/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141331431","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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 Phosphoproteomic Profiling of Mouse Sperm Maturation in Epididymis Revealed Kinases Important for Sperm Motility.","authors":"Xiangzheng Zhang, Haixia Tu, Xin Zhou, Bing Wang, Yueshuai Guo, Chenghao Situ, Yaling Qi, Yan Li, Xuejiang Guo","doi":"10.1016/j.mcpro.2024.100810","DOIUrl":"10.1016/j.mcpro.2024.100810","url":null,"abstract":"<p><p>Transcriptionally and translationally silent sperm undergo functional maturation during epididymis traverse, which provides sperm ability to move and is crucial for successful fertilization. However, the molecular mechanisms governing sperm maturation remain poorly understood, especially at the protein post-translational modification level. In this study, we conducted a comprehensive quantitative phosphoproteomic analysis of mouse epididymal sperm from different regions (caput, corpus, and cauda) to unveil the dynamics of protein phosphorylation during sperm maturation. We identified 6447 phosphorylation sites in 1407 phosphoproteins, and 345 phosphoproteins were differentially phosphorylated between caput and cauda sperm. Gene ontology and KEGG pathway analyses showed enrichment of differentially phosphorylated proteins in energy metabolism, sperm motility, and fertilization. Kinase substrate network analysis followed by inhibition assay and quantitative phosphoproteomics analysis showed that TSSK2 kinase is important for sperm motility and progressive motility. This study systemically characterized the intricate phosphorylation regulation during sperm maturation in the mouse epididymis, which can be a basis to elucidate sperm motility acquisition, and to offer potential targets for male contraception and the treatment of male infertility.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100810"},"PeriodicalIF":6.1,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11338950/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141559242","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DIA-Based Phosphoproteomics Identifies Early Phosphorylation Events in Response to EGTA and Mannitol in Arabidopsis.","authors":"Tian Sang, Chin-Wen Chen, Zhen Lin, Yu Ma, Yanyan Du, Pei-Yi Lin, Marco Hadisurya, Jian-Kang Zhu, Zhaobo Lang, W Andy Tao, Chuan-Chih Hsu, Pengcheng Wang","doi":"10.1016/j.mcpro.2024.100804","DOIUrl":"10.1016/j.mcpro.2024.100804","url":null,"abstract":"<p><p>Osmotic stress significantly hampers plant growth and crop yields, emphasizing the need for a thorough comprehension of the underlying molecular responses. Previous research has demonstrated that osmotic stress rapidly induces calcium influx and signaling, along with the activation of a specific subset of protein kinases, notably the Raf-like protein (RAF)-sucrose nonfermenting-1-related protein kinase 2 (SnRK2) kinase cascades within minutes. However, the intricate interplay between calcium signaling and the activation of RAF-SnRK2 kinase cascades remains elusive. Here, in this study, we discovered that Raf-like protein (RAF) kinases undergo hyperphosphorylation in response to osmotic shocks. Intriguingly, treatment with the calcium chelator EGTA robustly activates RAF-SnRK2 cascades, mirroring the effects of osmotic treatment. Utilizing high-throughput data-independent acquisition-based phosphoproteomics, we unveiled the global impact of EGTA on protein phosphorylation. Beyond the activation of RAFs and SnRK2s, EGTA treatment also activates mitogen-activated protein kinase cascades, Calcium-dependent protein kinases, and receptor-like protein kinases, etc. Through overlapping assays, we identified potential roles of mitogen-activated protein kinase kinase kinase kinases and receptor-like protein kinases in the osmotic stress-induced activation of RAF-SnRK2 cascades. Our findings illuminate the regulation of phosphorylation and cellular events by Ca²⁺ signaling, offering insights into the (exocellular) Ca²⁺ deprivation during early hyperosmolality sensing and signaling.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100804"},"PeriodicalIF":6.1,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11325057/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141432316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhanced In Situ Spatial Proteomics by Effective Combination of MALDI Imaging and LC-MS/MS.","authors":"Frederike Schäfer, Archana Tomar, Shogo Sato, Raffaele Teperino, Axel Imhof, Shibojyoti Lahiri","doi":"10.1016/j.mcpro.2024.100811","DOIUrl":"10.1016/j.mcpro.2024.100811","url":null,"abstract":"<p><p>Highly specialized cells are fundamental for the proper functioning of complex organs. Variations in cell-type-specific gene expression and protein composition have been linked to a variety of diseases. Investigation of the distinctive molecular makeup of these cells within tissues is therefore critical in biomedical research. Although several technologies have emerged as valuable tools to address this cellular heterogeneity, most workflows lack sufficient in situ resolution and are associated with high costs and extremely long analysis times. Here, we present a combination of experimental and computational approaches that allows a more comprehensive investigation of molecular heterogeneity within tissues than by either shotgun LC-MS/MS or MALDI imaging alone. We applied our pipeline to the mouse brain, which contains a wide variety of cell types that not only perform unique functions but also exhibit varying sensitivities to insults. We explored the distinct neuronal populations within the hippocampus, a brain region crucial for learning and memory that is involved in various neurological disorders. As an example, we identified the groups of proteins distinguishing the neuronal populations of the dentate gyrus (DG) and the cornu ammonis (CA) in the same brain section. Most of the annotated proteins matched the regional enrichment of their transcripts, thereby validating the method. As the method is highly reproducible, the identification of individual masses through the combination of MALDI-IMS and LC-MS/MS methods can be used for the much faster and more precise interpretation of MALDI-IMS measurements only. This greatly speeds up spatial proteomic analyses and allows the detection of local protein variations within the same population of cells. The method's general applicability has the potential to be used to investigate different biological conditions and tissues and a much higher throughput than other techniques making it a promising approach for clinical routine applications.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100811"},"PeriodicalIF":6.1,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11345593/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141600583","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Carrier-Based Quantitative Proteomics Method Applied to Biomarker Discovery in Pericardial Fluid.","authors":"Amanda J Campbell, Samir Cakar, Nicolai B Palstrøm, Lars P Riber, Lars M Rasmussen, Hans C Beck","doi":"10.1016/j.mcpro.2024.100812","DOIUrl":"10.1016/j.mcpro.2024.100812","url":null,"abstract":"<p><p>Data-dependent liquid chromatography tandem mass spectrometry is challenged by the large concentration range of proteins in plasma and related fluids. We adapted the SCoPE method from single-cell proteomics to pericardial fluid, where a myocardial tissue carrier was used to aid protein quantification. The carrier proteome and patient samples were labeled with distinct isobaric labels, which allowed separate quantification. Undepleted pericardial fluid from patients with type 2 diabetes mellitus and/or heart failure undergoing heart surgery was analyzed with either a traditional liquid chromatography tandem mass spectrometry method or with the carrier proteome. In total, 1398 proteins were quantified with a carrier, compared to 265 without, and a higher proportion of these proteins were of myocardial origin. The number of differentially expressed proteins also increased nearly four-fold. For patients with both heart failure and type 2 diabetes mellitus, pathway analysis of upregulated proteins demonstrated the enrichment of immune activation, blood coagulation, and stress pathways. Overall, our work demonstrates the applicability of a carrier for enhanced protein quantification in challenging biological matrices such as pericardial fluid, with potential applications for biomarker discovery. Mass spectrometry data are available via ProteomeXchange with identifier PXD053450.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100812"},"PeriodicalIF":6.1,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11387241/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141616854","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proximity Labeling Proteomics Reveals Kv1.3 Potassium Channel Immune Interactors in Microglia.","authors":"Christine A Bowen, Hai M Nguyen, Young Lin, Pritha Bagchi, Aditya Natu, Claudia Espinosa-Garcia, Erica Werner, Rashmi Kumari, Amanda Dabdab Brandelli, Prateek Kumar, Brendan R Tobin, Levi Wood, Victor Faundez, Heike Wulff, Nicholas T Seyfried, Srikant Rangaraju","doi":"10.1016/j.mcpro.2024.100809","DOIUrl":"10.1016/j.mcpro.2024.100809","url":null,"abstract":"<p><p>Microglia are resident immune cells of the brain and regulate its inflammatory state. In neurodegenerative diseases, microglia transition from a homeostatic state to a state referred to as disease-associated microglia (DAM). DAM express higher levels of proinflammatory signaling molecules, like STAT1 and TLR2, and show transitions in mitochondrial activity toward a more glycolytic response. Inhibition of Kv1.3 decreases the proinflammatory signature of DAM, though how Kv1.3 influences the response is unknown. Our goal was to identify the potential proteins interacting with Kv1.3 during transition to DAM. We utilized TurboID, a biotin ligase, fused to Kv1.3 to evaluate potential interacting proteins with Kv1.3 via mass spectrometry in BV-2 microglia following TLR4-mediated activation. Electrophysiology, Western blotting, and flow cytometry were used to evaluate Kv1.3 channel presence and TurboID biotinylation activity. We hypothesized that Kv1.3 contains domain-specific interactors that vary during a TLR4-induced inflammatory response, some of which are dependent on the PDZ-binding domain on the C terminus. We determined that the N terminus of Kv1.3 is responsible for trafficking Kv1.3 to the cell surface and mitochondria (e.g., NUDC, TIMM50). Whereas, the C terminus interacts with immune signaling proteins in a lipopolysaccharide-induced inflammatory response (e.g., STAT1, TLR2, and C3). There are 70 proteins that rely on the C-terminal PDZ-binding domain to interact with Kv1.3 (e.g., ND3, Snx3, and Sun1). Furthermore, we used Kv1.3 blockade to verify functional coupling between Kv1.3 and interferon-mediated STAT1 activation. Overall, we highlight that the Kv1.3 potassium channel functions beyond conducting the outward flux of potassium ions in an inflammatory context and that Kv1.3 modulates the activity of key immune signaling proteins, such as STAT1 and C3.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100809"},"PeriodicalIF":6.1,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469537","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comprehensive Glycomic and Proteomic Analysis of Mouse Striatum and Lateral Hypothalamus Following Repeated Exposures to Cocaine or Methamphetamine.","authors":"Manveen K Sethi, Riccardo Maccioni, John D Hogan, Tomoya Kawamura, Vez Repunte-Canonigo, Jihuan Chen, Joseph Zaia, Pietro Paolo Sanna","doi":"10.1016/j.mcpro.2024.100803","DOIUrl":"10.1016/j.mcpro.2024.100803","url":null,"abstract":"<p><p>Substance use disorder is a major concern, with few therapeutic options. Heparan sulfate (HS) and chondroitin sulfate (CS) interact with a plethora of growth factors and their receptors and have profound effects on cellular signaling. Thus, targeting these dynamic interactions might represent a potential novel therapeutic modality. In the present study, we performed mass spectrometry-based glycomic and proteomic analysis to understand the effects of cocaine and methamphetamine (METH) on HS, CS, and the proteome of two brain regions critically involved in drug addiction: the lateral hypothalamus and the striatum. We observed that cocaine and METH significantly alter HS and CS abundances as well as sulfate contents and composition. In particular, repeated METH or cocaine treatments reduced CS 4-O-sulfation and increased CS 6-O-sulfation. Since C4S and C6S exercise differential effects on axon growth, regeneration, and plasticity, these changes likely contribute to drug-induced neural plasticity in these brain regions. Notably, we observed that restoring these alterations by increasing CS 4-0 levels in the lateral hypothalamus by adeno-associated virus delivery of an shRNA to arylsulfatase B (N-acetylgalactosamine-4-sulfatase) ameliorated anxiety and prevented the expression of preference for cocaine in a novelty induced conditioned place preference test during cocaine withdrawal. Finally, proteomics analyses revealed a number of aberrant proteins in METH- and cocaine-treated versus saline-treated mice, including myelin proteolipid protein, calcium/calmodulin-dependent protein kinase type II subunit alpha, synapsin-2, tenascin-R, calnexin, annexin A7, hepatoma-derived growth factor, neurocan, and CSPG5, and oxidative phosphorylation among the top perturbed pathway. Taken together, these data support the role of HS, CS, and associated proteins in stimulants abuse and suggest that manipulation of HSPGs can represent a novel therapeutic strategy.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100803"},"PeriodicalIF":6.1,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11324981/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141331432","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Profiling Proteins and Phosphorylation Sites During T Cell Activation Using an Integrated Thermal Shift Assay.","authors":"Brandon M Gassaway, Edward L Huttlin, Emily M Huntsman, Tomer M Yaron-Barir, Jared L Johnson, Kiran Kurmi, Lewis C Cantley, Joao A Paulo, Alison E Ringel, Steven P Gygi, Marcia C Haigis","doi":"10.1016/j.mcpro.2024.100801","DOIUrl":"10.1016/j.mcpro.2024.100801","url":null,"abstract":"<p><p>T cell activation is a complex biological process of naive cells maturing into effector cells. Proteomic and phospho-proteomic approaches have provided critical insights into this process, yet it is not always clear how changes in individual proteins or phosphorylation sites have functional significance. Here, we developed the Phosphorylation Integrated Thermal Shift Assay (PITSA) that combines the measurement of protein or phosphorylation site abundance and thermal stability into a single tandem mass tags experiment and apply this method to study T cell activation. We quantified the abundance and thermal stability of over 7500 proteins and 5000 phosphorylation sites and identified significant differences in chromatin-related, TCR signaling, DNA repair, and proliferative phosphoproteins. PITSA may be applied to a wide range of biological contexts to generate hypotheses as to which proteins or phosphorylation sites are functionally regulated in a given system as well as the mechanisms by which this regulation may occur.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100801"},"PeriodicalIF":6.1,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11298636/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141331434","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhanced Proteomic Coverage in Tissue Microenvironment by Immune Cell Subtype Library-Assisted DIA-MS.","authors":"Jhih-Ci Yang, Tzi-Hui Hsu, Ciao-Syuan Chen, Jou-Hui Yu, Kuo-I Lin, Yu-Ju Chen","doi":"10.1016/j.mcpro.2024.100792","DOIUrl":"10.1016/j.mcpro.2024.100792","url":null,"abstract":"<p><p>Immune cells that infiltrate the tumor microenvironment (TME) play crucial roles in shaping cancer development and influencing clinical outcomes and therapeutic responses. However, obtaining a comprehensive proteomic snapshot of tumor-infiltrating immunity in clinical specimens is often hindered by small sample amounts and a low proportion of immune infiltrating cells in the TME. To enable in-depth and highly sensitive profiling of microscale tissues, we established an immune cell-enriched library-assisted strategy for data-independent acquisition mass spectrometry (DIA-MS). Firstly, six immune cell subtype-specific spectral libraries were established from sorted cluster of differentiation markers, CD8⁺, CD4⁺ T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, and macrophages in murine mesenteric lymph nodes (MLNs), covering 7815 protein groups with surface markers and immune cell-enriched proteins. The feasibility of microscale immune proteomic profiling was demonstrated on 1 μg tissue protein from the tumor of murine colorectal cancer (CRC) models using single-shot DIA; the immune cell-enriched library increased coverage to quantify 7419 proteins compared to directDIA analysis (6978 proteins). The enhancement enabled the mapping of 841 immune function-related proteins and exclusive identification of many low-abundance immune proteins, such as CD1D1, and CD244, demonstrating high sensitivity for immune landscape profiling. This approach was used to characterize the MLNs in CRC models, aiming to elucidate the mechanism underlying their involvement in cancer development within the TME. Even with a low percentage of immune cell infiltration (0.25-3%) in the tumor, our results illuminate downregulation in the adaptive immune signaling pathways (such as C-type lectin receptor signaling, and chemokine signaling), T cell receptor signaling, and Th1/Th2/Th17 cell differentiation, suggesting an immunosuppressive status in MLNs of CRC model. The DIA approach using the immune cell-enriched libraries showcased deep coverage and high sensitivity that can facilitate illumination of the immune proteomic landscape for microscale samples.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100792"},"PeriodicalIF":6.1,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11260568/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141174859","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}