Molecular & Cellular Proteomics最新文献

{"title":"Multiple Classes of Antigen Contribute to the Antigenic Landscape of Mesothelioma.","authors":"Kirti Pandey, Pouya Faridi, Rochelle Ayala, Y C Gary Lee, Ebony Rouse, Sanjay S G Krishna, Ian Dick, Alec Redwood, Bruce Robinson, Jenette Creaney, Anthony W Purcell","doi":"10.1016/j.mcpro.2025.100925","DOIUrl":"10.1016/j.mcpro.2025.100925","url":null,"abstract":"<p><p>Mesothelioma is an incurable, asbestos-exposure-related cancer that typically affects the lining or pleura of the lungs. Symptoms typically develop many decades after initial asbestos exposure, leaving an enduring legacy of disease. The current disease burden is peaking worldwide and thus there is a massive unmet clinical need for curative therapies. Recently, immune checkpoint blockade-based therapy has been adopted as a first-line of treatment for mesothelioma. Vaccine-induced augmentation of immune responses unleashed during checkpoint blockade may provide further clinical benefit in mesothelioma. In this study, we explore the human leukocyte antigen class I landscape (or immunopeptidome) of mesothelioma in patient-derived cell lines and clinical material (pleural effusion samples). We identify a range of peptide antigens derived from targets including cancer testis antigens, endogenous retroviruses as well as novel post-translational modification of peptides. This information will facilitate the characterization of the immune response to these antigens to determine which class of antigen is most immunogenic and has the potential to be tested in future vaccine studies.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100925"},"PeriodicalIF":6.1,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11929013/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143374170","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":"Proteogenomic Profiling Reveals Small ORFs and Functional Microproteins in Activated T Cells.","authors":"Yang Yang, Chuangmiao Chen, Kecheng Li, Yuanliang Zhang, Lei Chen, Jue Shi, Quanhua Mu, Yang Xu, Qian Zhao","doi":"10.1016/j.mcpro.2025.100914","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.100914","url":null,"abstract":"<p><p>Noncanonical micropeptides or called novel microproteins, i.e., polypeptides mostly under 10 kDa, are encoded by genomic sequences that have been previously annotated as noncoding but now known as small open reading frames (sORFs). The recent identification of microproteins encoded by sORFs has provided evidence that many sORFs encode functional microproteins that play crucial roles in various biological processes. T cell activation is a critical biological process for adaptive immune response. Understanding key players in this process will allow us to decipher the complex mechanisms as well as develop immunotherapy for treating a wide range of diseases. Although there have been extensive studies on canonical proteins in T cell activation, the novel microproteins in T cells and their roles have been uncharted water to date. Nascent proteins are defined as newly synthesized polypeptides emerged during the translation of mRNA. In this study, we combined nascent proteomics and quantitative proteomics to identify 411 novel microproteins in primary human T cells, including 83 nascent microproteins. We activated the T cell function with either PMA/Ionomycin (distal activation) or CD3/CD28 activating antibodies (proximal activation), and obtained a comprehensive canonical protein and microprotein profiles to pinpoint common and distinct differentially expressed proteins under these two activation conditions. After experimental testing, three microproteins numbered T1, T2 and T3 were found to be functional in regulating T cell activation. Bioinformatic and proteomic analyses suggested that T1 was functional related to immune as negative feedback to T cell activation. Our study not only established an integrated approach to uncover and elucidate novel microproteins but also highlight the significant role of microproteins in regulating T cell activation.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100914"},"PeriodicalIF":6.1,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143365507","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":"Filter-Aided Extracellular Vesicle Enrichment (FAEVEr) for Proteomics.","authors":"Jarne Pauwels, Tessa Van de Steene, Jana Van de Velde, Freya De Muyer, Danaë De Pauw, Femke Baeke, Sven Eyckerman, Kris Gevaert","doi":"10.1016/j.mcpro.2025.100907","DOIUrl":"10.1016/j.mcpro.2025.100907","url":null,"abstract":"<p><p>Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, are gaining substantial interest due to their involvement in cellular homeostasis and their contribution to disease pathology. The latter in particular has led to an exponential increase in interest in EVs as they are considered to be circulating packages containing potential biomarkers and are also a possible biological means to deliver drugs in a cell-specific manner. However, several challenges hamper straightforward proteome analysis of EVs as they are generally low abundant and reside in complex biological matrices. These matrices typically contain abundant proteins at concentrations that vastly exceed the concentrations of proteins found in the EV proteome. Therefore, extensive EV isolation and purification protocols are imperative and many have been developed, including (density) ultracentrifugation, size-exclusion, and precipitation methods. Here, we describe filter-aided extracellular vesicle enrichment (FAEVEr) as an approach based on 300 kDa molecular weight cutoff filtration that allows the processing of multiple samples in parallel within a reasonable time frame and at moderate cost. We demonstrate that FAEVEr is capable of quantitatively retaining EV particles on filters, while allowing extensive washing with the mild detergent Tween-20 to remove interfering non-EV proteins. The retained particles are directly lysed on the filter for a complete recovery of the EV protein cargo toward proteome analysis. Here, we validate and optimize FAEVEr on recombinant EV material and apply it on conditioned medium as well as on complex bovine serum, human plasma, and urine. Our results indicate that EVs isolated from MCF7 cells cultured with or without serum have a drastic different proteome because of nutrient deprivation.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100907"},"PeriodicalIF":6.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11872570/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143022968","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":"Molecular Display of the Animal Meta-Venome for Discovery of Novel Therapeutic Peptides.","authors":"Meng-Hsuan Hsiao, Yang Miao, Zixing Liu, Konstantin Schütze, Nathachit Limjunyawong, Daphne Chun-Che Chien, Wayne Denis Monteiro, Lee-Shin Chu, William Morgenlander, Sahana Jayaraman, Sung-Eun Jang, Jeffrey J Gray, Heng Zhu, Xinzhong Dong, Martin Steinegger, H Benjamin Larman","doi":"10.1016/j.mcpro.2024.100901","DOIUrl":"10.1016/j.mcpro.2024.100901","url":null,"abstract":"<p><p>Animal venoms, distinguished by their unique structural features and potent bioactivities, represent a vast and relatively untapped reservoir of therapeutic molecules. However, limitations associated with comprehensively constructing and expressing highly complex venom and venom-like molecule libraries have precluded their therapeutic evaluation via high-throughput screening. Here, we developed an innovative computational approach to design a highly diverse library of animal venoms and \"metavenoms\". We used programmable M13 hyperphage display to preserve critical disulfide-bonded structures for highly parallelized single-round biopanning with quantitation via high-throughput DNA sequencing. Our approach led to the discovery of Kunitz-type domain containing proteins that target the human itch receptor Mas-related G-protein coupled receptor member X4, which plays a crucial role in itch perception. Deep learning-based structural homology mining identified two endogenous human homologs, tissue factor pathway inhibitor (TFPI), and serine peptidase inhibitor, Kunitz type 2 (SPINT2), which exhibit agonist-dependent potentiation of Mas-related G-protein coupled receptor member X4. Highly multiplexed screening of animal venoms and metavenoms is therefore a promising approach to uncover new drug candidates.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100901"},"PeriodicalIF":6.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11833617/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142922071","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":"Multitiered Proteome Analysis Displays the Hyperpermeability of the Rheumatoid Synovial Compartment for Plasma Proteins.","authors":"Eva Maria Stork, Sofia Kalaidopoulou Nteak, Danique M H van Rijswijck, J Mirjam A Damen, Hans Ulrich Scherer, Rene E M Toes, Albert Bondt, Tom W J Huizinga, Albert J R Heck","doi":"10.1016/j.mcpro.2024.100900","DOIUrl":"10.1016/j.mcpro.2024.100900","url":null,"abstract":"<p><p>Rheumatoid arthritis (RA) is characterized by synovial hyperplasia and cartilage/bone destruction. RA affects the synovial joints, the synovial lining, and the permeability of the synovium. As the latter is of central relevance for the distribution of systemically delivered therapeutics into synovial fluid (SF), we here assessed the protein composition of paired plasma and SF of patients diagnosed with RA at three distinct levels of depth using mass spectrometric approaches: the \"total\" proteome, the \"total\" immunoglobulin G1 (IgG1) antibody repertoire, and the RA-specific anticitrullinated protein IgG1 autoantibody repertoire. The SF proteome was found to be dominated in numbers and concentration by plasma proteins, although we additionally detected several cartilage- and neutrophil-derived proteins of lower abundance. Strikingly, the plasma proteins were not only qualitatively reflected in SF but also quantitatively, independent of their size and/or other biochemical features. Also, the synovial \"total\" IgG1 and autoreactive anticitrullinated protein antibody IgG1 repertoire highly resembled the IgG1 repertoires detected in plasma within the same patient. Our comprehensive multilayer data thus reveals that the proteome, including the dominant, most abundant (auto)antibody clones, present in SF of RA patients is a direct reflection of the proteome present in blood, spiked by the local (immune) processes within the RA joint. We thus conclude that proteins directly pass from blood into SF of these joints without substantial bias. These findings thereby not only exemplify the use of in-depth multilayer proteome analyses to revisit basic concepts underlying RA pathology and to monitor the local (immune) processes destructive to cartilage but also provide evidence indicating that (protein-based) therapeutics may equally enter SF of swollen joints and that pharmacokinetic analyses of such therapeutics in blood are directly relevant to the synovial compartment.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100900"},"PeriodicalIF":6.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11821404/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142922072","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":"Early Life Exposure to Deltamethrin Impairs Synaptic Function by Altering the Brain-Derived Extracellular Vesicle Proteome.","authors":"Leandra Koff, Jessica Di Re, Subhash Chand, Yosef Avchalumov, Nghi M Nguyen, Timothy J Baumgartner, Aditya K Singh, Nana A Goode, Mate Marosi, Lance M Hallberg, Bill T Ameredes, Thomas A Green, Sowmya V Yelamanchili, Gurudutt Pendyala, Fernanda Laezza","doi":"10.1016/j.mcpro.2024.100902","DOIUrl":"10.1016/j.mcpro.2024.100902","url":null,"abstract":"<p><p>Pyrethroid pesticides have been associated with neurodevelopmental disorders including attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). While behavioral effects of pyrethroid exposure have been previously reported, the underlying mechanisms remain unclear. Here, we hypothesized that exposure to deltamethrin (DM), a widely used pyrethroid pesticide known for its neurotoxicity during early developmental stages, induces brain dysfunction through alterations in brain-derived extracellular vesicle (BDEV) signaling. Using a well-established rodent model of early life DM exposure within the recommended no observable effect level, we isolated BDEVs from postnatal 30-day-old vehicle-exposed (control) and DM-exposed mice using a differential sucrose density gradient. Following ZetaView nanoparticle tracking and electron microscopy characterization, quantitative mass spectrometry-based proteomics revealed 89 differentially expressed proteins (DEPs) in BDEVs from DM exposed animals compared to control BDEVs. Bioinformatic analysis identified convergence of DEPs on pathways associated with mitochondrial function and synaptic plasticity. PKH67-green conjugated BDEVs derived from either control or DM-exposed mice were bilaterally injected intracerebroventricularly into naive adult mice, and the brain distribution of labeled BDEVs was verified prior to extracellular field recording experiments. Strikingly, long-term potentiation (LTP) at CA3-CA1 hippocampal synapses, a functional correlate of learning and memory, was intact in control BDEVs but absent in naive mice receiving BDEVs from DM exposed mice. Notably, exogenously delivering LRRTM1, one of the DEPs found in DM BDEVs, disrupts synaptic transmission in CA1 neurons consistent with impaired LTP. Thus, differentially regulated signaling in BDEVs represents a novel mechanism of DM neurotoxicity.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100902"},"PeriodicalIF":6.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11847076/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142922070","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":"Chemical Glycoproteomic Profiling in Rice Seedlings Reveals N-glycosylation in the ERAD-L Machinery.","authors":"Cong Lei, Xilong Li, Wenjia Li, Zihan Chen, Simiao Liu, Bo Cheng, Yili Hu, Qitao Song, Yahong Qiu, Yilan Zhou, Xiangbing Meng, Hong Yu, Wen Zhou, Xing Chen, Jiayang Li","doi":"10.1016/j.mcpro.2024.100883","DOIUrl":"10.1016/j.mcpro.2024.100883","url":null,"abstract":"<p><p>As a ubiquitous and essential posttranslational modification occurring in both plants and animals, protein N-linked glycosylation regulates various important biological processes. Unlike the well-studied animal N-glycoproteomes, the landscape of rice N-glycoproteome remains largely unexplored. Here, by developing a chemical glycoproteomic strategy based on metabolic glycan labeling, we report a comprehensive profiling of the N-glycoproteome in rice seedlings. The rice seedlings are incubated with N-azidoacetylgalactosamine-a monosaccharide analog containing a bioorthogonal functional group-to metabolically label N-glycans, followed by conjugation with an affinity probe via click chemistry for the enrichment of the N-glycoproteins. Subsequent mass spectrometry analyses identify a total of 403 N-glycosylation sites and 673 N-glycosylated proteins, which are involved in various important biological processes. In particular, the core components of the endoplasmic reticulum-associated protein degradation machinery are N-glycosylated, and the N-glycosylation is important for the endoplasmic reticulum-associated protein degradation-L function. This work not only provides an invaluable resource for studying rice N-glycosylation but also demonstrates the applicability of metabolic glycan labeling in glycoproteomic profiling for crop species.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100883"},"PeriodicalIF":6.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11869521/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142693221","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":"Improving the Depth and Reliability of Glycopeptide Identification Using Protein Prospector.","authors":"Robert J Chalkley, Peter R Baker","doi":"10.1016/j.mcpro.2025.100903","DOIUrl":"10.1016/j.mcpro.2025.100903","url":null,"abstract":"<p><p>Glycosylation is the most common and diverse modification of proteins. It can affect protein function and stability and is associated with many diseases. While proteomic methods to study most post-translational modifications are now quite mature, glycopeptide analysis is still a challenge, particularly from the aspect of data analysis. Several software packages have been developed in the last few years that aim to support omic-level N-linked glycopeptide analysis. This study presents developments of Protein Prospector for the analysis of N-glycopeptide data. Results are compared to other software, showing that Protein Prospector reports many more glycoforms of glycopeptides than competing software. The advantages and disadvantages of considering glycan adducts are also evaluated, showing how considering them can correct previously wrong assignments.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100903"},"PeriodicalIF":6.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11851224/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142951613","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":"Electrophoresis-Correlative Ion Mobility Deepens Single-Cell Proteomics in Capillary Electrophoresis Mass Spectrometry.","authors":"Bowen Shen, Fei Zhou, Peter Nemes","doi":"10.1016/j.mcpro.2024.100892","DOIUrl":"10.1016/j.mcpro.2024.100892","url":null,"abstract":"<p><p>Detection of trace-sensitive signals is a current challenge in single-cell mass spectrometry (MS) proteomics. Separation prior to detection improves the fidelity and depth of proteome identification and quantification. We recently recognized capillary electrophoresis (CE) electrospray ionization (ESI) for ordering peptides into mass-to-charge (m/z)-dependent series, introducing electrophoresis-correlative (Eco) data-independent acquisition. Here, we demonstrate that these correlations based on electrophoretic mobility (μ_ef) in the liquid phase are transferred into the gas phase, essentially temporally sorting the peptide ions into charge-dependent ion mobility (IM, 1/K₀) trends (ρ > 0.97). Rather than sampling the entire IM region broadly, we pursued these predictable correlations to schedule narrower frames. Compared to classical data-dependent (dda) PASEF, Eco-framing significantly enhanced the resolution of IM MS (IMS) on a trapped IM mass spectrometer (timsTOF PRO). This approach returned ∼50% more proteins from HeLa proteome digests approximating to one-to-two cells, identifying ∼962 proteins from ∼200 pg in <20 min of effective electrophoresis, without match-between-runs. As a proof of principle, we deployed Eco-IMS to detect 1157 proteins by analyzing <4% of the total proteome content in single, yolk-laden embryonic stem cells (∼80-μm) that were isolated from the animal cap of the South African clawed frog (Xenopus laevis). Quantitative profiling of nine different blastomeres revealed detectable differences among these cells, which are normally fated to form the ectoderm but retain pluripotentiality. Eco-framing in the IM dimension effectively deepens the proteome sensitivity in IMS using ddaPASEF, facilitating the proteome-driven classification of differentiating cells, as demonstrated in the chordate frog embryo in this report.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100892"},"PeriodicalIF":6.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11875174/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142824204","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":"Site-Specific Competitive Kinase Inhibitor Target Profiling Using Phosphonate Affinity Tags.","authors":"Wouter van Bergen, Anneroos E Nederstigt, Albert J R Heck, Marc P Baggelaar","doi":"10.1016/j.mcpro.2025.100906","DOIUrl":"10.1016/j.mcpro.2025.100906","url":null,"abstract":"<p><p>Protein kinases are prime targets for drug development due to their involvement in various cancers. However, selective inhibition of kinases, while avoiding off-target effects remains a significant challenge for the development of protein kinase inhibitors. Activity-based protein profiling (ABPP), in combination with pan-kinase activity-based probes (ABPs) and mass spectrometry-based proteomics, enables the identification of kinase drug targets. Here, we extend existing ABPP strategies for kinase profiling with a site-specific analysis, allowing for protein kinase inhibitor target engagement profiling with amino acid specificity. The site-specific approach involves highly efficient enrichment of ABP-labeled peptides, resulting in a less complex peptide matrix, straightforward data analysis, and the screening of over ∼100 kinase active sites in a single LC-MS analysis. The complementary use of both trypsin and pepsin in parallel to generate the ABP-labeled peptides considerably expanded the coverage of kinases and pinpoint the exact binding sites. Using the site-specific strategy to examine the on- and off-targets of the Ephrin receptor (Eph) B4 inhibitor NVP-BHG712 showed binding to EphA2 with an IC₅₀ of 17 nM and EphB4 with an IC₅₀ of 20 nM. Next to the known targets, EphA2 and EphB4, NVP-BHG712 bound to the discoidin domain-containing receptor 1 with an IC₅₀ of 2.1 nM, suggesting that a discoidin domain-containing receptor 1-targeting regio-isomer of NVP-BHG712 was used. The promiscuity of XO44 toward ATP-binding pockets on nonkinase proteins facilitated the screening of additional off-target sites, revealing inosine-5'-monophosphate dehydrogenase 2 as a putative off-target. Expanding the search to other amino acids revealed that XO44, in addition to 745 lysines, also covalently linked 715 tyrosines, which significantly expands the competitive ABPP search space and highlights the added value of the site-specific method. Therefore, the presented approach, which can be fully automated with liquid handling platforms, provides a straightforward, valuable new approach for competitive site-specific kinase inhibitor target profiling.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100906"},"PeriodicalIF":6.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11889359/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008370","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}