Molecular & Cellular Proteomics最新文献

{"title":"High-Throughput Acoustic FFPE Proteomics Reveals ATP5IF1-Associated Mitochondrial Alterations in Acral Melanoma.","authors":"Diana Lashidua Fernández-Coto, Marisol Ayala, Ramiro Alonso, Alicia Palmqvist, Ma Fernanda García, Scarlett F Balderas-López, Henriett Oskolas, Lukas Christersson, Livia Fulöp, Balazs Szigeti, Victor H Olivera-Rodríguez, Matilda Marko-Varga, Peter Horvatovich, Johan Malm, Leticia Szadai, Roberto Herrera-Goepfert, Attila Marcell Szasz, Sergio Encarnación-Guevara, György Marko-Varga, Jeovanis Gil","doi":"10.1016/j.mcpro.2026.101620","DOIUrl":"10.1016/j.mcpro.2026.101620","url":null,"abstract":"<p><p>Formalin-fixed, paraffin-embedded (FFPE) archives underpin dermatopathology and translational oncology, enabling clinically annotated melanoma cohorts, but cohort-scale proteomics remains limited by labor and variability in upstream processing. We established a plate-scale, acoustic FFPE proteomics workflow and integrated it with AI-assisted digital pathology to support composition-aware molecular profiling from routine sections. The optimized pipeline reduces handling steps, is designed to improve reproducibility, and supports rapid parallel processing in a 96-well format. Deep data-independent acquisition MS of 40 primary melanomas spanning acral lentiginous, lentigo maligna, superficial spreading, and nodular subtypes quantified more than 8200 protein groups and a mean of 5200 proteins per tumor. Proteome profiles resolved melanoma subtypes and defined an acral lentiginous melanoma program enriched for translation/biogenesis and extracellular matrix/adhesion processes with relative depletion of lipid and fatty-acid metabolism and peroxisomal pathways. Supervised feature selection highlighted tenascin, periostin, eukaryotic initiation factor 4A-I, and ADP-ribosylation factor 4 and uncovered selective depletion of ATPase inhibitor, mitochondrial (ATP5IF1), a regulator of mitochondrial ATP synthase, in acral lentiginous melanoma. ATP5IF1 depletion persisted after adjustment for mitochondrial proxies and QuPath-derived tumor content, and coincided with higher glycolysis relative to Complex V. This pathology-integrated, high-throughput FFPE proteomics framework enables scalable retrospective discovery and suggests subtype-specific metabolic alterations consistent with mitochondrial remodeling in an underrepresented melanoma subtype.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101620"},"PeriodicalIF":6.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13452992/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148437468","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":"Proteoform Detection Across 17,000 Single Nuclei From Differentiating Skin Keratinocytes.","authors":"Vijaya Lakshmi Kanchustambham, Indira Pla, Tian Xu, Pei Su, Nickolas P Fisher, Michael A R Hollas, Paulina Markiv, Uday Agarwal, Monika Lysakowska, Jared O Kafader, Xiaomin Bao, Neil L Kelleher","doi":"10.1016/j.mcpro.2026.101621","DOIUrl":"10.1016/j.mcpro.2026.101621","url":null,"abstract":"<p><p>Advances in single-nucleus RNA sequencing have demonstrated advantages over single-cell transcriptomics by enabling capture of dynamic cellular states and transcriptional activity in rare or difficult-to-isolate cell types. To achieve analogous enhancements in intact protein profiling, we applied single-cell proteoform imaging mass spectrometry (scPiMS) to nuclei for label-free, proteoform-resolved analysis of >10⁴ human skin cells. Using scPiMS, we surveyed ∼400 proteoforms across over 17,000 single nuclei isolated from human epidermal keratinocytes spanning undifferentiated (UD) proliferative to differentiated (DF) states, generating single-nucleus proteoform maps of epidermal differentiation. Proteoforms were assigned using intact mass tag matching to a reference proteoform library generated from bulk top-down LC-MS/MS analyses of UD and DF nuclei and subsequently applied to single-nucleus proteoform assignment scores. Unsupervised clustering of single-nucleus proteoform imaging mass spectrometry (snPiMS) data resolved 12 distinct proteoform-defined clusters spanning a continuum from UD proliferative states to terminal differentiation. A progenitor-enriched cluster (cluster 4) exhibited elevated high-mobility group-17, acetylated H2A/H2B variants, and H2A.Z, consistent with open and developmentally poised chromatin. An early differentiating population (cluster 3) was marked by histone H3 bearing H3K4 acetylation together with H3K9 monomethylation, indicative of transcriptional activation. Progressive histone H4 methylation (H4K20me1, H4K20me2, and H4K20me3) reflected cell cycle-coupled modification of newly synthesized H4 in progenitor-like nuclei prior to differentiation, corresponding to clusters 5, 2, and 8, respectively. Cluster 1 was enriched in H3 proteoforms bearing H3K4me3 together with H3K9me1, consistent with an active yet transcriptionally poised chromatin state. Differentiation-committed states (cluster 0) exhibited H3 proteoforms containing H3K4 and H3K9 acetylation together with H3K36me2 reflecting transcriptionally active chromatin. Bulk histone post-translational modification profiling corroborated these trends, with UD nuclei enriched in H3K9me2 and H4K20me1 and DF nuclei enriched in H4K20me2, H3K79me2, H3K27me2/3, and H3K36me3. Together, snPiMS uniquely resolves combinatorial histone proteoforms within individual nuclei, revealing a continuous chromatin trajectory across epidermal differentiation.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101621"},"PeriodicalIF":6.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13487837/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148447988","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":"Exploring the CD38-Associated Surfaceome via Nanobody-Targeted TurboID.","authors":"Kangze Feng, Ying Wei, Shangcheng Yu, Wenxuan Hou, Meng Han, Yuling Chen, Yuxiang Tang, Haiteng Deng","doi":"10.1016/j.mcpro.2026.101623","DOIUrl":"10.1016/j.mcpro.2026.101623","url":null,"abstract":"<p><p>Cluster of differentiation (CD38) is a cell-surface glycoprotein whose molecular functions are closely linked to its surrounding protein environment. To date, omics-level understanding of CD38-associated surface proteins remains lacking. To investigate the CD38-proximal surfaceome in live adherent cell culture, we developed nanobody-targeted TurboID (NBID), a proximity labeling approach using protein of interest-specific nanobody-TurboID chimeras, and applied this approach to characterize CD38-proximal proteome on the cell surface. The approach was validated using epidermal growth factor receptor-targeting NTC construct, which recovered multiple known epidermal growth factor receptor-associated proteins. CD38-specific NTC was applied to A549 lung cancer cells and Tohoku Hospital Pediatrics-1 monocytic leukemia cells, and enriched multiple proteins involved in adhesion, ECM organization, and lipid raft-associated membrane domains. In A549 cells, proteins related to Wnt signaling were additionally enriched. NAD⁺ treatment did not significantly alter the CD38-proximal surfaceome in A549 cells. Total internal reflection fluorescence microscopy further demonstrated coclustering of several enriched candidates with CD38 on membrane protrusions. Functional assays showed that CD38 contributes to transendothelial migration of A549 cells. By combining NBID with stable isotope labeling by amino acids in cell culture, we further identified CD38-associated cadherin adhesion complexes involving N-cadherin and desmoglein-2 at the tumor-endothelial interface. Together, these results establish NBID as a robust platform for investigating surface protein environments and intercellular protein neighborhoods under native conditions. Furthermore, our findings reveal a CD38-associated membrane adhesion network and provide new insight into the role of CD38 in tumor cell migration and tumor-endothelial interactions.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101623"},"PeriodicalIF":6.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13486792/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148448023","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":"Multidimensional Proteomics Reveals the Pro-apoptotic Mechanism of Platycodin D: Targeting RFC4 to Regulate the Notch Signaling Axis in Non-Small Cell Lung Cancer.","authors":"Hongyu Zhang, Baojin Zhou, Jingwen Liu, Yan Ren","doi":"10.1016/j.mcpro.2026.101587","DOIUrl":"10.1016/j.mcpro.2026.101587","url":null,"abstract":"<p><p>Platycodin D (PD), a major bioactive saponin isolated from the traditional Chinese medicine Platycodon grandiflorus, has shown promising therapeutic potential against non-small cell lung cancer (NSCLC). However, the functional mechanisms of PD in NSCLC progression remains unclear. This study aimed to explore the pharmacological mechanism of PD against NSCLC. Thermal proteome profiling approach, molecular docking, cellular thermal shift assay and peptide-centric local stability assay were employed to identify the potential binding target of PD. Subsequent Western Blot and immunoprecipitation-Western Blot experiments were conducted to investigate the downstream signaling pathways of the target. Furthermore, proteomic and ubiquitinomic profiling of PD-treated cells were performed to investigate its functions on global. replication factor C subunit 4 (RFC4) was identified as a potential binding target of PD by thermal proteome profiling and their binding sites were further exposed by peptide-centric local stability assay. PD-RFC4 complex promotes the degradation of Notch1 and Notch3 by reducing nuclear entry of their domains. Compared with control treatment, the differentially expressed proteins induced by PD were found to be primarily involved in ferroptosis, ubiquitination, platinum drug resistance, and ribosome-related processes. The ubiquitin proteome analysis revealed that proteins associated with the Notch pathway underwent ubiquitin modifications. PD binds to RFC4 and inhibits its activity, leading to downregulation of the Notch signaling pathway, ultimately triggering cancer cell apoptosis. PD is a natural product with potential therapeutic value for NSCLC.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101587"},"PeriodicalIF":6.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13469972/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147959589","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":"Label-Free Targeted Proteomics Data Analysis Workflow Selection - Benchmarking AI-based and Data-Driven Approaches.","authors":"Daniel Fochtman, Łukasz Marczak, Monika Pietrowska, Joanna Polańska, Anna Wojakowska","doi":"10.1016/j.mcpro.2026.101631","DOIUrl":"10.1016/j.mcpro.2026.101631","url":null,"abstract":"<p><p>As global proteomics continues to advance, the number of identifiable proteins has increased substantially. However, this does not inherently ensure optimal quantitative performance. While targeted assays using isotope-labeled peptides can be developed, label-free strategies remain an attractive and cost-efficient option for methodological validation. Yet, systematic evaluations of data analysis workflows for label-free targeted proteomics, particularly those incorporating artificial intelligence (AI)-based tools, are still limited. Therefore, this study aimed to benchmark multiple data analysis approaches for label-free targeted proteomics as a validation framework for results obtained from global analyses. Missing-data imputation (MDI) strategies, including no MDI, k-nearest neighbors, data-driven, MSstats and AI-based methods, were evaluated, alongside consolidation and testing frameworks such as mathematical summation, best-peak selection, AI-based scaling with univariate statistics, p-value integration, MSstats Tukey's median polish or linear models and multivariate testing. Data-driven MDI combined with p-value integration consistently showed the strongest performance across accuracy, precision, specificity, and false-discovery rate, outperforming all other strategies. These findings demonstrate that careful selection of data analysis workflows can yield substantially improved quantitative outcomes compared with commonly used approaches such as simple mathematical summation. Although our conclusions are based on a controlled benchmarking dataset comprising three yeast proteins spiked into a constant human background using only one targeted approach, they can be generally applied as a default workflow for biomarker validation using the label-free approach.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101631"},"PeriodicalIF":6.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13520092/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579353","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":"Platelet Proteome Links Metabolism to Reactivity in Essential Thrombocythemia.","authors":"Xiomara Guerrero-Carreño, Sanne Smits, Alfonso Esteban Lasso, Martina Samiotaki, Virtu Calabuig-Navarro, Francisco José Iborra, Frida Rantanen, Alberto Álvarez-Larrán, Anna Angona Figueras, Beatriz Bellosillo, Adolfo J Sáez Marín, Valentín García-Gutiérrez, Dick H W Dekkers, Jeroen A A Demmers, Francisca Ferrer-Marín, Juan Carlos Hernández-Boluda, Antonios Matsakas, Celina Benavente Cuesta, Peter Vandenberghe, Petros Papadopoulos","doi":"10.1016/j.mcpro.2026.101617","DOIUrl":"10.1016/j.mcpro.2026.101617","url":null,"abstract":"<p><p>Essential thrombocythemia (ET) is a myeloproliferative neoplasm in which JAK2, CALR, and MPL mutations are associated with distinct clinical phenotypes. Here, we analyzed the platelet proteome of ET patients by MS, combined with functional assays, to investigate platelet activation. Compared with healthy controls, ET platelets showed altered abundance of mitochondrial and metabolic proteins, including reduced levels of tricarboxylic acid cycle enzymes and a relative enrichment of glycolytic signatures. Acetylsalicylic acid decreased the abundance of metabolic proteins in CALR Type1 platelets, whereas JAK2 V617F platelets showed only minor, heterogeneous increases restricted to a subset of proteins and samples. In functional assays, JAK2 V617F platelets showed aggregation responses similar to or lower than those of healthy controls, whereas untreated CALR Type2 platelets showed higher CD62P expression, indicating a more activated phenotype than the other ET groups. These findings indicate that ET platelets display mutation-associated proteomic and functional differences and suggest that an altered metabolic state may contribute to platelet reactivity.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101617"},"PeriodicalIF":6.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13450191/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148352453","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":"Genetic Rescue of Disrupted Synaptic Protein Interaction Network Dynamics Following SYNGAP1 Reactivation.","authors":"Vera Stamenkovic, Felicia Harsh, Breann Kniffen, Gavin Rumbaugh, Stephen E P Smith","doi":"10.1016/j.mcpro.2026.101614","DOIUrl":"10.1016/j.mcpro.2026.101614","url":null,"abstract":"<p><p>Synaptic protein interaction networks (PINs) dynamically translate neural activity into biochemical signals that regulate synaptic structure and plasticity. Disruption of these coordinated networks is a common feature of autism spectrum disorder (ASD) risk genes, yet it remains unclear whether the molecular organization of a perturbed network can be restored after development. Here, we examined how post-developmental re-expression of the synaptic Ras GTPase-activating protein SynGAP1 affects network structure and signaling dynamics in a conditional SynGAP1 haploinsufficient mouse. Quantitative multiplex co-immunoprecipitation (QMI) across development revealed that SynGAP haploinsufficiency selectively reduced SynGAP-containing complexes without broadly disrupting NMDA-dependent network responses. Tamoxifen-inducible re-expression of SynGAP at postnatal day 21 fully restored both steady-state and activity-dependent interactions within the SynGAP module in hippocampus, and additionally normalized secondary alterations in Shank-Homer scaffolding complexes in somatosensory cortex. These data demonstrate that biochemical restoration of a disrupted synaptic network is achievable, even after early developmental windows have closed. Our findings suggest that while critical periods may constrain functional recovery, molecular network normalization remains possible through genetic reactivation of haploinsufficient synaptic regulators.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101614"},"PeriodicalIF":6.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13427445/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148339351","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":"Proteome Profiling Reveals NQO2 Activity Contributing to Proteasome Inhibitor Resistance in Multiple Myeloma Cell Lines.","authors":"Chien-Yun Lee, Seungbin Han, Larissa Haertle, Umair Munawar, Cornelia Vogt, Johanna Streubel, Andrej Besse, Lenka Besse, Christoph Driessen, Hermann Einsele, Johannes Waldschmidt, Martin Kortüm, Bernhard Kuster","doi":"10.1016/j.mcpro.2026.101615","DOIUrl":"10.1016/j.mcpro.2026.101615","url":null,"abstract":"<p><p>Proteasome inhibitors (PIs) are frontline therapies for multiple myeloma (MM). Although MM patients initially respond to PIs, resistance frequently emerges. While all PIs nominally target the same proteasomal catalytic subunit (PSMB5), the extent to which resistance mechanisms are the same or different among different PIs or between patients is poorly understood. To address this, we performed proteome and phosphoproteome profiling of 12 MM cell line models, comprising four parental lines (AMO-1, ARH77, L363, and RPMI8226) paired with lines that acquired resistance to bortezomib (BTZ) or carfilzomib (CFZ). Over 7000 proteins and up to 10,000 phosphopeptides were identified per cell line, enabling a comprehensive comparative analysis of shared and cell line-specific resistance signatures at the protein level. Data analysis revealed surprisingly few changes in the phosphoproteome but substantial reprogramming of the proteome in most models. Beyond known adaptations such as the overexpression of the PI target PSMB5 and the drug efflux transporter ABCB1, we identified the oxidoreductases NQO1 and NQO2 as significantly upregulated proteins under chronic proteotoxic stress across several models. Pharmacological follow up in PI resistant AMO-1 cells showed that NQO2 inhibition by imatinib fully restored CFZ sensitivity, validating NQO2 as a contributor to resistance formation in this model system.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101615"},"PeriodicalIF":6.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13396865/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148339330","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":"Alovudine Targets mtDNA-dependent Proteins Encoded by Genes Recurrently Amplified in Metastatic Breast Cancer Cohorts.","authors":"Jessica Brandi, Gloria Gatto, Federica Sotgia, Michael P Lisanti, Daniela Cecconi","doi":"10.1016/j.mcpro.2026.101622","DOIUrl":"10.1016/j.mcpro.2026.101622","url":null,"abstract":"<p><p>Previously, we demonstrated that high levels of mitochondrial DNA (mtDNA) were functionally associated with \"stemness\" and aggressive phenotypic behaviors in human breast cancer cells, including spontaneous metastasis. More specifically, we showed that treatment with Alovudine induced mtDNA-depletion in MDA-MB-231 cells and prevented their ability to form colonies in vitro and metastasize in vivo. To better understand the underlying mechanism(s) and identify candidate mtDNA-dependent mitochondrial protein biomarkers relevant to metastatic breast cancer, Alovudine-treated MDA-MB-231 cells were subjected to proteomics analysis. For comparison purposes, mtDNA-depleted cells (MDA-MB-231 and MCF-7) were generated and also subjected to proteomics analysis. Intersection of these three distinct data sets revealed that a small number of proteins were commonly downregulated in mtDNA-depleted cells and Alovudine-treated cells. Remarkably, many of these nuclear-encoded mitochondrial genes (>20) exhibited recurrent genomic amplification across metastatic breast cancer cohorts. Therefore, the action of a single drug, namely Alovudine, was sufficient to effectively suppress the expression of a large number of mitochondrial proteins associated with i) gene amplification and ii) cancer cell metastasis, in human breast cancer patients. These findings may have important clinical implications for the development of new therapeutics targeting advanced breast cancer.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101622"},"PeriodicalIF":6.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13486814/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148447967","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":"Integrated Multi-Omics Analysis Uncovers Molecular Dysregulation in Glucosylceramide Synthase-Deficient Human Induced Pluripotent Stem Cells.","authors":"Julia Beimdiek, Karsten Cirksena, Charlotte Rossdam, Smilla Brand-Merseburger, Astrid Oberbeck, Philippe V Barbosa, Ana Tzvetkova, Malte Juchem, Christian Bär, Thomas Thum, Axel Schambach, Andreas Pich, Britta Brügger, Andreas W Kuss, Falk F R Buettner","doi":"10.1016/j.mcpro.2026.101630","DOIUrl":"10.1016/j.mcpro.2026.101630","url":null,"abstract":"<p><p>Glycosphingolipids are membrane lipids characterized by highly diverse glycosylation patterns. They are involved in numerous cellular processes and can be linked to diseases. With the aim to investigate their role in early mammalian developmental processes in vitro, we knocked out the UDP-glucose ceramide glucosyltransferase gene (UGCG) encoding the enzyme glucosylceramide synthase (UGCG) in human induced pluripotent stem cells (hiPSCs) by CRISPR/Cas9. The functional impairment of UGCG was confirmed by glycomic profiling. UGCG KO hiPSCs displayed normal morphology, growth behavior, and expression of stem cell markers compared to WT hiPSCs. Furthermore, the KO cells maintained pluripotency, as evidenced by their capacity for in vitro differentiation into all three embryonic germ layers and their ability to form teratomas in vivo. Quantitative proteomic analysis of explanted teratoma derived from WT and UGCG KO hiPSCs revealed differential expression of numerous proteins, with notable enrichment of pathways associated with signal transduction or protein localization and transport. Phosphorylation profiling of key kinases and their targets uncovered reduced activation of STAT family transcription factors in UGCG KO hiPSCs. Complementary transcriptomic profiling combined with Ingenuity Pathway Analysis of differentially expressed genes between WT and UGCG KO hiPSCs, as well as their ectodermal derivatives, revealed a predicted positive enrichment of proteins associated with signaling pathways in UGCG KO hiPSCs. Global lipidomic profiling showed a significant increase in sphingomyelin levels in UGCG KO hiPSCs and their ectodermally differentiated derivatives, whereas total ceramide content remained comparable between UGCG KO and WT cells. In summary, despite the absence of overt phenotypic changes, our findings demonstrate that glycosphingolipid deficiency induces multiple molecular perturbations in hiPSCs.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101630"},"PeriodicalIF":6.3,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13520899/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579364","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}