Molecular & Cellular Proteomics最新文献

Chronic myeloid leukemia (CML) resistance to BCR-ABL tyrosine kinase inhibitors (TKIs) can arise from ABL kinase domain mutations, BCR-ABL fusion gene amplification, or kinase-independent mechanisms. To investigate imatinib-resistance, we performed quantitative mass spectrometry comparing the proteome and phosphoproteome of K562 cells (a standard CML model) and ImR cells, an imatinib-resistant K562 derivative that also exhibits cross-resistance to second- and third-generation BCR-ABL TKIs. In addition to revealing global proteome and phosphoproteome changes associated with drug resistance, we identified LIN28A-a multi-functional RNA-binding protein-as a critical mediator of imatinib resistance. LIN28A was significantly overexpressed and hyperphosphorylated in ImR cells. Depleting LIN28A via shRNA restored imatinib sensitivity, while its ectopic expression in parental K562 cells induced imatinib resistance. Mechanistically, LIN28A coordinates an extensive kinase-substrate network regulating proliferation, survival, and metabolism to drive resistance. Notably, pharmacological inhibition of LIN28A-dependent kinases (PKC, AKT, SGK1, and RPS6K) suppressed ImR proliferation. Midostaurin, a clinical PKC/FLT3 inhibitor used in FLT3-ITD-positive AML, potently re-sensitized ImR cells to imatinib. Our findings suggest that targeting LIN28A and its downstream effectors, particularly PKC, could overcome resistance to imatinib and next-generation BCR-ABL inhibitors.

In recent years, arboviral infections have surged dramatically due to the geographic expansion of Aedes and Culex mosquitoes, their main vector mosquitoes. Despite significant efforts to uncover arbovirus-host interactions and viral protein effector functions in mammals, systematic studies aiming to characterize virus-vector interactions in arthropods are largely missing, and the functions and cellular targets of many arboviral proteins in mosquitoes remain elusive. Here, we applied a multi-omic approach to systematically evaluate the ability of arboviral capsids to interact with the Ae. aegypti proteome. This extensive multi-modal atlas across 12 pathogenic arboviral species spanning three viral genera revealed shared and distinct host factor specificities, uncovering species-, genus- and vector preference-specific patterns of host usage in mosquitoes. Functional phenotypic screening of 110 newly discovered host proteins across three prototypic arboviruses (La Crosse virus, dengue virus and West Nile virus) identified several novel host dependency factors, including a new role for the chromatin-remodeling Brahma complex in orthoflavivirus replication. Using a combination of biochemical and sequencing approaches, we characterized the cellular determinants of these interactions and profiled their functional consequences on the chromatin landscape. Altogether, this study provides a multi-layered repository to categorize and characterize arboviral capsid effector functions in invertebrates, providing important cues on novel mechanisms of transcriptional regulation via capsid-mediated modulation of chromatin accessibility in insects.

Thyroid cancer comprises a heterogeneous group of malignancies with distinct clinical outcomes and molecular features, including papillary thyroid carcinoma (PTC), poorly differentiated thyroid carcinoma (PDTC), and anaplastic thyroid carcinoma (ATC). This study aimed to delineate the molecular and immune landscapes of these subtypes and identify potential biomarkers for the aggressive forms, ATC and PDTC. We assembled a well-annotated cohort of 120 formalin-fixed paraffin-embedded (FFPE) samples, including 35 ATC, 18 PDTC, 37 PTC cases, and 30 adjacent normal tissues (N) paired with PTC, collected over the past decade from multiple hospitals. To our knowledge, this represents the largest clinical ATC/PDTC cohort subjected to multi-omics profiling and the first comprehensive proteomic analysis of these aggressive thyroid cancers. Using 4D-DIA proteomics on 118 tumors (ATC 34, PDTC 18, PTC 36, and N 30), integrated with total RNA-seq on 69 samples (ATC 10, PDTC 5, PTC 31, and N 23), we revealed substantial molecular similarities between ATC and PDTC, both markedly distinct from PTC and adjacent normal tissues. ATC and PDTC exhibited significant enrichment in immune-related and metabolic pathways, with transcriptomic data indicating aggressive phenotypes and pronounced immunosuppression. Distinct immune landscapes of ATC and PDTC were revealed with neutrophil extracellular trap (NET) formation and M0 macrophage accumulation as key immunosuppressive mechanisms. Notably, Fc fragment of IgG receptor IIa (FCGR2A, or CD32) was identified as a promising biomarker for ATC, implicating a functional link between immune evasion and tumor aggressiveness. Our findings provide a comprehensive molecular and immunological characterization of thyroid cancer subtypes, offering novel insights into the pathogenesis of ATC and PDTC, and identifying potential targets for diagnosis and precision therapy.

Extracellular vesicles (EVs), including exosomes and microvesicles, act as transmitters of various biological signals through cell-cell communication. Although EVs derived from immune response cells have been partially studied, the characteristics of EVs mediated by NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation remain unclear. Here, we characterize inflammatory EVs, termed infosomes, derived from NLRP3 inflammasome-activated macrophages, which play a role in inducing inflammation. Proteomic analysis revealed that EVs production was increased in macrophages with activated NLRP3 inflammasomes and that these EVs were enriched with marker proteins involved in metabolism, membrane structure, and cytoskeletal organization. Furthermore, significantly increased proteins were associated with signaling pathways and biological processes related to immune response, phagocytosis, endocytosis, and neurodegenerative diseases. Crucially, these alterations in EV secretion and molecular composition were dependent on NLRP3 and its subsequent inflammasome activity. Functionally, these infosomes were shown to amplify the expression of inflammatory factors in both macrophages and endothelial cells (HUVECs). These findings provide insights into the biological roles of infosomes, suggesting that EVs generated and loaded by NLRP3 inflammasome activation act as key biological mediators that disseminate and amplify inflammatory responses through cell-cell communication. This highlights their potential as novel biomarkers and therapeutic targets for inflammatory diseases.

Peripartum cardiomyopathy (PPCM) is a rare form of acute heart failure that develops in women toward the end of pregnancy or early postpartum. No effective, specific treatment for PPCM is available and heart transplantation or mechanical circulatory support may be required in severe cases where drug treatment for heart failure is insufficient. The mechanisms through which the disease progresses are not well understood, and despite similar clinical characteristics to dilated cardiomyopathy of other etiologies (nonperipartum cardiomyopathy; NPCM) it is not known how the molecular remodeling differs between these groups. We aimed to provide insight into the human PPCM heart using unbiased methodologies, and to use changes occurring within the heart tissue to facilitate biomarker discovery. We obtained heart tissue from female patients with end-stage disease receiving either heart transplantation or left ventricular assist device implantation, or from organ donors without heart disease as a control group. We performed deep proteomics, single nucleus transcriptomics and spatial transcriptomics, providing a comprehensive map of the molecular phenotype in advanced PPCM compared to both control and NPCM hearts. Consistent with similarities in the clinical phenotypes of PPCM and NPCM, we observed regulation of canonical markers of end-stage heart failure in both PPCM and NPCM hearts in comparison to controls. Among the changes specific to PPCM and that were consistently observed across multiple data types and cohorts was an upregulation of chymase and carboxypeptidase A3, consistent with mast cell proliferation/activation. Analysis of the proteome of peripheral blood serum from a larger cohort of patients with PPCM and controls showed that chymase was strongly predictive of cardiomyopathy in peripartum women. PPCM heart tissue is characterized by increased mast cell proteins chymase and carboxypeptidase A3. Chymase may have clinical utility as a biomarker for the diagnosis of cardiomyopathy in peripartum women.

Acute kidney injury (AKI), characterized by a rapid decline in renal function, has high mortality rates and frequently progresses to chronic kidney disease (CKD). A major contributor to AKI is ischemia-reperfusion injury (IRI). However, the global molecular changes underlying the AKI-to-CKD transition post-IRI remain to be fully elucidated. Using 4D label-free proteomic and phosphoproteomic analyses in a murine unilateral IRI model at 1 hour, 1 day, 3 days, 7 days, and 28 days post-injury, we systematically identified dysregulated proteins, phosphoproteins, and signaling pathways involved in the progression from AKI to CKD. Critically, these analyses consistently revealed the enrichment and sustained activation of NF-κB signaling, a key pathway driving inflammatory and fibrotic responses, across multiple time points. In addition, we identified significant impairment of fatty acid β-oxidation (FAO). Notably, our omics analysis specifically identified the dedicator of cytokinesis (Dock) protein family, with Dock2 emerging as a prime candidate due to its known immune regulatory functions. Dock2 expression showed significant upregulation post-IRI and was found predominantly localized to injured tubular epithelial cells (TECs). Functional validation demonstrated that Dock2 knockdown attenuated pro-inflammatory responses in TECs by inhibiting IKKβ-mediated NF-κB activation in vitro. Consistently, pharmacological inhibition of Dock2 by CPYPP ameliorated renal tubular injury, inflammation, and fibrosis in vivo. To our knowledge, this is the first study to reveal the role and mechanism of Dock2 in the AKI-to-CKD progression post-IRI. In conclusion, our findings delineate molecular mechanisms underpinning the transition from AKI to CKD and nominate Dock2 as a promising therapeutic target for mitigating this process.

High-throughput proteomics is critical for understanding biological processes, enabling large-scale studies such as biomarker discovery and systems biology. However, current mass spectrometry technologies face limitations in speed, sensitivity, and scalability for analyzing large sample cohorts. The Thermo Scientific Orbitrap Astral Zoom mass spectrometer (MS) was developed to address these limitations by improving acquisition speed, ion utilization, and spectral processing, which are all essential for advancing proteome depth in high-throughput proteomics. The Orbitrap Astral Zoom MS achieves ultra-fast MS/MS scan rates of up to 270 Hz with enhanced ion utilization through pre-accumulation, enabling the identification of ∼100,000 unique peptides and >8400 proteins in a single 300 samples-per-day analysis of human cell lysate. The optimized system reduces analysis time by 40%, achieves near-complete proteome coverage (>12,000 proteins) in 2.7 h, and enables ultra-high-throughput workflows, identifying >7000 proteins in a 500 samples-per-day method with exceptional reproducibility (pairwise Pearson correlations >0.99). These advancements establish the Orbitrap Astral Zoom MS among the fastest and most sensitive instruments under the tested conditions, significantly enhancing speed, sensitivity, and scalability, paving the way for routine large-scale proteomics with applications in clinical research and systems biology.

Small extracellular vesicles (sEVs), lipid-bilayer delimited particles (50-200 nm) released by cells, are emerging as a promising class of liquid biopsy biomarkers for elusive cancers, such as high-grade serous ovarian cancer (HGSOC). HGSOC originates from the fallopian tube (FT), progressing from p53 signatures to a precursor lesion known as serous tubal intraepithelial carcinoma (STIC). We hypothesize that sEVs contribute to ovarian cancer pathogenesis, carry cargo reflective of their site of origin, and serve as diagnostic biomarkers for early detection. To test this, we established a case-control cohort using archival plasma samples from 30 HGSOC patients (10 early stage [ES] and 20 late stage [LS]) and 40 healthy controls (HC). sEVs were enriched by size-exclusion chromatography and profiled by LC-MS/MS. Across all samples, 1078 EV-associated proteins (exoproteins) were identified, including 52 upregulated in ES HGSOC versus HC and 59 upregulated in LS HGSOC versus HCs (log₂ fold change >1, p < 0.05). Upregulated EV proteins were prioritized based on FT origin and tissue expression in STIC lesions. Seven candidate biomarkers (MYL6, GSTP1, TTYH3, PRDX6, MUC1, MYH14, and PTGS1) were validated by immunohistochemistry in FT tissue harboring STIC lesions and in HGSOC tissues, as well as by Western blotting in FT/HGSOC cell-derived EVs. These findings suggest that circulating exoproteins upregulated in ES cancer disease reflect precursor lesions. A four-protein combinatorial panel (MUC1, MYL6, TTYH3, and GSTP1), selected using Akaike Information Criterion, yielded an area under the curve (AUC) of 0.975 and 90% sensitivity at 95% specificity for distinguishing ES HGSOC versus HC. In addition, increased MUC1 levels in circulating sEVs were confirmed by immunoassay (AUC = 0.840 for ES HGSOC versus HC; AUC = 0.860 for LS HGSOC versus HC, p < 0.05). In summary, our sEV proteomic analysis of ES HGSOC reveals exobiomarkers associated with early FT lesions, offering a promising avenue for detecting disease while it remains confined to the FT.

Extracellular vesicles (EVs) have gained increasing attention with their intriguing biological functions and their molecular cargoes serving as potential biomarkers for various diseases, including cancers. A relatively lower abundance of EV proteins compared to cellular counterparts necessitates sensitive and accurate quantitative proteomic strategies. Multiplexed proteomics combined with data-independent acquisition (mDIA) has shown promise for improving sensitivity and quantification over traditional DDA and label-free methods. Despite this, mDIA pipelines that utilize various types of spectral libraries and search software suites have not been thoroughly evaluated with EV proteome samples. In this study, we aim to establish a robust mDIA pipeline based on dimethyl labeling for quantitative proteomics of EVs. EVs were isolated using the extracellular vesicle total recovery and purification (EVtrap) technique and processed directly through an on-bead one-pot sample preparation workflow to obtain digested peptides. We evaluated different mDIA pipelines, including library-free and library-based DIA on the timsTOF HT platform. Results showed that library-based DIA, with project-specific spectral libraries generated from StageTip-based fractionation, outperformed other pipelines in protein identification and quantification. We demonstrated for the first time EV proteome landscape changes caused by the IDH1 mutation and inhibitor treatment in intrahepatic cholangiocarcinoma, highlighting the utility of mDIA in EV-based biomarker discovery.

The development of targeted assays that monitor biomedically relevant proteins is an important step in bridging discovery experiments to large scale clinical studies. Targeted assays are currently unable to scale to hundreds or thousands of targets. We demonstrate the generation of large-scale assays using a novel hybrid nominal mass instrument. The scale of these assays is achievable with the Stellar mass spectrometer through the accommodation of shifting retention times by real-time alignment, while being sensitive and fast enough to handle many concurrent targets. Assays were constructed using precursor information from gas-phase fractionation data-independent acquisition (DIA). We demonstrate the ability to schedule methods from orbitrap and linear ion trap acquired gas-phase fractionation DIA library, and compare the quantification of a matrix-matched calibration curve from orbitrap DIA and linear ion trap parallel reaction monitoring (PRM). Two applications of these proposed workflows are shown with a cerebrospinal fluid neurodegenerative disease protein PRM assay and with a Mag-Net enriched plasma extracellular vesicle protein survey PRM assay. In cerebrospinal fluid, our assay targets proteins discovered previously to be associated with Alzheimer's disease in a small independent sample set. For the Mag-Net enriched plasma survey assay, we observe that proteins selected based on their measurement robustness are still able to capture differences in abundance across disease groups in a small sample set. These highlight the application of highly multiplex, targeted protein assays in clinical research.