Project description:Spatial tissue proteomics integrating whole-slide imaging, laser microdissection and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states in (patho)physiology. To be applicable to large patient cohorts and low sample input amounts, including single-cell applications, loss-minimized and streamlined end-to-end workflows are key. We here introduce an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE® robotic system, which has the capacity to process 192 samples in three hours. Following laser microdissection collection directly into the proteoCHIP LF 48 or EVO 96 chip, our optimized protocol facilitates lysis, formalin de-crosslinking and tryptic digest of low-input archival tissue samples. The seamless integration with the Evosep ONE LC system by centrifugation allows ‘on-the-fly’ sample clean-up, particularly pertinent for laser microdissected workflows. We validate our method in human tonsil archival tissue, where we profile proteomes of spatially-defined B-cell, T-cell and epithelial microregions of 4,000 µm2 to a depth of ~2,000 proteins and with high cell type specificity. We finally provide detailed equipment templates and experimental guidelines for broad accessibility.
Project description:The experiment was performed to identify autophagy targets in wildtype and autophagy-deficient forebrain excitatory neurons. Therefore, neurons were isolated from the cortex, hippocampus and striatum of 2-3 weeks old Atg5flox/flox:CamKIIα-Cretg/wt:tdTomato+ (KO) and Atg5wt/wt:CamKIIα-Cretg/wt:tdTomato+ (WT) mice. Neurons in suspension were FACS sorted and excitatory forebrain neurons expressing tdTomato were forwarded to global proteome analysis assessed by LC-MS/MS.
Project description:Spatial tissue proteomics combining microscopy-based cell phenotyping with ultrasensitive mass spectrometry (MS)-based proteomics is an emerging and powerful concept for the study of cell function and heterogeneity in health and disease. However, optimized workflows that preserve morphological information for image-based phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected archival tissue, are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low input formalin-fixed, paraffin-embedded (FFPE) material. Benchmarking in the murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions with high quantitative reproducibility. Applied to human tonsil, we profiled 146 microregions including spatially defined T and B lymphocyte niches and quantified cell-type specific markers, cytokines, immune cell regulators and transcription factors. These rich data also highlighted proteome dynamics in spatially defined zones of activated germinal centers, illuminating sites undergoing active B-cell proliferation and somatic hypermutation. Our results demonstrate the power of spatially-resolved proteomics for tissue phenotyping by integrating high-content imaging, laser microdissection, and ultrasensitive mass spectrometry. This approach has broad implications for a wide range of biomedical applications, including early disease profiling, drug target discovery and biomarker research.
Project description:The glucocorticoid receptor (GR) is a crucial drug target in multiple myeloma as its activation with glucocorticoids effectively triggers myeloma cell death. However, as high-dose glucocorticoids are also associated with deleterious side effects, novel approaches are urgently needed to improve GR’s action in myeloma. Here we reveal a functional crosstalk between GR and the mineralocorticoid receptor (MR) that culminates in improved myeloma cell killing. We show that the GR agonist Dexamethasone (Dex) downregulates MR levels in a GR-dependent way in myeloma cells. Co-treatment of Dex with the MR antagonist Spironolactone (Spi) enhances Dex-induced cell killing in primary, newly diagnosed GC-sensitive myeloma cells, while in a relapsed GC-resistant setting, Spi alone induces distinct myeloma cell killing. On a mechanistic level, we find that a GR-MR crosstalk is arising from an endogenous interaction between GR and MR in myeloma cells. Quantitative dimerization assays show that Spi reduces Dex-induced GR-MR heterodimerization and completely abolishes Dex-induced MR MR homodimerization but leaves GR-GR homodimerization intact. Unbiased transcriptomics further reveals that c-myc and many of its target genes are downregulated most by Dex and Spi combined, while proteomics analyses identify that several metabolic hallmarks are modulated most by this combination treatment. Finally, we identified a subset of Dex+Spi downregulated genes and proteins that may predict prognosis in the CoMMpass patient cohort. Our study demonstrates that GR-MR crosstalk is therapeutically relevant in myeloma as it provides novel strategies towards glucocorticoid-based dose-reduction.
Project description:The combination of short liquid chromatography (LC) gradients and data independent acquisition (DIA) by mass spectrometry (MS) has proven its huge potential for high-throughput proteomics. However, the optimization of isolation window schemes resulting in a certain number of data points per peak (DPPP) is understudied, although it is one of the most important parameters for the outcome of this methodology. In this study, we show that substantially reducing the number of data points per peak (DPPP) for short gradient DIA massively increases protein identifications while maintaining quantitative precision. This is due to a large increase in the number of precursors identified, which keeps the number of data points per protein almost constant even at long cycle times. When proteins are inferred from its precursors, quantitative precision is maintained at low DPPP while greatly increasing proteomic depth. This strategy enabled us quantifying 6018 HeLa proteins (> 80,000 precursor identifications) with coefficients of variation below 20% in 30 min using a Q Exactive HF, which corresponds to a throughput of 29 samples per day. This indicates that the potential of high-throughput DIA-MS has not been fully exploited yet.
Project description:Background: Primary graft dysfunction (PGD) remains a challenge to lung transplantation (LTx) recipients as a leading cause of poor early outcomes. New methods are needed for the rapid detection of PGD and the measurement of particle flow rate (PFR) from exhaled breath is a novel means to monitor disease. Methods: 22 recipient pigs underwent orthotopic left LTx and were evaluated for PGD on the third post-operative day. Exhaled breath particles (EBPs) and PFR were measured on mechanical ventilation. EBPs were evaluated with mass spectrometry and the proteome was compared to tissue biopsies and bronchoalveolar lavage fluid (BALF). Findings were confirmed in EBPs from 11 human transplant recipients. Results: 9 recipients developed PGD and had significantly higher PFR (686.4 (449.7-8824.0) particles per minute (ppm)) compared to recipients without PGD (116.6 (79.7-307.4) ppm, p=0.0005). From proteomic analysis, porcine and human EBP proteins recapitulated the BAL and adherens and tight junction proteins were underexpressed in PGD tissue. Conclusions: Histological and proteomic analysis found significant changes to the alveolar-capillary barrier to explain the increased PFR in recipients with PGD. Combined with the similarity of proteomic profiles between EBPs and BALF, exhaled breath measurement is proposed as a rapid and non-invasive bedside measurement of PGD.
Project description:Embryonic stem (ES) cells and embryos reversibly pause via chemical mTOR inhibition. In this study, we investigate the tissue-specific response to mTORi-induced pausing in ES and trophoblast stem (TS) cells. To resolve the sequential rewiring of the proteome, we conducted a time-series proteomics experiment at 1, 3, 6, 12, 24, and 48 hours upon induction of pausing, and at 1, 3, 6, 12, 24, and 48 hours upon release of pausing in ES and TS cells. We find that ES, but not TS cells pause reversibly. To optimise developmental pausing conditions, we reasoned that by understanding the difference in pausing response of ES and TS cells, we could identify which pathways are essential for pausing. We found that KEGG pathways related to amino acid degradation, fatty acid degradation, and DNA repair are upregulated in ES cells, but downregulated in TS cells during entry into pausing. Moreover, by targeted metabolomics, we found a depletion of short chain carnitines in the paused ES cells. To extend the length of developmental pausing, we supplemented paused embryos with L-carnitine. The L-carnitine supplementation facilitates lipid usage and prolongs the pausing length by 19 days through the establishment of a more dormant state.
Project description:Hyperactive TLR7 signaling has long been appreciated as a driver of autoimmune disease in mouse models by breaking tolerance to self-nucleic acids1-5. Recently, the first monogenic mutations within TLR7 or its associated regulator Unc93b16,7 have been identified as causative agents of human lupus. The unifying feature of these mutations is TLR7 gain-of-function resulting from increased ligand binding. TLR7 is an intracellular transmembrane receptor, localized to late endosomes, that senses RNA breakdown products within these hydrolytic compartments8,9. Hence, its function depends on a complex interplay between specialized organelles, transport mechanisms and membrane interactions. Whether perturbations of any of these endosome-related processes can give rise to TLR7 gain-of-function and facilitate self-reactivity has not been investigated. Here we show that a dysregulated endosomal compartment can result in TLR7 gain-of-function and lupus disease in humans. Mechanistically, the late endosomal protein complex BORC-Arl8b controls TLR7 protein levels by mediating the receptor's final sorting step towards lysosomal degradation. A direct interaction between Arl8b and Unc93b1 is required to regulate the turnover of TLR7. We identified an amino acid insertion in Unc93b1 in a patient with childhood-onset lupus, which results in loss of interaction with the BORC-Arl8b complex and an accumulation of functional TLR7. Our results highlight the importance of an intact endomembrane system to prevent autoimmune disease. Disrupting the proper progression of TLR7 through its endocytic life cycle is sufficient to break immunological tolerance to nucleic acids. Our work expands the repertoire of cellular mechanisms important to restrict pathological TLR7 activity. Identifying and stratifying lupus patients based on a TLR7-driven pathology opens the way for precision medicine specifically targeting TLR7.
Project description:The first macrophages that seed the developing heart originate from the yolk sac during fetal life. While murine studies reveal important homeostatic and reparative functions in adults, we know little about their roles in the earliest stages of human heart development due to a lack of accessible tissue. Generation of bioengineered human cardiac microtissues from pluripotent stem cells models these first steps in cardiac tissue development, however macrophages have not been included in these studies. To bridge these gaps, we differentiated human embryonic stem cells (hESCs) into primitive LYVE1+ macrophages (hESC-macrophages; akin to yolk sac macrophages) that stably engrafted within cardiac microtissues composed of hESC-cardiomyocytes and fibroblasts to study reciprocal interactions. Engraftment induced a tissue resident macrophage gene program resembling human fetal cardiac macrophages, enriched in efferocytic pathways. Functionally, hESC-macrophages induced production and maturation of cardiomyocyte sarcomeric proteins, and enhanced contractile force, relaxation kinetics, and electrical properties. Mechanistically, the primary effect of hESC-macrophages was during the stressful events surrounding early microtissue formation, where they engaged in phosphatidylserine dependent ingestion of apoptotic cardiomyocyte cargo, which reinforced core resident macrophage identity, reduced microtissue stress and drove hESC-cardiomyocytes to become more similar to human ventricular cardiomyocytes found in early development, both transcriptionally and metabolically. Inhibiting efferocytosis of hESC-cardiomyocytes by hESC-macrophages led to increased cell stress, impaired sarcomeric protein maturation and reduced cardiac microtissue function (contraction and relaxation). Taken together, macrophage-engineered human cardiac microtissues represent a considerably improved model for human heart development, and reveal a major beneficial, yet previously unappreciated role for human primitive macrophages in enhancing cardiac tissue function.
Project description:The first macrophages that seed the developing heart originate from the yolk sac during fetal life. While murine studies reveal important homeostatic and reparative functions in adults, we know little about their roles in the earliest stages of human heart development due to a lack of accessible tissue. Generation of bioengineered human cardiac microtissues from pluripotent stem cells models these first steps in cardiac tissue development, however macrophages have not been included in these studies. To bridge these gaps, we differentiated human embryonic stem cells (hESCs) into primitive LYVE1+ macrophages (hESC-macrophages; akin to yolk sac macrophages) that stably engrafted within cardiac microtissues composed of hESC-cardiomyocytes and fibroblasts to study reciprocal interactions. Engraftment induced a tissue resident macrophage gene program resembling human fetal cardiac macrophages, enriched in efferocytic pathways. Functionally, hESC-macrophages induced production and maturation of cardiomyocyte sarcomeric proteins, and enhanced contractile force, relaxation kinetics, and electrical properties. Mechanistically, the primary effect of hESC-macrophages was during the stressful events surrounding early microtissue formation, where they engaged in phosphatidylserine dependent ingestion of apoptotic cardiomyocyte cargo, which reinforced core resident macrophage identity, reduced microtissue stress and drove hESC-cardiomyocytes to become more similar to human ventricular cardiomyocytes found in early development, both transcriptionally and metabolically. Inhibiting efferocytosis of hESC-cardiomyocytes by hESC-macrophages led to increased cell stress, impaired sarcomeric protein maturation and reduced cardiac microtissue function (contraction and relaxation). Taken together, macrophage-engineered human cardiac microtissues represent a considerably improved model for human heart development, and reveal a major beneficial, yet previously unappreciated role for human primitive macrophages in enhancing cardiac tissue function.