ABSTRACT
Proteins carry out the vast majority of functions in all biological domains, but for technological reasons their large-scale investigation has lagged behind the study of genomes. Since the first essentially complete eukaryotic proteome was reported1, advances in mass-spectrometry-based proteomics2 have enabled increasingly comprehensive identification and quantification of the human proteome3-6. However, there have been few comparisons across species7,8, in stark contrast with genomics initiatives9. Here we use an advanced proteomics workflow-in which the peptide separation step is performed by a microstructured and extremely reproducible chromatographic system-for the in-depth study of 100 taxonomically diverse organisms. With two million peptide and 340,000 stringent protein identifications obtained in a standardized manner, we double the number of proteins with solid experimental evidence known to the scientific community. The data also provide a large-scale case study for sequence-based machine learning, as we demonstrate by experimentally confirming the predicted properties of peptides from Bacteroides uniformis. Our results offer a comparative view of the functional organization of organisms across the entire evolutionary range. A remarkably high fraction of the total proteome mass in all kingdoms is dedicated to protein homeostasis and folding, highlighting the biological challenge of maintaining protein structure in all branches of life. Likewise, a universally high fraction is involved in supplying energy resources, although these pathways range from photosynthesis through iron sulfur metabolism to carbohydrate metabolism. Generally, however, proteins and proteomes are remarkably diverse between organisms, and they can readily be explored and functionally compared at www.proteomesoflife.org.
Subject(s)
Classification , Deep Learning , Peptides/chemistry , Peptides/isolation & purification , Proteome/chemistry , Proteome/isolation & purification , Proteomics/methods , Animals , Bacteroides/chemistry , Bacteroides/classification , Carbohydrate Metabolism , Chromatography , Glycolysis , Homeostasis , Ion Transport , Iron-Sulfur Proteins/metabolism , Oxidation-Reduction , Photosynthesis , Protein Biosynthesis , Protein Folding , Proteolysis , Species SpecificityABSTRACT
BACKGROUND & AIMS: Colonic adenomatous polyps, or adenomas, are frequent precancerous lesions and the origin of most cases of colorectal adenocarcinoma. However, we know from epidemiologic studies that although most colorectal cancers (CRCs) originate from adenomas, only a small fraction of adenomas (3%-5%) ever progress to cancer. At present, there are no molecular markers to guide follow-up surveillance programs. METHODS: We profiled, by mass spectrometry-based proteomics combined with machine learning analysis, a selected cohort of formalin-fixed, paraffin-embedded high-grade (HG) adenomas with long clinical follow-up, collected as part of the Danish national screening program. We grouped subjects in the cohort according to their subsequent history of findings: a nonmetachronous advanced neoplasia group (G0), with no new HG adenomas or CRCs up to 10 years after polypectomy, and a metachronous advanced neoplasia group (G1) where individuals developed a new HG adenoma or CRC within 5 years of diagnosis. RESULTS: We generated a proteome dataset from 98 selected HG adenoma samples, including 20 technical replicates, of which 45 samples belonged to the nonmetachronous advanced neoplasia group and 53 to the metachronous advanced neoplasia group. The clear distinction of these 2 groups seen in a uniform manifold approximation and projection plot indicated that the information contained within the abundance of the â¼5000 proteins was sufficient to predict the future occurrence of HG adenomas or development of CRC. CONCLUSIONS: We performed an in-depth analysis of quantitative proteomic data from 98 resected adenoma samples using various novel algorithms and statistical packages and found that their proteome can predict development of metachronous advanced lesions and progression several years in advance.
Subject(s)
Adenoma , Colonic Polyps , Colorectal Neoplasms , Neoplasms, Second Primary , Humans , Proteome , Proteomics , Colorectal Neoplasms/pathology , Colonic Polyps/pathology , Adenoma/pathology , Neoplasms, Second Primary/pathology , Colonoscopy , Risk FactorsABSTRACT
Undifferentiated small round cell sarcomas (USRS) of bone and soft tissue are a group of tumors with heterogenic genomic alterations sharing similar morphology. In the present study, we performed a comparative large-scale proteomic analysis of USRS (n = 42) with diverse genomic translocations including classic Ewing sarcomas with EWSR1::FLI1 fusions (n = 24) or EWSR1::ERG fusions (n = 4), sarcomas with an EWSR1 rearrangement (n = 2), CIC::DUX4 fusion (n = 8), as well as tumors classified as USRS with no genetic data available (n = 4). Proteins extracted from formalin-fixed, paraffin-embedded pretherapeutic biopsies were analyzed qualitatively and quantitatively using shotgun mass spectrometry (MS). More than 8000 protein groups could be quantified using data-independent acquisition. Unsupervised hierarchical cluster analysis based on proteomic data allowed stratification of the 42 cases into distinct groups reflecting the different molecular genotypes. Protein signatures that significantly correlated with the respective genomic translocations were identified and used to generate a heatmap of all 42 sarcomas with assignment of cases with unknown molecular genetic data to either the EWSR1- or CIC-rearranged groups. MS-based prediction of sarcoma subtypes was molecularly confirmed in 2 cases where next-generation sequencing was technically feasible. MS also detected proteins routinely used in the immunohistochemical approach for the differential diagnosis of USRS. BCL11B highly expressed in Ewing sarcomas, and BACH2 as well as ETS-1 highly expressed in CIC::DUX4-associated sarcomas, were among proteins identified by the present proteomic study, and were chosen for immunohistochemical confirmation of MS data in our study cohort. Differential expressions of these 3 markers in the 2 genetic groups were further validated in an independent cohort of n = 34 USRS. Finally, our proteomic results point toward diverging signaling pathways in the different USRS subgroups.
Subject(s)
Biomarkers, Tumor , Proteomics , RNA-Binding Protein EWS , Sarcoma, Small Cell , Translocation, Genetic , Humans , RNA-Binding Protein EWS/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/analysis , Sarcoma, Small Cell/genetics , Sarcoma, Small Cell/pathology , Sarcoma, Small Cell/diagnosis , Female , Male , Adult , Adolescent , Young Adult , Soft Tissue Neoplasms/genetics , Soft Tissue Neoplasms/pathology , Soft Tissue Neoplasms/diagnosis , Middle Aged , Oncogene Proteins, Fusion/genetics , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Bone Neoplasms/diagnosis , Child , Calmodulin-Binding Proteins/genetics , RNA-Binding Proteins/genetics , Repressor Proteins/geneticsABSTRACT
Biomarkers are of central importance for assessing the health state and to guide medical interventions and their efficacy; still, they are lacking for most diseases. Mass spectrometry (MS)-based proteomics is a powerful technology for biomarker discovery but requires sophisticated bioinformatics to identify robust patterns. Machine learning (ML) has become a promising tool for this purpose. However, it is sometimes applied in an opaque manner and generally requires specialized knowledge. To enable easy access to ML for biomarker discovery without any programming or bioinformatics skills, we developed "OmicLearn" (http://OmicLearn.org), an open-source browser-based ML tool using the latest advances in the Python ML ecosystem. Data matrices from omics experiments are easily uploaded to an online or a locally installed web server. OmicLearn enables rapid exploration of the suitability of various ML algorithms for the experimental data sets. It fosters open science via transparent assessment of state-of-the-art algorithms in a standardized format for proteomics and other omics sciences.
Subject(s)
Ecosystem , Proteomics , Proteomics/methods , Biomarkers/analysis , Algorithms , Machine LearningABSTRACT
Deeper understanding of liver pathophysiology would benefit from a comprehensive quantitative proteome resource at cell type resolution to predict outcome and design therapy. Here, we quantify more than 150,000 sequence-unique peptides aggregated into 10,000 proteins across total liver, the major liver cell types, time course of primary cell cultures, and liver disease states. Bioinformatic analysis reveals that half of hepatocyte protein mass is comprised of enzymes and 23% of mitochondrial proteins, twice the proportion of other liver cell types. Using primary cell cultures, we capture dynamic proteome remodeling from tissue states to cell line states, providing useful information for biological or pharmaceutical research. Our extensive data serve as spectral library to characterize a human cohort of non-alcoholic steatohepatitis and cirrhosis. Dramatic proteome changes in liver tissue include signatures of hepatic stellate cell activation resembling liver cirrhosis and providing functional insights. We built a web-based dashboard application for the interactive exploration of our resource (www.liverproteome.org).
Subject(s)
Non-alcoholic Fatty Liver Disease , Proteome , Humans , Liver/metabolism , Liver Cirrhosis/metabolism , Non-alcoholic Fatty Liver Disease/metabolism , Proteome/metabolism , ProteomicsABSTRACT
BACKGROUND: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. METHODS: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. RESULTS: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. CONCLUSIONS: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.
Subject(s)
Hypoplastic Left Heart Syndrome/genetics , Organogenesis/genetics , Genetic Heterogeneity , HumansABSTRACT
Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Herein we describe a MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low-input cancer tissue isolated by laser microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent acquisition (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total workup took less than a day. We observed a moderate trend towards lower protein identification in long-term stored samples (>15 years), but clustering into distinct proteomic subtypes was independent of archival time. Our results underscore the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and they prove the feasibility of analyzing large tissue cohorts in a robust, timely, and streamlined manner. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Subject(s)
Neoplasms/pathology , Proteome/metabolism , Proteomics , Chromatography, Liquid/methods , Cohort Studies , Humans , Paraffin Embedding/methods , Proteomics/methods , Tandem Mass Spectrometry/methods , Tissue Fixation/methodsABSTRACT
Non-alcoholic fatty liver disease (NAFLD) affects 25% of the population and can progress to cirrhosis with limited treatment options. As the liver secretes most of the blood plasma proteins, liver disease may affect the plasma proteome. Plasma proteome profiling of 48 patients with and without cirrhosis or NAFLD revealed six statistically significantly changing proteins (ALDOB, APOM, LGALS3BP, PIGR, VTN, and AFM), two of which are already linked to liver disease. Polymeric immunoglobulin receptor (PIGR) was significantly elevated in both cohorts by 170% in NAFLD and 298% in cirrhosis and was further validated in mouse models. Furthermore, a global correlation map of clinical and proteomic data strongly associated DPP4, ANPEP, TGFBI, PIGR, and APOE with NAFLD and cirrhosis. The prominent diabetic drug target DPP4 is an aminopeptidase like ANPEP, ENPEP, and LAP3, all of which are up-regulated in the human or mouse data. Furthermore, ANPEP and TGFBI have potential roles in extracellular matrix remodeling in fibrosis. Thus, plasma proteome profiling can identify potential biomarkers and drug targets in liver disease.
Subject(s)
Biomarkers/blood , Liver Cirrhosis/blood , Non-alcoholic Fatty Liver Disease/blood , Proteome , Proteomics , Animals , Cohort Studies , Female , Gene Expression Profiling , Humans , Liver/metabolism , Liver Cirrhosis/drug therapy , Liver Cirrhosis/metabolism , Male , Mass Spectrometry , Mice , Mice, Inbred C57BL , Middle Aged , Non-alcoholic Fatty Liver Disease/drug therapy , Non-alcoholic Fatty Liver Disease/metabolismABSTRACT
RATIONALE: Structural and electrophysiological remodeling of the atria are recognized consequences of sustained atrial arrhythmias, such as atrial fibrillation. The identification of underlying key molecules and signaling pathways has been challenging because of the changing cell type composition during structural remodeling of the atria. OBJECTIVE: Thus, the aims of our study were (1) to search for transcription factors and downstream target genes, which are involved in atrial structural remodeling, (2) to characterize the significance of the transcription factor ETV1 (E twenty-six variant 1) in atrial remodeling and arrhythmia, and (3) to identify ETV1-dependent gene regulatory networks in atrial cardiac myocytes. METHODS AND RESULTS: The transcription factor ETV1 was significantly upregulated in atrial tissue from patients with permanent atrial fibrillation. Mice with cardiac myocyte-specific overexpression of ETV1 under control of the myosin heavy chain promoter developed atrial dilatation, fibrosis, thrombosis, and arrhythmia. Cardiac myocyte-specific ablation of ETV1 in mice did not alter cardiac structure and function at baseline. Treatment with Ang II (angiotensin II) for 2 weeks elicited atrial remodeling and fibrosis in control, but not in ETV1-deficient mice. To identify ETV1-regulated genes, cardiac myocytes were isolated and purified from mouse atrial tissue. Active cis-regulatory elements in mouse atrial cardiac myocytes were identified by chromatin accessibility (assay for transposase-accessible chromatin sequencing) and the active chromatin modification H3K27ac (chromatin immunoprecipitation sequencing). One hundred seventy-eight genes regulated by Ang II in an ETV1-dependent manner were associated with active cis-regulatory elements containing ETV1-binding sites. Various genes involved in Ca2+ handling or gap junction formation ( Ryr2, Jph2, Gja5), potassium channels ( Kcnh2, Kcnk3), and genes implicated in atrial fibrillation ( Tbx5) were part of this ETV1-driven gene regulatory network. The atrial ETV1-dependent transcriptome in mice showed a significant overlap with the human atrial proteome of patients with permanent atrial fibrillation. CONCLUSIONS: This study identifies ETV1 as an important component in the pathophysiology of atrial remodeling associated with atrial arrhythmias.
Subject(s)
Arrhythmias, Cardiac/genetics , Atrial Remodeling , DNA-Binding Proteins/genetics , Gene Regulatory Networks , Transcription Factors/genetics , Animals , Arrhythmias, Cardiac/metabolism , Arrhythmias, Cardiac/pathology , Cells, Cultured , Chromatin Assembly and Disassembly/genetics , Connexins/genetics , Connexins/metabolism , DNA-Binding Proteins/metabolism , Humans , Mice , Myocytes, Cardiac/metabolism , Potassium Channels/genetics , Potassium Channels/metabolism , Transcription Factors/metabolism , TranscriptomeABSTRACT
To further integrate mass spectrometry (MS)-based proteomics into biomedical research and especially into clinical settings, high throughput and robustness are essential requirements. They are largely met in high-flow rate chromatographic systems for small molecules but these are not sufficiently sensitive for proteomics applications. Here we describe a new concept that delivers on these requirements while maintaining the sensitivity of current nano-flow LC systems. Low-pressure pumps elute the sample from a disposable trap column, simultaneously forming a chromatographic gradient that is stored in a long storage loop. An auxiliary gradient creates an offset, ensuring the re-focusing of the peptides before the separation on the analytical column by a single high-pressure pump. This simplified design enables robust operation over thousands of sample injections. Furthermore, the steps between injections are performed in parallel, reducing overhead time to a few minutes and allowing analysis of more than 200 samples per day. From fractionated HeLa cell lysates, deep proteomes covering more than 130,000 sequence unique peptides and close to 10,000 proteins were rapidly acquired. Using this data as a library, we demonstrate quantitation of 5200 proteins in only 21 min. Thus, the new system - termed Evosep One - analyzes samples in an extremely robust and high throughput manner, without sacrificing in depth proteomics coverage.
Subject(s)
Chromatography, Liquid/methods , Proteomics/methods , Blood Proteins/metabolism , HeLa Cells , Humans , Proteome/metabolism , Ultraviolet RaysABSTRACT
Glioblastoma multiformes (GBMs) are high-grade astrocytomas and the most common brain malignancies. Primary GBMs are often associated with disturbed RAS signaling, and expression of oncogenic HRAS results in a malignant phenotype in glioma cell lines. Secondary GBMs arise from lower-grade astrocytomas, have slower progression than primary tumors, and contain IDH1 mutations in over 70% of cases. Despite significant amount of accumulating genomic and transcriptomic data, the fundamental mechanistic differences of gliomagenesis in these two types of high-grade astrocytoma remain poorly understood. Only a few studies have attempted to investigate the proteome, phosphorylation signaling, and epigenetic regulation in astrocytoma. In the present study, we applied quantitative phosphoproteomics to identify the main signaling differences between oncogenic HRAS and mutant IDH1-driven glioma cells as models of primary and secondary GBM, respectively. Our analysis confirms the driving roles of the MAPK and PI3K/mTOR signaling pathways in HRAS driven cells and additionally uncovers dysregulation of other signaling pathways. Although a subset of the signaling changes mediated by HRAS could be reversed by a MEK inhibitor, dual inhibition of MEK and PI3K resulted in more complete reversal of the phosphorylation patterns produced by HRAS expression. In contrast, cells expressing mutant IDH1 did not show significant activation of MAPK or PI3K/mTOR pathways. Instead, global downregulation of protein expression was observed. Targeted proteomic analysis of histone modifications identified significant histone methylation, acetylation, and butyrylation changes in the mutant IDH1 expressing cells, consistent with a global transcriptional repressive state. Our findings offer novel mechanistic insight linking mutant IDH1 associated inhibition of histone demethylases with specific histone modification changes to produce global transcriptional repression in secondary glioblastoma. Our proteomic datasets are available for download and provide a comprehensive catalogue of alterations in protein abundance, phosphorylation, and histone modifications in oncogenic HRAS and IDH1 driven astrocytoma cells beyond the transcriptomic level.
Subject(s)
Brain Neoplasms/pathology , Glioblastoma/pathology , Isocitrate Dehydrogenase/genetics , Phosphoproteins/analysis , Proteomics/methods , Proto-Oncogene Proteins p21(ras)/genetics , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Cell Line, Tumor , Glioblastoma/genetics , Glioblastoma/metabolism , Histone Code , Histones/metabolism , Humans , Mitogen-Activated Protein Kinases/metabolism , Mutation , Phosphatidylinositol 3-Kinases/metabolism , Protein Interaction Maps , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
The interconnected PI3K and MAPK signaling pathways are commonly perturbed in cancer. Dual inhibition of these pathways by the small-molecule PI3K inhibitor pictilisib (GDC-0941) and the MEK inhibitor cobimetinib (GDC-0973) suppresses cell proliferation and induces cell death better than either single agent in several preclinical models. Using mass spectrometry-based phosphoproteomics, we have identified the RING finger E3 ubiquitin ligase RNF157 as a target at the intersection of PI3K and MAPK signaling. We demonstrate that RNF157 phosphorylation downstream of the PI3K and MAPK pathways influences the ubiquitination and stability of RNF157 during the cell cycle in an anaphase-promoting complex/cyclosome-CDH1-dependent manner. Deletion of these phosphorylation-targeted residues on RNF157 disrupts binding to CDH1 and protects RNF157 from ubiquitination and degradation. Expression of the cyclin-dependent kinase 2 (CDK2), itself a downstream target of PI3K/MAPK signaling, leads to increased phosphorylation of RNF157 on the same residues modulated by PI3K and MAPK signaling. Inhibition of PI3K and MEK in combination or of CDK2 by their respective small-molecule inhibitors reduces RNF157 phosphorylation at these residues and attenuates RNF157 interaction with CDH1 and its subsequent degradation. Knockdown of endogenous RNF157 in melanoma cells leads to late S phase and G2/M arrest and induces apoptosis, the latter further potentiated by concurrent PI3K/MEK inhibition, consistent with a role for RNF157 in the cell cycle. We propose that RNF157 serves as a novel node integrating oncogenic signaling pathways with the cell cycle machinery and promoting optimal cell cycle progression in transformed cells.
Subject(s)
Apoptosis , MAP Kinase Signaling System , Melanoma/metabolism , Phosphatidylinositol 3-Kinase/metabolism , Protein Processing, Post-Translational , Signal Transduction , Ubiquitin-Protein Ligases/metabolism , Amino Acid Substitution , Antigens, CD , Apoptosis/drug effects , Cadherins/antagonists & inhibitors , Cadherins/genetics , Cadherins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Stability/drug effects , Gene Deletion , Gene Expression Regulation, Neoplastic/drug effects , Humans , MAP Kinase Signaling System/drug effects , Melanoma/drug therapy , Melanoma/enzymology , Melanoma/pathology , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Point Mutation , Protein Processing, Post-Translational/drug effects , RNA Interference , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , S Phase/drug effects , Signal Transduction/drug effects , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitination/drug effectsABSTRACT
Despite the recent success of antibody-drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking, and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody-FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs.
Subject(s)
Biological Transport , Fluorescence Resonance Energy Transfer , Immunoconjugates/pharmacokinetics , Cell Membrane Permeability , Cross-Linking Reagents/chemistry , Humans , Immunoconjugates/metabolism , PeptidesABSTRACT
BACKGROUND: Sympathetic system abnormalities have been reported in sepsis-related cardiac dysfunction. The present study aimed at evaluating the potential of the norepinephrine radiolabeled analogue [123I]-meta-iodobenzylguanidine (123I-MIBG) for the noninvasive assessment of modifications in cardiac sympathetic activity occurring in lipopolysaccharide (LPS)-induced experimental acute sepsis by single-photon emission computed tomographic imaging (SPECT). METHODS AND RESULTS: Sepsis was induced in male Wistar rats by intraperitoneal injection of 10 mg·kg-1 lipopolysaccharide (n = 16), whereas control animals (n = 7) were injected with vehicle (NaCl 0.9%). Echocardiography in LPS-injected animals (n = 8) demonstrated systolic and diastolic cardiac dysfunction. 123I-MIBG was injected 1 hour after LPS or vehicle administration (n = 8 and 7, respectively), and in vivo SPECT imaging was performed early and late (20 and 180 minutes) after tracer injection prior to animal euthanasia and ex vivo assessment of 123I-MIBG biodistribution. Global and 17-segment SPECT image analysis indicated that early 123I-MIBG activity was not affected by LPS treatment, whereas late cardiac tracer activity was significantly decreased in LPS-treated animals. Consequently, the cardiac washout of 123I-MIBG was significantly higher in LPS-treated (63.3% ± 4.0%) than that in control animals (56.7% ± 5.8%) (P < .05). CONCLUSION: Sepsis-induced modifications in cardiac sympathetic nervous system activity were evidenced by noninvasive in vivo 123I-MIBG SPECT imaging.
Subject(s)
3-Iodobenzylguanidine/pharmacokinetics , Heart/diagnostic imaging , Shock, Septic/physiopathology , Sympathetic Nervous System/physiopathology , Tomography, Emission-Computed, Single-Photon , Animals , Lipopolysaccharides/chemistry , Male , Norepinephrine/blood , Prognosis , Rats , Rats, Wistar , Receptors, Adrenergic/metabolism , Tissue DistributionABSTRACT
The RAS-RAF-MEK-ERK (MAPK) pathway is prevalently perturbed in cancer. Recent large-scale sequencing initiatives profiled thousands of tumors providing insight into alterations at the DNA and RNA levels. These efforts confirmed that key nodes of the MAPK pathway, in particular KRAS and BRAF, are among the most frequently altered proteins in cancer. The establishment of targeted therapies, however, has proven difficult. To decipher the underlying challenges, it is essential to decrypt the phosphorylation network spanned by the MAPK core axis. Using mass spectrometry we identified 2241 phosphorylation sites on 1020 proteins, and measured their responses to inhibition of MEK or ERK. Multiple phosphorylation patterns revealed previously undetected feedback, as upstream signaling nodes, including receptor kinases, showed changes at the phosphorylation level. We provide a dataset rich in potential therapeutic targets downstream of the MAPK cascade. By integrating TCGA (The Cancer Genome Atlas) data, we highlight some downstream phosphoproteins that are frequently altered in cancer. All MS data have been deposited in the ProteomeXchange with identifier PXD003908 (http://proteomecentral.proteomexchange.org/dataset/PXD003908).
Subject(s)
Colonic Neoplasms/genetics , Feedback, Physiological , Gene Expression Regulation, Neoplastic , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase Kinases/genetics , Neoplasm Proteins/genetics , Phosphoproteins/genetics , Amino Acid Sequence , Antineoplastic Agents/pharmacology , Atlases as Topic , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Gene Expression Profiling , HCT116 Cells , Humans , Internet , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Proteomics/methods , SoftwareABSTRACT
The PI3K pathway is commonly activated in cancer. Only a few studies have attempted to explore the spectrum of phosphorylation signaling downstream of the PI3K cascade. Such insight, however, is imperative to understand the mechanisms responsible for oncogenic phenotypes. By applying MS-based phosphoproteomics, we mapped 2509 phosphorylation sites on 1096 proteins, and quantified their responses to activation or inhibition of PIK3CA using isogenic knock-in derivatives and a series of targeted inhibitors. We uncovered phosphorylation changes in a wide variety of proteins involved in cell growth and proliferation, many of which have not been previously associated with PI3K signaling. A significant update of the posttranslational modification database PHOSIDA (http://www.phosida.com) allows efficient use of the data. All MS data have been deposited in the ProteomeXchange with identifier PXD003899 (http://proteomecentral.proteomexchange.org/dataset/PXD003899).
Subject(s)
Cell Transformation, Neoplastic/genetics , Epithelial Cells/metabolism , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphoproteins/genetics , Protein Processing, Post-Translational , Antineoplastic Agents/pharmacology , Cell Line , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Class I Phosphatidylinositol 3-Kinases , Colon/cytology , Colon/drug effects , Colon/metabolism , Databases, Genetic , Epithelial Cells/cytology , Epithelial Cells/drug effects , Humans , Internet , Mutation , Neoplasm Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/metabolism , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Proteomics/methods , Signal Transduction , SoftwareABSTRACT
BACKGROUND: Many cancer cells show distorted epigenetic landscapes. The Cancer Genome Atlas (TCGA) project profiles thousands of tumors, allowing the discovery of somatic alterations in the epigenetic machinery and the identification of potential cancer drivers among members of epigenetic protein families. METHODS: We integrated mutation, expression, and copy number data from 5943 tumors from 13 cancer types to train a classification model that predicts the likelihood of being an oncogene (OG), tumor suppressor (TSG) or neutral gene (NG). We applied this predictor to epigenetic regulator genes (ERGs), and used differential expression and correlation network analysis to identify dysregulated ERGs along with co-expressed cancer genes. Furthermore, we quantified global proteomic changes by mass spectrometry after EZH2 inhibition. RESULTS: Mutation-based classifiers uncovered the OG-like profile of DNMT3A and TSG-like profiles for several ERGs. Differential gene expression and correlation network analyses revealed that EZH2 is the most significantly over-expressed ERG in cancer and is co-regulated with a cell cycle network. Proteomic analysis showed that EZH2 inhibition induced down-regulation of cell cycle regulators in lymphoma cells. CONCLUSIONS: Using classical driver genes to train an OG/TSG predictor, we determined the most predictive features at the gene level. Our predictor uncovered one OG and several TSGs among ERGs. Expression analyses elucidated multiple dysregulated ERGs including EZH2 as member of a co-expressed cell cycle network.
Subject(s)
Computational Biology , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Neoplasms/genetics , Neoplasms/metabolism , Enhancer of Zeste Homolog 2 Protein , Genes, Tumor Suppressor , Humans , Oncogenes , Polycomb Repressive Complex 2/genetics , Proteome/geneticsABSTRACT
Acute type A aortic dissection (ATAAD) constitutes a life-threatening aortic pathology with significant morbidity and mortality. Without surgical intervention the usual mortality rate averages between 1 and 2% per hour. Thus, an early diagnosis of ATAAD is of pivotal importance to direct the affected patients to the appropriate treatment. Preceding tests to find an appropriate biomarker showed among others an increased aggrecan (ACAN) mRNA expression in aortic tissue of ATAAD patients. As a consequence, we investigated whether ACAN is a potential biomarker for diagnosing ATAAD. Mean ACAN protein concentration showed a significantly higher plasma concentration in ATAAD patients (38.59 ng/mL, n = 33) compared to plasma of patients with thoracic aortic aneurysms (4.45 ng/mL, n = 13), patients with myocardial infarction (11.77 ng/mL, n = 18) and healthy volunteers (8.05 ng/mL, n = 12). Cardiac enzymes like creatine kinase MB and cardiac troponin T showed no correlation with ACAN levels in ATAAD patients. Receiver-operator characteristics (ROC) curve analysis for ATAAD patients versus control subjects an optimum discrimination limit of ACAN plasma levels at 14.3 ng/mL with a corresponding sensitivity of 97% and specificity of 81%. According to our findings ACAN is a reliable potential biomarker in plasma samples to detect ATAAD with high sensitivity and specificity.
Subject(s)
Aggrecans/blood , Aortic Aneurysm, Thoracic/diagnosis , Aortic Dissection/diagnosis , Myocardial Infarction/diagnosis , Acute Disease , Aged , Aortic Dissection/blood , Aortic Dissection/etiology , Aortic Aneurysm, Thoracic/blood , Biomarkers/blood , Creatine Kinase, MB Form/blood , Diagnosis, Differential , Female , Healthy Volunteers , Humans , Male , Middle Aged , Myocardial Infarction/blood , ROC Curve , Retrospective Studies , Troponin T/bloodABSTRACT
A deeper understanding of COVID-19 on human molecular pathophysiology is urgently needed as a foundation for the discovery of new biomarkers and therapeutic targets. Here we applied mass spectrometry (MS)-based proteomics to measure serum proteomes of COVID-19 patients and symptomatic, but PCR-negative controls, in a time-resolved manner. In 262 controls and 458 longitudinal samples of 31 patients, hospitalized for COVID-19, a remarkable 26% of proteins changed significantly. Bioinformatics analyses revealed co-regulated groups and shared biological functions. Proteins of the innate immune system such as CRP, SAA1, CD14, LBP, and LGALS3BP decreased early in the time course. Regulators of coagulation (APOH, FN1, HRG, KNG1, PLG) and lipid homeostasis (APOA1, APOC1, APOC2, APOC3, PON1) increased over the course of the disease. A global correlation map provides a system-wide functional association between proteins, biological processes, and clinical chemistry parameters. Importantly, five SARS-CoV-2 immunoassays against antibodies revealed excellent correlations with an extensive range of immunoglobulin regions, which were quantified by MS-based proteomics. The high-resolution profile of all immunoglobulin regions showed individual-specific differences and commonalities of potential pathophysiological relevance.
Subject(s)
COVID-19 , Proteome , Antibodies, Viral , Aryldialkylphosphatase , Humans , Proteomics , SARS-CoV-2 , SeroconversionABSTRACT
Human skin provides both physical integrity and immunological protection from the external environment using functionally distinct layers, cell types and extracellular matrix. Despite its central role in human health and disease, the constituent proteins of skin have not been systematically characterized. Here, we combine advanced tissue dissection methods, flow cytometry and state-of-the-art proteomics to describe a spatially-resolved quantitative proteomic atlas of human skin. We quantify 10,701 proteins as a function of their spatial location and cellular origin. The resulting protein atlas and our initial data analyses demonstrate the value of proteomics for understanding cell-type diversity within the skin. We describe the quantitative distribution of structural proteins, known and previously undescribed proteins specific to cellular subsets and those with specialized immunological functions such as cytokines and chemokines. We anticipate that this proteomic atlas of human skin will become an essential community resource for basic and translational research ( https://skin.science/ ).