RESUMEN
Protein structures are essential to understanding cellular processes in molecular detail. While advances in artificial intelligence revealed the tertiary structure of proteins at scale, their quaternary structure remains mostly unknown. We devise a scalable strategy based on AlphaFold2 to predict homo-oligomeric assemblies across four proteomes spanning the tree of life. Our results suggest that approximately 45% of an archaeal proteome and a bacterial proteome and 20% of two eukaryotic proteomes form homomers. Our predictions accurately capture protein homo-oligomerization, recapitulate megadalton complexes, and unveil hundreds of homo-oligomer types, including three confirmed experimentally by structure determination. Integrating these datasets with omics information suggests that a majority of known protein complexes are symmetric. Finally, these datasets provide a structural context for interpreting disease mutations and reveal coiled-coil regions as major enablers of quaternary structure evolution in human. Our strategy is applicable to any organism and provides a comprehensive view of homo-oligomerization in proteomes.
Asunto(s)
Inteligencia Artificial , Proteínas , Proteoma , Humanos , Proteínas/química , Proteínas/genética , Archaea/química , Archaea/genética , Eucariontes/química , Eucariontes/genética , Bacterias/química , Bacterias/genéticaRESUMEN
Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of â¼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Células Eucariotas/metabolismo , Redes Neurales de la Computación , Proteoma/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
A generalizable strategy with programmable site specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and ß-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation and established linkages for chromatin acylation mark-defined proteome, genome, and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr's gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a platform technology for elucidating the "metabolites-modification-regulation" axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones.
Asunto(s)
Cromatina , Proteoma , Acilación , Mapeo Cromosómico , Histonas , Supervivencia CelularRESUMEN
In poikilotherms, temperature changes challenge the integration of physiological function. Within the complex nervous systems of the behaviorally sophisticated coleoid cephalopods, these problems are substantial. RNA editing by adenosine deamination is a well-positioned mechanism for environmental acclimation. We report that the neural proteome of Octopus bimaculoides undergoes massive reconfigurations via RNA editing following a temperature challenge. Over 13,000 codons are affected, and many alter proteins that are vital for neural processes. For two highly temperature-sensitive examples, recoding tunes protein function. For synaptotagmin, a key component of Ca2+-dependent neurotransmitter release, crystal structures and supporting experiments show that editing alters Ca2+ binding. For kinesin-1, a motor protein driving axonal transport, editing regulates transport velocity down microtubules. Seasonal sampling of wild-caught specimens indicates that temperature-dependent editing occurs in the field as well. These data show that A-to-I editing tunes neurophysiological function in response to temperature in octopus and most likely other coleoids.
Asunto(s)
Octopodiformes , Proteoma , Animales , Proteoma/metabolismo , Octopodiformes/genética , Edición de ARN , Temperatura , Sistema Nervioso/metabolismo , Adenosina Desaminasa/metabolismo , ARN/metabolismoRESUMEN
Functional genomic strategies have become fundamental for annotating gene function and regulatory networks. Here, we combined functional genomics with proteomics by quantifying protein abundances in a genome-scale knockout library in Saccharomyces cerevisiae, using data-independent acquisition mass spectrometry. We find that global protein expression is driven by a complex interplay of (1) general biological properties, including translation rate, protein turnover, the formation of protein complexes, growth rate, and genome architecture, followed by (2) functional properties, such as the connectivity of a protein in genetic, metabolic, and physical interaction networks. Moreover, we show that functional proteomics complements current gene annotation strategies through the assessment of proteome profile similarity, protein covariation, and reverse proteome profiling. Thus, our study reveals principles that govern protein expression and provides a genome-spanning resource for functional annotation.
Asunto(s)
Proteoma , Proteómica , Proteómica/métodos , Proteoma/metabolismo , Genómica/métodos , Genoma , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here, we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules (SGs), uncovering a role for SGs in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID offers a powerful approach for distinguishing protein populations based on compartment or cell type of origin.
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Mitocondrias , Proteoma , Proteoma/metabolismo , Mitocondrias/metabolismo , Nucléolo Celular/metabolismo , Espectrometría de Masas/métodos , Regulación de la Expresión GénicaRESUMEN
Neurons build synaptic contacts using different protein combinations that define the specificity, function, and plasticity potential of synapses; however, the diversity of synaptic proteomes remains largely unexplored. We prepared synaptosomes from 7 different transgenic mouse lines with fluorescently labeled presynaptic terminals. Combining microdissection of 5 different brain regions with fluorescent-activated synaptosome sorting (FASS), we isolated and analyzed the proteomes of 18 different synapse types. We discovered â¼1,800 unique synapse-type-enriched proteins and allocated thousands of proteins to different types of synapses (https://syndive.org/). We identify shared synaptic protein modules and highlight the proteomic hotspots for synapse specialization. We reveal unique and common features of the striatal dopaminergic proteome and discover the proteome signatures that relate to the functional properties of different interneuron classes. This study provides a molecular systems-biology analysis of synapses and a framework to integrate proteomic information for synapse subtypes of interest with cellular or circuit-level experiments.
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Encéfalo , Proteoma , Sinapsis , Animales , Ratones , Encéfalo/metabolismo , Ratones Transgénicos , Proteoma/metabolismo , Proteómica , Sinapsis/metabolismo , Sinaptosomas/metabolismoRESUMEN
To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5.
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Proteoma , Espermatozoides , Animales , Masculino , Ratones , Axonema/química , Microscopía por Crioelectrón/métodos , Flagelos/metabolismo , Microtúbulos/metabolismo , Semen , Espermatozoides/química , Proteoma/análisisRESUMEN
Metals are essential components in life processes and participate in many important biological processes. Dysregulation of metal homeostasis is correlated with many diseases. Metals are also frequently incorporated into diagnosis and therapeutics. Understanding of metal homeostasis under (patho)physiological conditions and the molecular mechanisms of action of metallodrugs in biological systems has positive impacts on human health. As an emerging interdisciplinary area of research, metalloproteomics involves investigating metal-protein interactions in biological systems at a proteome-wide scale, has received growing attention, and has been implemented into metal-related research. In this review, we summarize the recent advances in metalloproteomics methodologies and applications. We also highlight emerging single-cell metalloproteomics, including time-resolved inductively coupled plasma mass spectrometry, mass cytometry, and secondary ion mass spectrometry. Finally, we discuss future perspectives in metalloproteomics, aiming to attract more original research to develop more advanced methodologies, which could be utilized rapidly by biochemists or biologists to expand our knowledge of how metal functions in biology and medicine.
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Investigación Biomédica , Metaloproteínas , Humanos , Metaloproteínas/análisis , Metaloproteínas/química , Metaloproteínas/genética , Metales/análisis , Metales/química , Proteoma/genética , Proteómica/métodosRESUMEN
Technologies for counting protein molecules are enabling single-cell proteomics at increasing depth and scale. New advances in single-molecule methods by Brinkerhoff and colleagues promise to further increase the sensitivity of protein analysis and motivate questions about scaling up the counting of the human proteome.
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Proteoma , Proteómica , Humanos , NanotecnologíaRESUMEN
Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.
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Enfermedad de Alzheimer , Proteoma , Ratones , Humanos , Animales , Proteoma/análisis , Proteómica/métodos , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides , Espectrometría de Masas , Placa AmiloideRESUMEN
Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.
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Tejido Adiposo Pardo , Proteoma , Humanos , Ratones , Animales , Tejido Adiposo Pardo/metabolismo , Proteoma/metabolismo , Termogénesis/fisiología , Adiposidad , Obesidad/metabolismo , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. Proportions of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.
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COVID-19/inmunología , COVID-19/patología , Activación de Complemento , Proteoma , SARS-CoV-2/inmunología , Linfocitos T Citotóxicos/inmunología , Transcriptoma , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/virología , Factores Quimiotácticos/metabolismo , Citotoxicidad Inmunológica , Células Endoteliales/virología , Femenino , Humanos , Activación de Linfocitos , Masculino , Microvasos/virología , Persona de Mediana Edad , Monocitos/metabolismo , Neutrófilos/metabolismo , Receptores de IgG/metabolismo , Análisis de la Célula Individual , Adulto JovenRESUMEN
The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis.
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Evolución Biológica , Hepatocitos/metabolismo , Macrófagos/metabolismo , Proteogenómica , Animales , Núcleo Celular/metabolismo , Hígado Graso/genética , Hígado Graso/patología , Homeostasis , Humanos , Macrófagos del Hígado/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Lípidos/química , Hígado/metabolismo , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Modelos Biológicos , Células Mieloides/metabolismo , Obesidad/patología , Proteoma/metabolismo , Transducción de Señal , Transcriptoma/genéticaRESUMEN
Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism, and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design, and personalized medicine approaches for COVID-19.
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Biomarcadores/sangre , COVID-19/patología , Proteoma/análisis , Adulto , Proteínas Sanguíneas/metabolismo , COVID-19/sangre , COVID-19/virología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Femenino , Humanos , Gripe Humana/sangre , Gripe Humana/patología , Linfocitos/inmunología , Linfocitos/metabolismo , Aprendizaje Automático , Masculino , Persona de Mediana Edad , Proteína Quinasa 14 Activada por Mitógenos/genética , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Análisis de Componente Principal , SARS-CoV-2/aislamiento & purificación , Sepsis/sangre , Sepsis/patología , Índice de Severidad de la Enfermedad , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Mitochondria are multifaceted organelles with key roles in anabolic and catabolic metabolism, bioenergetics, cellular signalling and nutrient sensing, and programmed cell death processes. Their diverse functions are enabled by a sophisticated set of protein components encoded by the nuclear and mitochondrial genomes. The extent and complexity of the mitochondrial proteome remained unclear for decades. This began to change 20 years ago when, driven by the emergence of mass spectrometry-based proteomics, the first draft mitochondrial proteomes were established. In the ensuing decades, further technological and computational advances helped to refine these 'maps', with current estimates of the core mammalian mitochondrial proteome ranging from 1,000 to 1,500 proteins. The creation of these compendia provided a systemic view of an organelle previously studied primarily in a reductionist fashion and has accelerated both basic scientific discovery and the diagnosis and treatment of human disease. Yet numerous challenges remain in understanding mitochondrial biology and translating this knowledge into the medical context. In this Roadmap, we propose a path forward for refining the mitochondrial protein map to enhance its discovery and therapeutic potential. We discuss how emerging technologies can assist the detection of new mitochondrial proteins, reveal their patterns of expression across diverse tissues and cell types, and provide key information on proteoforms. We highlight the power of an enhanced map for systematically defining the functions of its members. Finally, we examine the utility of an expanded, functionally annotated mitochondrial proteome in a translational setting for aiding both diagnosis of mitochondrial disease and targeting of mitochondria for treatment.
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Enfermedades Mitocondriales , Proteoma , Animales , Humanos , Proteoma/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Orgánulos/metabolismo , Enfermedades Mitocondriales/metabolismo , Espectrometría de Masas , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mamíferos/metabolismoRESUMEN
Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins. The second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome whose structure encodes protein function, localization, and complex membership. Comparison across cell lines validates thousands of interactions and reveals extensive customization. Whereas shared interactions reside in core complexes and involve essential proteins, cell-specific interactions link these complexes, "rewiring" subnetworks within each cell's interactome. Interactions covary among proteins of shared function as the proteome remodels to produce each cell's phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization.
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Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/genética , Proteoma/genética , Biología Computacional/métodos , Células HCT116/metabolismo , Células HEK293/metabolismo , Humanos , Espectrometría de Masas/métodos , Mapas de Interacción de Proteínas/fisiología , Proteoma/metabolismo , Proteómica/métodosRESUMEN
Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer's disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.
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Envejecimiento/metabolismo , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Autofagia Mediada por Chaperones/fisiología , Neuronas/metabolismo , Proteostasis , Envejecimiento/patología , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Quinasa de la Caseína I/genética , Autofagia Mediada por Chaperones/genética , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Neuronas/patología , ProteomaRESUMEN
The molecular pathology of multi-organ injuries in COVID-19 patients remains unclear, preventing effective therapeutics development. Here, we report a proteomic analysis of 144 autopsy samples from seven organs in 19 COVID-19 patients. We quantified 11,394 proteins in these samples, in which 5,336 were perturbed in the COVID-19 patients compared to controls. Our data showed that cathepsin L1, rather than ACE2, was significantly upregulated in the lung from the COVID-19 patients. Systemic hyperinflammation and dysregulation of glucose and fatty acid metabolism were detected in multiple organs. We also observed dysregulation of key factors involved in hypoxia, angiogenesis, blood coagulation, and fibrosis in multiple organs from the COVID-19 patients. Evidence for testicular injuries includes reduced Leydig cells, suppressed cholesterol biosynthesis, and sperm mobility. In summary, this study depicts a multi-organ proteomic landscape of COVID-19 autopsies that furthers our understanding of the biological basis of COVID-19 pathology.
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COVID-19/metabolismo , Regulación de la Expresión Génica , Proteoma/biosíntesis , Proteómica , SARS-CoV-2/metabolismo , Autopsia , COVID-19/patología , COVID-19/terapia , Femenino , Humanos , Masculino , Especificidad de ÓrganosRESUMEN
Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology.