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Cell Nov 2023Neurons build synaptic contacts using different protein combinations that define the specificity, function, and plasticity potential of synapses; however, the diversity...
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 ∼1,800 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.
Topics: Animals; Mice; Brain; Mice, Transgenic; Proteome; Proteomics; Synapses; Synaptosomes
PubMed: 37918396
DOI: 10.1016/j.cell.2023.09.028 -
Journal of Proteome Research Jul 2023Mass spectrometry is unmatched in its versatility for studying practically any aspect of the proteome. Because the foundations of mass spectrometry-based proteomics are...
Mass spectrometry is unmatched in its versatility for studying practically any aspect of the proteome. Because the foundations of mass spectrometry-based proteomics are complex and span multiple scientific fields, proteomics can be perceived as having a high barrier to entry. This tutorial is intended to be an accessible illustrated guide to the technical details of a relatively simple quantitative proteomic experiment. An attempt is made to explain the relevant concepts to those with limited knowledge of mass spectrometry and a basic understanding of proteins. An experimental overview is provided, from the beginning of sample preparation to the analysis of protein group quantities, with explanations of how the data are acquired, processed, and analyzed. A selection of advanced topics is briefly surveyed and works for further reading are cited. To conclude, a brief discussion of the future of proteomics is given, considering next-generation protein sequencing technologies that may complement mass spectrometry to create a fruitful future for proteomics.
Topics: Proteomics; Mass Spectrometry; Proteome; Specimen Handling
PubMed: 37260118
DOI: 10.1021/acs.jproteome.2c00838 -
Nature Metabolism Apr 2024Surviving long periods without food has shaped human evolution. In ancient and modern societies, prolonged fasting was/is practiced by billions of people globally for...
Surviving long periods without food has shaped human evolution. In ancient and modern societies, prolonged fasting was/is practiced by billions of people globally for religious purposes, used to treat diseases such as epilepsy, and recently gained popularity as weight loss intervention, but we still have a very limited understanding of the systemic adaptions in humans to extreme caloric restriction of different durations. Here we show that a 7-day water-only fast leads to an average weight loss of 5.7 kg (±0.8 kg) among 12 volunteers (5 women, 7 men). We demonstrate nine distinct proteomic response profiles, with systemic changes evident only after 3 days of complete calorie restriction based on in-depth characterization of the temporal trajectories of ~3,000 plasma proteins measured before, daily during, and after fasting. The multi-organ response to complete caloric restriction shows distinct effects of fasting duration and weight loss and is remarkably conserved across volunteers with >1,000 significantly responding proteins. The fasting signature is strongly enriched for extracellular matrix proteins from various body sites, demonstrating profound non-metabolic adaptions, including extreme changes in the brain-specific extracellular matrix protein tenascin-R. Using proteogenomic approaches, we estimate the health consequences for 212 proteins that change during fasting across ~500 outcomes and identified putative beneficial (SWAP70 and rheumatoid arthritis or HYOU1 and heart disease), as well as adverse effects. Our results advance our understanding of prolonged fasting in humans beyond a merely energy-centric adaptions towards a systemic response that can inform targeted therapeutic modulation.
Topics: Humans; Caloric Restriction; Proteome; Female; Male; Fasting; Adult; Weight Loss; Proteomics; Adaptation, Physiological
PubMed: 38429390
DOI: 10.1038/s42255-024-01008-9 -
Molecular & Cellular Proteomics : MCP Jul 2023Accurate biomarkers are a crucial and necessary precondition for precision medicine, yet existing ones are often unspecific and new ones have been very slow to enter the...
Accurate biomarkers are a crucial and necessary precondition for precision medicine, yet existing ones are often unspecific and new ones have been very slow to enter the clinic. Mass spectrometry (MS)-based proteomics excels by its untargeted nature, specificity of identification, and quantification, making it an ideal technology for biomarker discovery and routine measurement. It has unique attributes compared to affinity binder technologies, such as OLINK Proximity Extension Assay and SOMAscan. In in a previous review in 2017, we described technological and conceptual limitations that had held back success. We proposed a 'rectangular strategy' to better separate true biomarkers by minimizing cohort-specific effects. Today, this has converged with advances in MS-based proteomics technology, such as increased sample throughput, depth of identification, and quantification. As a result, biomarker discovery studies have become more successful, producing biomarker candidates that withstand independent verification and, in some cases, already outperform state-of-the-art clinical assays. We summarize developments over the last years, including the benefits of large and independent cohorts, which are necessary for clinical acceptance. Shorter gradients, new scan modes, and multiplexing are about to drastically increase throughput, cross-study integration, and quantification, including proxies for absolute levels. We have found that multiprotein panels are inherently more robust than current single analyte tests and better capture the complexity of human phenotypes. Routine MS measurement in the clinic is fast becoming a viable option. The full set of proteins in a body fluid (global proteome) is the most important reference and the best process control. Additionally, it increasingly has all the information that could be obtained from targeted analysis although the latter may be the most straightforward way to enter regular use. Many challenges remain, not least of a regulatory and ethical nature, but the outlook for MS-based clinical applications has never been brighter.
Topics: Humans; Proteomics; Mass Spectrometry; Biomarkers; Proteome; Body Fluids
PubMed: 37209816
DOI: 10.1016/j.mcpro.2023.100577 -
Nature Methods Oct 2023Single-cell proteomics by mass spectrometry is emerging as a powerful and unbiased method for the characterization of biological heterogeneity. So far, it has been...
Single-cell proteomics by mass spectrometry is emerging as a powerful and unbiased method for the characterization of biological heterogeneity. So far, it has been limited to cultured cells, whereas an expansion of the method to complex tissues would greatly enhance biological insights. Here we describe single-cell Deep Visual Proteomics (scDVP), a technology that integrates high-content imaging, laser microdissection and multiplexed mass spectrometry. scDVP resolves the context-dependent, spatial proteome of murine hepatocytes at a current depth of 1,700 proteins from a cell slice. Half of the proteome was differentially regulated in a spatial manner, with protein levels changing dramatically in proximity to the central vein. We applied machine learning to proteome classes and images, which subsequently inferred the spatial proteome from imaging data alone. scDVP is applicable to healthy and diseased tissues and complements other spatial proteomics and spatial omics technologies.
Topics: Animals; Mice; Proteome; Mass Spectrometry; Proteomics; Laser Capture Microdissection
PubMed: 37783884
DOI: 10.1038/s41592-023-02007-6 -
Nature Chemical Biology Nov 2023Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive...
Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events affect protein function, however, remains challenging. Here we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation. The resulting atlas, which covers more than 13,800 cysteines on more than 1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in more than 160 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.
Topics: Humans; Cysteine; Proteomics; Gene Editing; Proteome; Neoplasms; Nuclear Proteins
PubMed: 37783940
DOI: 10.1038/s41589-023-01428-w -
Molecular Systems Biology Sep 2023Single-cell proteomics aims to characterize biological function and heterogeneity at the level of proteins in an unbiased manner. It is currently limited in proteomic...
Single-cell proteomics aims to characterize biological function and heterogeneity at the level of proteins in an unbiased manner. It is currently limited in proteomic depth, throughput, and robustness, which we address here by a streamlined multiplexed workflow using data-independent acquisition (mDIA). We demonstrate automated and complete dimethyl labeling of bulk or single-cell samples, without losing proteomic depth. Lys-N digestion enables five-plex quantification at MS1 and MS2 level. Because the multiplexed channels are quantitatively isolated from each other, mDIA accommodates a reference channel that does not interfere with the target channels. Our algorithm RefQuant takes advantage of this and confidently quantifies twice as many proteins per single cell compared to our previous work (Brunner et al, PMID 35226415), while our workflow currently allows routine analysis of 80 single cells per day. Finally, we combined mDIA with spatial proteomics to increase the throughput of Deep Visual Proteomics seven-fold for microdissection and four-fold for MS analysis. Applying this to primary cutaneous melanoma, we discovered proteomic signatures of cells within distinct tumor microenvironments, showcasing its potential for precision oncology.
Topics: Humans; Proteome; Melanoma; Proteomics; Precision Medicine; Skin Neoplasms; Tumor Microenvironment
PubMed: 37602975
DOI: 10.15252/msb.202211503 -
Science (New York, N.Y.) Apr 2024Chemical modulation of proteins enables a mechanistic understanding of biology and represents the foundation of most therapeutics. However, despite decades of research,...
Chemical modulation of proteins enables a mechanistic understanding of biology and represents the foundation of most therapeutics. However, despite decades of research, 80% of the human proteome lacks functional ligands. Chemical proteomics has advanced fragment-based ligand discovery toward cellular systems, but throughput limitations have stymied the scalable identification of fragment-protein interactions. We report proteome-wide maps of protein-binding propensity for 407 structurally diverse small-molecule fragments. We verified that identified interactions can be advanced to active chemical probes of E3 ubiquitin ligases, transporters, and kinases. Integrating machine learning binary classifiers further enabled interpretable predictions of fragment behavior in cells. The resulting resource of fragment-protein interactions and predictive models will help to elucidate principles of molecular recognition and expedite ligand discovery efforts for hitherto undrugged proteins.
Topics: Humans; Drug Discovery; Ligands; Machine Learning; Protein Binding; Proteome; Proteomics; Small Molecule Libraries; Ubiquitin-Protein Ligases
PubMed: 38662832
DOI: 10.1126/science.adk5864 -
Current Opinion in Biotechnology Apr 2024In recent years, single-cell proteomics (SCP) has advanced significantly, enabling the analysis of thousands of proteins within single mammalian cells. This progress is... (Review)
Review
In recent years, single-cell proteomics (SCP) has advanced significantly, enabling the analysis of thousands of proteins within single mammalian cells. This progress is driven by advances in experimental design, with maturing label-free and multiplexed methods, optimized sample preparation, and innovations in separation techniques, including ultra-low-flow nanoLC. These factors collectively contribute to improved sensitivity, throughput, and reproducibility. Cutting-edge mass spectrometry platforms and data acquisition approaches continue to play a critical role in enhancing data quality. Furthermore, the exploration of spatial proteomics with single-cell resolution offers significant promise for understanding cellular interactions, giving rise to various phenotypes. SCP has far-reaching applications in cancer research, biomarker discovery, and developmental biology. Here, we provide a critical review of recent advances in the field of SCP.
Topics: Animals; Proteomics; Reproducibility of Results; Mass Spectrometry; Proteins; Proteome; Mammals
PubMed: 38359605
DOI: 10.1016/j.copbio.2024.103077 -
Cell Systems Nov 2023Spatial proteomics combining microscopy-based cell phenotyping with ultrasensitive mass-spectrometry-based proteomics is an emerging and powerful concept to study cell...
Spatial proteomics combining microscopy-based cell phenotyping with ultrasensitive mass-spectrometry-based proteomics is an emerging and powerful concept to study cell function and heterogeneity in (patho)physiology. However, optimized workflows that preserve morphological information for phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected tissue are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low-input archival material. Benchmarking in murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions. Applied to human tonsil, we profiled 146 microregions including T and B lymphocyte niches and quantified cell-type-specific markers, cytokines, and transcription factors. These data also highlighted proteome dynamics within activated germinal centers, illuminating sites undergoing B cell proliferation and somatic hypermutation. This approach has broad implications in biomedicine, including early disease profiling and drug target and biomarker discovery. A record of this paper's transparent peer review process is included in the supplemental information.
Topics: Humans; Animals; Mice; Proteome; Proteomics; Mass Spectrometry
PubMed: 37909047
DOI: 10.1016/j.cels.2023.10.003