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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 -
Archives of Disease in Childhood.... Jun 2021Proteomics is the study of a large number of proteins in biological systems. We aim to introduce the complex field to paediatricians and present some recent examples of...
Proteomics is the study of a large number of proteins in biological systems. We aim to introduce the complex field to paediatricians and present some recent examples of applications to paediatric problems. Various approaches have been used to study proteomes. The current mainstay is tandem mass spectrometry of enzymatically digested proteins ('bottom-up proteomics'), and we describe the experimental and computational approach further. Proteomics can offer advantages over transcriptomics by giving direct information about proteins rather than RNA; however, typically data are obtained at lower depth and the confident identification of mass spectra can be challenging. Proteomics frequently complements transcriptomics and other -omics. Used effectively, proteomics offers promise to help answer important clinical and biological questions.
Topics: Child; Humans; Proteome; Proteomics; Tandem Mass Spectrometry; Transcriptome
PubMed: 32241812
DOI: 10.1136/archdischild-2019-317434 -
Current Protein & Peptide Science 2021In the current omics-age of research, major developments have been made in technologies that attempt to survey the entire repertoire of genes, transcripts, proteins, and... (Review)
Review
In the current omics-age of research, major developments have been made in technologies that attempt to survey the entire repertoire of genes, transcripts, proteins, and metabolites present within a cell. While genomics has led to a dramatic increase in our understanding of such things as disease morphology and how organisms respond to medications, it is critical to obtain information at the proteome level since proteins carry out most of the functions within the cell. The primary tool for obtaining proteome-wide information on proteins within the cell is mass spectrometry (MS). While it has historically been associated with the protein identification, developments over the past couple of decades have made MS a robust technology for protein quantitation as well. Identifying quantitative changes in proteomes is complicated by its dynamic nature and the inability of any technique to guarantee complete coverage of every protein within a proteome sample. Fortunately, the combined development of sample preparation and MS methods have made it capable of quantitatively comparing many thousands of proteins obtained from cells and organisms.
Topics: Chromatography, Liquid; Humans; Isotope Labeling; Peptide Mapping; Peptides; Proteome; Proteomics; Software; Staining and Labeling; Tandem Mass Spectrometry
PubMed: 32957902
DOI: 10.2174/1389203721666200921153513 -
Molecular Systems Biology Mar 2022Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular...
Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10-fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable-core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease.
Topics: Mass Spectrometry; Protein Processing, Post-Translational; Proteome; Proteomics; Workflow
PubMed: 35226415
DOI: 10.15252/msb.202110798 -
Annual Review of Plant Biology May 2022Proteins are intimately involved in executing and controlling virtually all cellular processes. To understand the molecular mechanisms that underlie plant phenotypes, it... (Review)
Review
Proteins are intimately involved in executing and controlling virtually all cellular processes. To understand the molecular mechanisms that underlie plant phenotypes, it is essential to investigate protein expression, interactions, and modifications, to name a few. The proteome is highly dynamic in time and space, and a plethora of protein modifications, protein interactions, and network constellations are at play under specific conditions and developmental stages. Analysis of proteomes aims to characterize the entire protein complement of a particular cell type, tissue, or organism-a challenging task, given the dynamic nature of the proteome. Modern mass spectrometry-based proteomics technology can be used to address this complexity at a system-wide scale by the global identification and quantification of thousands of proteins. In this review, we present current methods and technologies employed in mass spectrometry-based proteomics and provide examples of dynamic changes in the plant proteome elucidated by proteomic approaches.
Topics: Mass Spectrometry; Plants; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 35138880
DOI: 10.1146/annurev-arplant-102620-031308 -
EMBO Molecular Medicine Apr 2021The correspondence of cell state changes in diseased organs to peripheral protein signatures is currently unknown. Here, we generated and integrated single-cell...
The correspondence of cell state changes in diseased organs to peripheral protein signatures is currently unknown. Here, we generated and integrated single-cell transcriptomic and proteomic data from multiple large pulmonary fibrosis patient cohorts. Integration of 233,638 single-cell transcriptomes (n = 61) across three independent cohorts enabled us to derive shifts in cell type proportions and a robust core set of genes altered in lung fibrosis for 45 cell types. Mass spectrometry analysis of lung lavage fluid (n = 124) and plasma (n = 141) proteomes identified distinct protein signatures correlated with diagnosis, lung function, and injury status. A novel SSTR2+ pericyte state correlated with disease severity and was reflected in lavage fluid by increased levels of the complement regulatory factor CFHR1. We further discovered CRTAC1 as a biomarker of alveolar type-2 epithelial cell health status in lavage fluid and plasma. Using cross-modal analysis and machine learning, we identified the cellular source of biomarkers and demonstrated that information transfer between modalities correctly predicts disease status, suggesting feasibility of clinical cell state monitoring through longitudinal sampling of body fluid proteomes.
Topics: Biomarkers; Bronchoalveolar Lavage Fluid; Calcium-Binding Proteins; Humans; Proteome; Proteomics; Pulmonary Fibrosis
PubMed: 33650774
DOI: 10.15252/emmm.202012871 -
EMBO Molecular Medicine Nov 2019Plasma and serum are rich sources of information regarding an individual's health state, and protein tests inform medical decision making. Despite major investments, few...
Plasma and serum are rich sources of information regarding an individual's health state, and protein tests inform medical decision making. Despite major investments, few new biomarkers have reached the clinic. Mass spectrometry (MS)-based proteomics now allows highly specific and quantitative readout of the plasma proteome. Here, we employ Plasma Proteome Profiling to define quality marker panels to assess plasma samples and the likelihood that suggested biomarkers are instead artifacts related to sample handling and processing. We acquire deep reference proteomes of erythrocytes, platelets, plasma, and whole blood of 20 individuals (> 6,000 proteins), and compare serum and plasma proteomes. Based on spike-in experiments, we determine sample quality-associated proteins, many of which have been reported as biomarker candidates as revealed by a comprehensive literature survey. We provide sample preparation guidelines and an online resource ( www.plasmaproteomeprofiling.org) to assess overall sample-related bias in clinical studies and to prevent costly miss-assignment of biomarker candidates.
Topics: Bias; Biomarkers; Female; Germany; Healthy Volunteers; Humans; Male; Plasma; Proteome; Proteomics; Specimen Handling
PubMed: 31566909
DOI: 10.15252/emmm.201910427 -
Nature Communications Jul 2022The dia-PASEF technology uses ion mobility separation to reduce signal interferences and increase sensitivity in proteomic experiments. Here we present a two-dimensional...
The dia-PASEF technology uses ion mobility separation to reduce signal interferences and increase sensitivity in proteomic experiments. Here we present a two-dimensional peak-picking algorithm and generation of optimized spectral libraries, as well as take advantage of neural network-based processing of dia-PASEF data. Our computational platform boosts proteomic depth by up to 83% compared to previous work, and is specifically beneficial for fast proteomic experiments and those with low sample amounts. It quantifies over 5300 proteins in single injections recorded at 200 samples per day throughput using Evosep One chromatography system on a timsTOF Pro mass spectrometer and almost 9000 proteins in single injections recorded with a 93-min nanoflow gradient on timsTOF Pro 2, from 200 ng of HeLa peptides. A user-friendly implementation is provided through the incorporation of the algorithms in the DIA-NN software and by the FragPipe workflow for spectral library generation.
Topics: Data Analysis; Humans; Mass Spectrometry; Peptides; Proteome; Proteomics
PubMed: 35803928
DOI: 10.1038/s41467-022-31492-0 -
Biomolecules Jun 2023In the last two decades, our knowledge of synaptic proteomes and their relationship to normal brain function and neuropsychiatric disorders has been expanding rapidly... (Review)
Review
In the last two decades, our knowledge of synaptic proteomes and their relationship to normal brain function and neuropsychiatric disorders has been expanding rapidly through the use of more powerful neuroproteomic approaches. However, mass spectrometry (MS)-based neuroproteomic studies of synapses still require cell-type, spatial, and temporal proteome information. With the advancement of sample preparation and MS techniques, we have just begun to identify and understand proteomes within a given cell type, subcellular compartment, and cell-type-specific synapse. Here, we review the progress and limitations of MS-based neuroproteomics of synapses in the mammalian CNS and highlight the recent applications of these approaches in studying neuropsychiatric disorders such as major depressive disorder and substance use disorders. Combining neuroproteomic findings with other omics studies can generate an in-depth, comprehensive map of synaptic proteomes and possibly identify new therapeutic targets and biomarkers for several central nervous system disorders.
Topics: Animals; Brain; Depressive Disorder, Major; Mammals; Proteome; Proteomics; Synapses
PubMed: 37371578
DOI: 10.3390/biom13060998