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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 -
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 -
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 -
Genome Biology Sep 2023Quantitative proteomics is an indispensable tool in life science research. However, there is a lack of reference materials for evaluating the reproducibility of...
BACKGROUND
Quantitative proteomics is an indispensable tool in life science research. However, there is a lack of reference materials for evaluating the reproducibility of label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based measurements among different instruments and laboratories.
RESULTS
Here, we develop the Quartet standard as a proteome reference material with built-in truths, and distribute the same aliquots to 15 laboratories with nine conventional LC-MS/MS platforms across six cities in China. Relative abundance of over 12,000 proteins on 816 mass spectrometry files are obtained and compared for reproducibility among the instruments and laboratories to ultimately generate proteomics benchmark datasets. There is a wide dynamic range of proteomes spanning about 7 orders of magnitude, and the injection order has marked effects on quantitative instead of qualitative characteristics.
CONCLUSION
Overall, the Quartet offers valuable standard materials and data resources for improving the quality control of proteomic analyses as well as the reproducibility and reliability of research findings.
Topics: Chromatography, Liquid; Proteomics; Reproducibility of Results; Tandem Mass Spectrometry; Proteome
PubMed: 37674236
DOI: 10.1186/s13059-023-03048-y -
Molecular Systems Biology Oct 2014In order to obtain a systems-level understanding of a complex biological system, detailed proteome information is essential. Despite great progress in proteomics...
In order to obtain a systems-level understanding of a complex biological system, detailed proteome information is essential. Despite great progress in proteomics technologies, thorough interrogation of the proteome from quantity-limited biological samples is hampered by inefficiencies during processing. To address these challenges, here we introduce a novel protocol using paramagnetic beads, termed Single-Pot Solid-Phase-enhanced Sample Preparation (SP3). SP3 provides a rapid and unbiased means of proteomic sample preparation in a single tube that facilitates ultrasensitive analysis by outperforming existing protocols in terms of efficiency, scalability, speed, throughput, and flexibility. To illustrate these benefits, characterization of 1,000 HeLa cells and single Drosophila embryos is used to establish that SP3 provides an enhanced platform for profiling proteomes derived from sub-microgram amounts of material. These data present a first view of developmental stage-specific proteome dynamics in Drosophila at a single-embryo resolution, permitting characterization of inter-individual expression variation. Together, the findings of this work position SP3 as a superior protocol that facilitates exciting new directions in multiple areas of proteomics ranging from developmental biology to clinical applications.
Topics: Animals; Drosophila melanogaster; HeLa Cells; Humans; Magnetic Phenomena; Mass Spectrometry; Proteome; Proteomics
PubMed: 25358341
DOI: 10.15252/msb.20145625 -
Annual Review of Virology Sep 2021The abundance, localization, modifications, and protein-protein interactions of many host cell and virus proteins can change dynamically throughout the course of any... (Review)
Review
The abundance, localization, modifications, and protein-protein interactions of many host cell and virus proteins can change dynamically throughout the course of any viral infection. Studying these changes is critical for a comprehensive understanding of how viruses replicate and cause disease, as well as for the development of antiviral therapeutics and vaccines. Previously, we developed a mass spectrometry-based technique called quantitative temporal viromics (QTV), which employs isobaric tandem mass tags (TMTs) to allow precise comparative quantification of host and virus proteomes through a whole time course of infection. In this review, we discuss the utility and applications of QTV, exemplified by numerous studies that have since used proteomics with a variety of quantitative techniques to study virus infection through time.
Topics: Mass Spectrometry; Proteome; Proteomics; Viral Proteins; Viruses
PubMed: 34129369
DOI: 10.1146/annurev-virology-091919-104458 -
International Journal of Molecular... Jan 2017n/a.
n/a.
Topics: Plant Proteins; Plants; Proteome; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 28054969
DOI: 10.3390/ijms18010088 -
Annual Review of Genomics and Human... Aug 2022Proteins are the molecular effectors of the information encoded in the genome. Proteomics aims at understanding the molecular functions of proteins in their biological... (Review)
Review
Proteins are the molecular effectors of the information encoded in the genome. Proteomics aims at understanding the molecular functions of proteins in their biological context. In contrast to transcriptomics and genomics, the study of proteomes provides deeper insight into the dynamic regulatory layers encoded at the protein level, such as posttranslational modifications, subcellular localization, cell signaling, and protein-protein interactions. Currently, mass spectrometry (MS)-based proteomics is the technology of choice for studying proteomes at a system-wide scale, contributing to clinical biomarker discovery and fundamental molecular biology. MS technologies are continuously being developed to fulfill the requirements of speed, resolution, and quantitative accuracy, enabling the acquisition of comprehensive proteomes. In this review, we present how MS technology and acquisition methods have evolved to meet the requirements of cutting-edge proteomics research, which is describing the human proteome and its dynamic posttranslational modifications with unprecedented depth. Finally, we provide a perspective on studying proteomes at single-cell resolution.
Topics: Genome; Humans; Mass Spectrometry; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 35440146
DOI: 10.1146/annurev-genom-112921-024948 -
Molecular & Cellular Proteomics : MCP Mar 2012Deep proteomic analysis of mammalian cell lines would yield an inventory of the building blocks of the most commonly used systems in biological research. Mass... (Comparative Study)
Comparative Study
Deep proteomic analysis of mammalian cell lines would yield an inventory of the building blocks of the most commonly used systems in biological research. Mass spectrometry-based proteomics can identify and quantify proteins in a global and unbiased manner and can highlight the cellular processes that are altered between such systems. We analyzed 11 human cell lines using an LTQ-Orbitrap family mass spectrometer with a "high field" Orbitrap mass analyzer with improved resolution and sequencing speed. We identified a total of 11,731 proteins, and on average 10,361 ± 120 proteins in each cell line. This very high proteome coverage enabled analysis of a broad range of processes and functions. Despite the distinct origins of the cell lines, our quantitative results showed surprisingly high similarity in terms of expressed proteins. Nevertheless, this global similarity of the proteomes did not imply equal expression levels of individual proteins across the 11 cell lines, as we found significant differences in expression levels for an estimated two-third of them. The variability in cellular expression levels was similar for low and high abundance proteins, and even many of the most highly expressed proteins with household roles showed significant differences between cells. Metabolic pathways, which have high redundancy, exhibited variable expression, whereas basic cellular functions such as the basal transcription machinery varied much less. We harness knowledge of these cell line proteomes for the construction of a broad coverage "super-SILAC" quantification standard. Together with the accompanying paper (Schaab, C. MCP 2012, PMID: 22301388) (17) these data can be used to obtain reference expression profiles for proteins of interest both within and across cell line proteomes.
Topics: Cells, Cultured; Chromatography, Liquid; Computational Biology; Humans; Isotope Labeling; Peptide Fragments; Proteome; Proteomics; Tandem Mass Spectrometry
PubMed: 22278370
DOI: 10.1074/mcp.M111.014050