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Molecular & Cellular Proteomics : MCP May 2024We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation,...
We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach.
Topics: Humans; Proteome; Tandem Mass Spectrometry; Proteomics; Time Factors
PubMed: 38579929
DOI: 10.1016/j.mcpro.2024.100760 -
Circulation Research Jan 2022Rapidly changing and transient protein-protein interactions regulate dynamic cellular processes in the cardiovascular system. Traditional methods, including affinity... (Review)
Review
Rapidly changing and transient protein-protein interactions regulate dynamic cellular processes in the cardiovascular system. Traditional methods, including affinity purification and mass spectrometry, have revealed many macromolecular complexes in cardiomyocytes and the vasculature. Yet these methods often fail to identify in vivo or transient protein-protein interactions. To capture these interactions in living cells and animals with subsequent mass spectrometry identification, enzyme-catalyzed proximity labeling techniques have been developed in the past decade. Although the application of this methodology to cardiovascular research is still in its infancy, the field is developing rapidly, and the promise is substantial. In this review, we outline important concepts and discuss how proximity proteomics has been applied to study physiological and pathophysiological processes relevant to the cardiovascular system.
Topics: Animals; Humans; Myocardium; Protein Interaction Mapping; Protein Interaction Maps; Proteome; Proteomics
PubMed: 35050691
DOI: 10.1161/CIRCRESAHA.121.319810 -
Biochimica Et Biophysica Acta. Proteins... Jul 2021Single-cell analysis came to change the way we look at cell populations. RNA sequencing of single cells allowed us to appreciate the diversity of cell types in the human... (Review)
Review
Single-cell analysis came to change the way we look at cell populations. RNA sequencing of single cells allowed us to appreciate the diversity of cell types in the human brain in an unprecedented manner and its power to reveal cell-type specific changes in cell populations has just begun to be explored. In this context, looking at the proteome of single cells promises to bring functional information and contribute to completing the picture. The potential of single cell proteome, in developing a better understanding of the intricate connections between the very diverse cell populations in the brain, is huge. Whereas early approaches to address single-cell proteome have identified hundreds of proteins, today, techniques combining isobaric labelling and LC-MS can lead to the identification of thousands of proteins. In this review, we describe methods which have been used to identify and quantify proteins from single cells and propose that the application of isobaric labeling and label-free quantitative proteomics approach for single-cell analysis is ready to provide useful information for the neurobiology field.
Topics: Animals; Chromatography, Liquid; Humans; Neurobiology; Proteome; Proteomics; Single-Cell Analysis; Tandem Mass Spectrometry
PubMed: 33845200
DOI: 10.1016/j.bbapap.2021.140658 -
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 & Cellular Proteomics : MCP Nov 2019The most straightforward applications of proteomics database searching involve intracellular proteins. Although intracellular gene products number in the thousands,... (Review)
Review
The most straightforward applications of proteomics database searching involve intracellular proteins. Although intracellular gene products number in the thousands, their well-defined post-translational modifications (PTMs) makes database searching practical. By contrast, cell surface and extracellular matrisome proteins pass through the secretory pathway where many become glycosylated, modulating their physicochemical properties, adhesive interactions, and diversifying their functions. Although matrisome proteins number only a few hundred, their high degree of complex glycosylation multiplies the number of theoretical proteoforms by orders of magnitude. Given that extracellular networks that mediate cell-cell and cell-pathogen interactions in physiology depend on glycosylation, it is important to characterize the proteomes, glycomes, and glycoproteomes of matrisome molecules that exist in a given biological context. In this review, we summarize proteomics approaches for characterizing matrisome molecules, with an emphasis on applications to brain diseases. We demonstrate the availability of methods that should greatly increase the availability of information on matrisome molecular structure associated with health and disease.
Topics: Animals; Biomarkers; Extracellular Matrix; Extracellular Matrix Proteins; Glycomics; Glycoproteins; Glycosylation; Humans; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 31471497
DOI: 10.1074/mcp.R119.001543 -
Molecular & Cellular Proteomics : MCP Aug 2023The human proteome comprises of all of the proteins produced by the sequences translated from the human genome with additional modifications in both sequence and... (Review)
Review
The human proteome comprises of all of the proteins produced by the sequences translated from the human genome with additional modifications in both sequence and function caused by nonsynonymous variants and posttranslational modifications including cleavage of the initial transcript into smaller peptides and polypeptides. The UniProtKB database (www.uniprot.org) is the world's leading high-quality, comprehensive and freely accessible resource of protein sequence and functional information and presents a summary of experimentally verified, or computationally predicted, functional information added by our expert biocuration team for each protein in the proteome. Researchers in the field of mass spectrometry-based proteomics both consume and add to the body of data available in UniProtKB, and this review highlights the information we provide to this community and the knowledge we in turn obtain from groups via deposition of large-scale datasets in public domain databases.
Topics: Humans; Proteomics; Proteome; Databases, Protein; Amino Acid Sequence; Peptides
PubMed: 37301379
DOI: 10.1016/j.mcpro.2023.100591 -
NeuroLINCS Proteomics: Defining human-derived iPSC proteomes and protein signatures of pluripotency.Scientific Data Jan 2023The National Institute of Health (NIH) Library of integrated network-based cellular signatures (LINCS) program is premised on the generation of a publicly available data...
The National Institute of Health (NIH) Library of integrated network-based cellular signatures (LINCS) program is premised on the generation of a publicly available data resource of cell-based biochemical responses or "signatures" to genetic or environmental perturbations. NeuroLINCS uses human inducible pluripotent stem cells (hiPSCs), derived from patients and healthy controls, and differentiated into motor neuron cell cultures. This multi-laboratory effort strives to establish i) robust multi-omic workflows for hiPSC and differentiated neuronal cultures, ii) public annotated data sets and iii) relevant and targetable biological pathways of spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Here, we focus on the proteomics and the quality of the developed workflow of hiPSC lines from 6 individuals, though epigenomics and transcriptomics data are also publicly available. Known and commonly used markers representing 73 proteins were reproducibly quantified with consistent expression levels across all hiPSC lines. Data quality assessments, data levels and metadata of all 6 genetically diverse human iPSCs analysed by DIA-MS are parsable and available as a high-quality resource to the public.
Topics: Humans; Induced Pluripotent Stem Cells; Motor Neurons; Pluripotent Stem Cells; Proteome; Proteomics
PubMed: 36631473
DOI: 10.1038/s41597-022-01687-7 -
Current Opinion in Chemical Biology Feb 2020Understanding the molecular mechanisms of endogenous and environmental metabolites is crucial for basic biology and drug discovery. With the genome, proteome, and... (Review)
Review
Understanding the molecular mechanisms of endogenous and environmental metabolites is crucial for basic biology and drug discovery. With the genome, proteome, and metabolome of many organisms being readily available, researchers now have the opportunity to dissect how key metabolites regulate complex cellular pathways in vivo. Nonetheless, characterizing the specific and functional protein targets of key metabolites associated with specific cellular phenotypes remains a major challenge. Innovations in chemical biology are now poised to address this fundamental limitation in physiology and disease. In this review, we highlight recent advances in chemoproteomics for targeted and proteome-wide analysis of metabolite-protein interactions that have enabled the discovery of unpredicted metabolite-protein interactions and facilitated the development of new small molecule therapeutics.
Topics: Humans; Metabolome; Metabolomics; Proteins; Proteome; Proteomics
PubMed: 31790852
DOI: 10.1016/j.cbpa.2019.10.008 -
Nature Biotechnology Jan 2023Current mass spectrometry methods enable high-throughput proteomics of large sample amounts, but proteomics of low sample amounts remains limited in depth and...
Current mass spectrometry methods enable high-throughput proteomics of large sample amounts, but proteomics of low sample amounts remains limited in depth and throughput. To increase the throughput of sensitive proteomics, we developed an experimental and computational framework, called plexDIA, for simultaneously multiplexing the analysis of peptides and samples. Multiplexed analysis with plexDIA increases throughput multiplicatively with the number of labels without reducing proteome coverage or quantitative accuracy. By using three-plex non-isobaric mass tags, plexDIA enables quantification of threefold more protein ratios among nanogram-level samples. Using 1-hour active gradients, plexDIA quantified ~8,000 proteins in each sample of labeled three-plex sets and increased data completeness, reducing missing data more than twofold across samples. Applied to single human cells, plexDIA quantified ~1,000 proteins per cell and achieved 98% data completeness within a plexDIA set while using ~5 minutes of active chromatography per cell. These results establish a general framework for increasing the throughput of sensitive and quantitative protein analysis.
Topics: Humans; Proteomics; Mass Spectrometry; Peptides; Chromatography, Liquid; Proteome
PubMed: 35835881
DOI: 10.1038/s41587-022-01389-w -
Journal of Proteome Research Dec 2023Top-down proteomics (TDP) aims to identify and profile intact protein forms (proteoforms) extracted from biological samples. True proteoform characterization requires... (Review)
Review
Top-down proteomics (TDP) aims to identify and profile intact protein forms (proteoforms) extracted from biological samples. True proteoform characterization requires that both the base protein sequence be defined and any mass shifts identified, ideally localizing their positions within the protein sequence. Being able to fully elucidate proteoform profiles lends insight into characterizing proteoform-unique roles, and is a crucial aspect of defining protein structure-function relationships and the specific roles of different (combinations of) protein modifications. However, defining and pinpointing protein post-translational modifications (PTMs) on intact proteins remains a challenge. Characterization of (heavily) modified proteins (>∼30 kDa) remains problematic, especially when they exist in a population of similarly modified, or kindred, proteoforms. This issue is compounded as the number of modifications increases, and thus the number of theoretical combinations. Here, we present our perspective on the challenges of analyzing kindred proteoform populations, focusing on annotation of protein modifications on an "average" protein. Furthermore, we discuss the technical requirements to obtain high quality fragmentation spectral data to robustly define site-specific PTMs, and the fact that this is tempered by the time requirements necessary to separate proteoforms in advance of mass spectrometry analysis.
Topics: Tandem Mass Spectrometry; Proteomics; Proteins; Protein Processing, Post-Translational; Amino Acid Sequence; Proteome
PubMed: 37937372
DOI: 10.1021/acs.jproteome.3c00416