Spatial single-cell mass spectrometry defines zonation of the hepatocyte proteome

Nature Methods, Год журнала: 2023, Номер 20(10), С. 1530 - 1536

Опубликована: Окт. 1, 2023

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 complex tissues would greatly enhance insights. Here we describe single-cell Deep Visual Proteomics (scDVP), technology that integrates high-content imaging, laser microdissection multiplexed spectrometry. scDVP resolves context-dependent, spatial proteome murine hepatocytes at current depth 1,700 proteins from cell slice. Half was differentially regulated in manner, with protein levels changing dramatically proximity central vein. We applied machine learning classes images, which subsequently inferred imaging data alone. applicable healthy diseased complements other omics technologies.

Язык: Английский

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MS-Based Proteomics of Body Fluids: The End of the Beginning

Molecular & Cellular Proteomics, Год журнала: 2023, Номер 22(7), С. 100577 - 100577

Опубликована: Май 19, 2023

Accurate biomarkers are a crucial and necessary precondition for precision medicine, yet existing ones often unspecific new have been very slow to enter the clinic. Mass spectrometry (MS)-based proteomics excels by its untargeted nature, specificity of identification, quantification, making it an ideal technology biomarker discovery routine measurement. It has unique attributes compared affinity binder technologies, such as OLINK Proximity Extension Assay SOMAscan. In in previous review 2017, we described technological conceptual limitations that had held back success. We proposed 'rectangular strategy' better separate true minimizing cohort-specific effects. Today, this converged with advances MS-based technology, increased sample throughput, depth quantification. As result, studies become more successful, producing candidates withstand independent verification and, some cases, already outperform state-of-the-art clinical assays. summarize developments over last years, including benefits large cohorts, which acceptance. Shorter gradients, scan modes, multiplexing about drastically increase cross-study integration, proxies absolute levels. found multiprotein panels inherently robust than current single analyte tests capture complexity human phenotypes. Routine MS measurement clinic is fast becoming viable option. The full set proteins body fluid (global proteome) most important reference best process control. Additionally, increasingly all information could be obtained from targeted analysis although latter may straightforward way regular use. Many challenges remain, not least regulatory ethical but outlook applications never brighter.

Язык: Английский

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Acquisition and Analysis of DIA-Based Proteomic Data: A Comprehensive Survey in 2023

Molecular & Cellular Proteomics, Год журнала: 2024, Номер 23(2), С. 100712 - 100712

Опубликована: Янв. 4, 2024

Data-independent acquisition (DIA) mass spectrometry (MS) has emerged as a powerful technology for high-throughput, accurate and reproducible quantitative proteomics. This review provides comprehensive overview of recent advances in both the experimental computational methods DIA proteomics, from data schemes to analysis strategies software tools. are categorized based on design precursor isolation windows, highlighting wide-window, overlapping-window, narrow-window, scanning quadrupole-based, parallel accumulation-serial fragmentation (PASEF)-enhanced methods. For analysis, major classified into spectrum reconstruction, sequence-based search, library-based de novo sequencing sequencing-independent approaches. A wide array tools implementing these reviewed, with details their overall workflows scoring approaches at different steps. The generation optimization spectral libraries, which critical resources also discussed. Publicly available benchmark datasets covering global proteomics phosphoproteomics summarized facilitate performance evaluation various workflows. Continued synergistic developments versatile components expected further enhance power DIA-based

Язык: Английский

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Automated single-cell proteomics providing sufficient proteome depth to study complex biology beyond cell type classifications

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Июль 8, 2024

Abstract The recent technological and computational advances in mass spectrometry-based single-cell proteomics have pushed the boundaries of sensitivity throughput. However, reproducible quantification thousands proteins within a single cell remains challenging. To address some those limitations, we present dedicated sample preparation chip, proteoCHIP EVO 96 that directly interfaces with Evosep One. This, combination Bruker timsTOF demonstrates double identifications without manual handling newest generation Ultra identifies up to 4000 an average 3500 protein groups per HEK-293T carrier or match-between runs. Our workflow spans 4 orders magnitude, over 50 E3 ubiquitin-protein ligases, profiles key regulatory upon small molecule stimulation. This study 96-based provides sufficient proteome depth complex biology beyond cell-type classifications.

Язык: Английский

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Instrumentation at the Leading Edge of Proteomics

Analytical Chemistry, Год журнала: 2024, Номер 96(20), С. 7976 - 8010

Опубликована: Май 13, 2024

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTInstrumentation at the Leading Edge of ProteomicsTrenton M. Peters-ClarkeTrenton Peters-ClarkeDepartment Chemistry, University Wisconsin─Madison, Madison, Wisconsin 53706, United StatesDepartment Biomolecular StatesMore by Trenton Peters-ClarkeView Biographyhttps://orcid.org/0000-0002-9153-2525, Joshua J. CoonJoshua CoonDepartment StatesMorgridge Institute for Research, 53715, CoonView Biographyhttps://orcid.org/0000-0002-0004-8253, and Nicholas Riley*Nicholas RileyDepartment Washington, Seattle, Washington 98195, States*Email: [email protected]More RileyView Biographyhttps://orcid.org/0000-0002-1536-2966Cite this: Anal. Chem. 2024, 96, 20, 7976–8010Publication Date (Web):May 13, 2024Publication History Received6 October 2023Accepted19 April 2024Revised17 2024Published online13 May inissue 21 2024https://pubs.acs.org/doi/10.1021/acs.analchem.3c04497https://doi.org/10.1021/acs.analchem.3c04497review-articleACS PublicationsCopyright © 2024 American Chemical SocietyRequest reuse permissionsArticle Views2104Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are COUNTER-compliant sum full text article downloads since November 2008 (both PDF HTML) across all institutions individuals. These metrics regularly updated to reflect usage leading up last few days.Citations number other articles citing this article, calculated Crossref daily. Find more information about citation counts.The Altmetric Attention Score is a quantitative measure attention that research has received online. Clicking on donut icon will load page altmetric.com with additional details score social media presence given article. how calculated. Share Add toView InAdd Full Text ReferenceAdd Description ExportRISCitationCitation abstractCitation referencesMore Options onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Dissociation,Ions,Mass spectrometry,Peptides proteins,Proteomics Get e-Alerts

Язык: Английский

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Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry

ACS Measurement Science Au, Год журнала: 2024, Номер 4(4), С. 338 - 417

Опубликована: Июнь 4, 2024

Proteomics is the large scale study of protein structure and function from biological systems through identification quantification."Shotgun proteomics" or "bottom-up prevailing strategy, in which proteins are hydrolyzed into peptides that analyzed by mass spectrometry.Proteomics studies can be applied to diverse ranging simple proteoforms, protein-protein interactions, structural alterations, absolute relative quantification, post-translational modifications, stability.To enable this range different experiments, there strategies for proteome analysis.The nuances how proteomic workflows differ may challenging understand new practitioners.Here, we provide a comprehensive overview proteomics methods.We cover biochemistry basics extraction interpretation orthogonal validation.We expect Review will serve as handbook researchers who field bottom-up proteomics.

Язык: Английский

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Spatial proteomics identifies JAKi as treatment for a lethal skin disease

Nature, Год журнала: 2024, Номер 635(8040), С. 1001 - 1009

Опубликована: Окт. 16, 2024

Abstract Toxic epidermal necrolysis (TEN) is a fatal drug-induced skin reaction triggered by common medications and an emerging public health issue 1–3 . Patients with TEN undergo severe sudden detachment caused keratinocyte cell death. Although molecular mechanisms that drive death have been proposed, the main drivers remain unknown, there no effective therapy for 4–6 Here, to systematically map changes are associated identify potential druggable targets, we utilized deep visual proteomics, which provides single-cell-based, cell-type-resolution proteomics 7,8 We analysed formalin-fixed, paraffin-embedded archived tissue biopsies of three types cutaneous drug reactions varying severity quantified more than 5,000 proteins in keratinocytes skin-infiltrating immune cells. This revealed marked enrichment type I II interferon signatures compartment patients TEN, as well phosphorylated STAT1 activation. Targeted inhibition pan-JAK inhibitor tofacitinib vitro reduced keratinocyte-directed cytotoxicity. In vivo oral administration tofacitinib, baricitinib or JAK1-specific inhibitors abrocitinib upadacitinib ameliorated clinical histological disease two distinct mouse models TEN. Crucially, treatment JAK (JAKi) was safe rapid re-epithelialization recovery seven study uncovers JAK/STAT signalling pathways key pathogenic demonstrates targeted JAKi curative therapy.

Язык: Английский

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Enhanced sensitivity and scalability with a Chip-Tip workflow enables deep single-cell proteomics

Nature Methods, Год журнала: 2025, Номер unknown

Опубликована: Янв. 16, 2025

Single-cell proteomics (SCP) promises to revolutionize biomedicine by providing an unparalleled view of the proteome in individual cells. Here, we present a high-sensitivity SCP workflow named Chip-Tip, identifying >5,000 proteins HeLa It also facilitated direct detection post-translational modifications single cells, making need for specific modification-enrichment unnecessary. Our study demonstrates feasibility processing up 120 label-free samples per day. An optimized tissue dissociation buffer enabled effective single-cell disaggregation drug-treated cancer cell spheroids, refining overall analysis. Analyzing nondirected human-induced pluripotent stem differentiation, consistently quantified markers OCT4 and SOX2 cells lineage such as GATA4 (endoderm), HAND1 (mesoderm) MAP2 (ectoderm) different embryoid body sets benchmark sensitivity throughput, with broad applications basic biology identification type-specific therapeutic targets.

Язык: Английский

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Rapid, One-Step Sample Processing for Label-Free Single-Cell Proteomics

Journal of the American Society for Mass Spectrometry, Год журнала: 2023, Номер 34(8), С. 1701 - 1707

Опубликована: Июль 6, 2023

Sample preparation for single-cell proteomics is generally performed in a one-pot workflow with multiple dispensing and incubation steps. These hours-long processes can be labor intensive lead to long sample-to-answer times. Here we report sample method that achieves cell lysis, protein denaturation, digestion 1 h using commercially available high-temperature-stabilized proteases single reagent step. Four different one-step compositions were evaluated, the mixture providing highest proteome coverage was compared previously employed multistep workflow. The increases relative previous while minimizing input possibility of human error. We also recovery between used microfabricated glass nanowell chips injection-molded polypropylene found provided improved coverage. Combined, substrates enabled identification an average nearly 2400 proteins per standard data-dependent Orbitrap mass spectrometers. advances greatly simplify broaden accessibility no compromise terms

Язык: Английский

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What’s new in single-cell proteomics

Current Opinion in Biotechnology, Год журнала: 2024, Номер 86, С. 103077 - 103077

Опубликована: Фев. 14, 2024

Язык: Английский

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