Single-cell proteomics enabled by next-generation sequencing or mass spectrometry

Nature Methods, Journal Year: 2023, Volume and Issue: 20(3), P. 363 - 374

Published: March 1, 2023

Language: Английский

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Exploration of cell state heterogeneity using single-cell proteomics through sensitivity-tailored data-independent acquisition

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: Sept. 22, 2023

Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount material encapsulated in a single cell however, raises significant technical challenges molecular profiling. Due extensive optimization efforts, single-cell proteomics by Mass Spectrometry (scp-MS) has emerged as powerful tool facilitate proteome profiling from ultra-low amounts input, although further development needed realize its full potential. To this end, we carry out comprehensive orbitrap-based data-independent acquisition (DIA) for proteomics. Notably, find difference between optimal DIA methods high- low-load samples. We improve our low-input method relying on high-resolution MS1 quantification, thus enhancing sensitivity more efficiently utilizing available mass analyzer time. With input tailored method, are able accommodate long injection times high resolution, while keeping the scan cycle time low enough ensure robust quantification. Finally, demonstrate capability approach mouse embryonic stem culture conditions, showcasing global proteomes highlighting differences key metabolic enzyme expression subclusters.

Language: Английский

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

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: July 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.

Language: Английский

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Instrumentation at the Leading Edge of Proteomics

Analytical Chemistry, Journal Year: 2024, Volume and Issue: 96(20), P. 7976 - 8010

Published: May 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

Language: Английский

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Recent advances in the field of single-cell proteomics

Translational Oncology, Journal Year: 2022, Volume and Issue: 27, P. 101556 - 101556

Published: Oct. 19, 2022

The field of single-cell omics is rapidly progressing. Although DNA and RNA sequencing-based methods have dominated the to date, global proteome profiling has also entered main stage. Single-cell proteomics was facilitated by advancements in different aspects mass spectrometry (MS)-based proteomics, such as instrument design, sample preparation, chromatography ion mobility. (scp-MS) moved beyond being a mere technical development, now able deliver actual biological application been successfully applied characterize cell states. Here, we review some key developments scp-MS, provide background field, discuss various available foresee possible future directions.

Language: Английский

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Slice-PASEF: fragmenting all ions for maximum sensitivity in proteomics

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2022, Volume and Issue: unknown

Published: Oct. 31, 2022

Abstract We present Slice-PASEF, a novel mass spectrometry technology based on trapped ion mobility separation of ions. Slice-PASEF allows to achieve the theoretical maximum MS/MS sensitivity and boosts proteomics low sample amounts. Leveraging we show, for first time, that comprehensive profiling single cell-level peptide amounts is possible using ultra-fast microflow chromatography general-purpose spectrometer, allowing quantification 1417 proteins from 200 picograms HeLa cell standard an Evosep One LC system coupled timsTOF Pro 2, at samples per day throughput. implemented module in our DIA-NN data processing software, make it readily available community.

Language: Английский

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Label‐free single cell proteomics utilizing ultrafast LC and MS instrumentation: A valuable complementary technique to multiplexing

PROTEOMICS, Journal Year: 2023, Volume and Issue: 23(13-14)

Published: Feb. 19, 2023

The ability to map a proteomic fingerprint transcriptomic data would master the understanding of how gene expression translates into actual phenotype. In contrast nucleic acid sequencing, in vitro protein amplification is impossible and no single cell workflow has been established as gold standard yet. Advances microfluidic sample preparation, multi-dimensional separation, sophisticated acquisition strategies, intelligent analysis algorithms have resulted major improvements successfully analyze such tiny amounts with steadily boosted performance. However, among broad variation published approaches, it commonly accepted that highest possible sensitivity, robustness, throughput are still most urgent needs for field. While many labs focused on multiplexing achieve these goals, label-free SCP highly promising strategy well whenever high dynamic range unbiased accurate quantification needed. We here focus recent advances single-cell mass spectrometry workflows try guide our readers choose best method or combinations methods their specific applications. further highlight which techniques propitious future applications but also limitations we foresee

Language: Английский

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

Journal of the American Society for Mass Spectrometry, Journal Year: 2023, Volume and Issue: 34(8), P. 1701 - 1707

Published: July 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

Language: Английский

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High-Throughput Single-Cell Proteomic Analysis of Organ-Derived Heterogeneous Cell Populations by Nanoflow Dual-Trap Single-Column Liquid Chromatography

Analytical Chemistry, Journal Year: 2023, Volume and Issue: 95(24), P. 9145 - 9150

Published: June 8, 2023

Identification and proteomic characterization of rare cell types within complex organ-derived mixtures is best accomplished by label-free quantitative mass spectrometry. High throughput required to rapidly survey hundreds thousands individual cells adequately represent populations. Here we present parallelized nanoflow dual-trap single-column liquid chromatography (nanoDTSC) operating at 15 min total run time per with peptides quantified over 11.5 using standard commercial components, thus offering an accessible efficient LC solution analyze 96 single day. At this throughput, nanoDTSC 1000 proteins in cardiomyocytes heterogeneous populations from the aorta.

Language: Английский

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Easy and Accessible Workflow for Label-Free Single-Cell Proteomics

Journal of the American Society for Mass Spectrometry, Journal Year: 2023, Volume and Issue: 34(10), P. 2374 - 2380

Published: Aug. 18, 2023

Single-cell proteomics (SCP) can provide information that is unattainable through either bulk-scale protein measurements or single-cell profiling of other omes. Maximizing proteome coverage often requires custom instrumentation, consumables, and reagents for sample processing separations, which has limited the accessibility SCP to a small number specialized laboratories. Commercial platforms have become available cell isolation preparation, but high cost these technical expertise required their operation place them out reach many interested Here, we assessed new HP D100 Single Cell Dispenser label-free SCP. The low-cost instrument proved highly accurate reproducible dispensing in range from 200 nL 2 μL. We used isolate prepare single cells within 384-well PCR plates. When well plates were immediately centrifuged following again after reagent dispensing, found ∼97% wells identified software as containing indeed provided expected cell. This commercial dispenser combined with one-step provides very rapid easy-to-use workflow no reduction relative nanowell-based workflow, also facilitate autosampling unmodified instrumentation. samples analyzed using home-packed 30 μm i.d. nanoLC columns commercially 50 columns. resulted ∼35% fewer proteins. However, plate-based preparation platform, presented fully relatively alternative separation, should greatly broaden

Language: Английский

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