دورية أكاديمية
An Automated Nanowell-Array Workflow for Quantitative Multiplexed Single-Cell Proteomics Sample Preparation at High Sensitivity.
| العنوان: | An Automated Nanowell-Array Workflow for Quantitative Multiplexed Single-Cell Proteomics Sample Preparation at High Sensitivity. |
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| المؤلفون: | Ctortecka C; Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. Electronic address: cctortec@broadinstitute.org., Hartlmayr D; Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria; Cellenion SASU, Lyon, France., Seth A; Cellenion SASU, Lyon, France., Mendjan S; Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Vienna, Austria., Tourniaire G; Cellenion SASU, Lyon, France., Udeshi ND; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA., Carr SA; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. Electronic address: scarr@broad.mit.edu., Mechtler K; Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria; Cellenion SASU, Lyon, France; Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Vienna, Austria; The Gregor Mendel Institute of Molecular Plant Biology of the Austrian Academy of Sciences (GMI), Vienna BioCenter (VBC), Vienna, Austria. Electronic address: Karl.mechtler@imp.ac.at. |
| المصدر: | Molecular & cellular proteomics : MCP [Mol Cell Proteomics] 2023 Dec; Vol. 22 (12), pp. 100665. Date of Electronic Publication: 2023 Oct 14. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology Country of Publication: United States NLM ID: 101125647 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1535-9484 (Electronic) Linking ISSN: 15359476 NLM ISO Abbreviation: Mol Cell Proteomics Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: 2021- : [New York, NY] : Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology Original Publication: Bethesda, MD : American Society for Biochemistry and Molecular Biology, [2002- |
| مواضيع طبية MeSH: | Proteomics*/methods , Proteome*/metabolism, Humans ; Workflow ; Mass Spectrometry/methods |
| مستخلص: | Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lags. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation including multiplexed labeling up to 16-plex with high sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE, reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1500 protein groups per TMT10-plex with reduced or eliminated need for a carrier proteome. We evaluated the effect of wider precursor isolation windows at single-cell input levels and found that using 2 Da isolation windows increased overall sensitivity without significantly impacting interference. Using the dedicated mass spectrometry acquisition strategies detailed here, we identified on average close to 2000 proteins per TMT10-plex across 170 multiplexed single cells that readily distinguished human cell types. Overall, our workflow combines highly efficient sample preparation, chromatographic and ion mobility-based filtering, rapid wide-window data-dependent acquisition analysis, and intelligent data analysis for optimal multiplexed single-cell proteomics. This versatile and automated proteoCHIP-based sample preparation approach is sufficiently sensitive to drive biological applications of single-cell proteomics and can be readily adopted by proteomics laboratories. Competing Interests: Conflict of interest D. H. (since 02/2022), A. S. and G. T. are employees of Cellenion. S. A. C. is a member of the scientific advisory boards of Kymera, PTM BioLabs, Seer and PrognomIQ. All other authors declare that they have no conflicts of interest with the contents of this article. (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.) |
| معلومات مُعتمدة: | P 35045 Austria FWF_ Austrian Science Fund FWF; P01 CA206978 United States CA NCI NIH HHS; U01 CA271402 United States CA NCI NIH HHS; U24 CA270823 United States CA NCI NIH HHS |
| فهرسة مساهمة: | Keywords: automated sample preparation; high field asymmetric waveform ion mobility spectrometry; isobaric peptide labeling; proteoCHIP; single-cell proteomics |
| المشرفين على المادة: | 0 (Proteome) |
| تواريخ الأحداث: | Date Created: 20231015 Date Completed: 20231222 Latest Revision: 20240130 |
| رمز التحديث: | 20240130 |
| مُعرف محوري في PubMed: | PMC10684380 |
| DOI: | 10.1016/j.mcpro.2023.100665 |
| PMID: | 37839701 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 1535-9484 |
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| DOI: | 10.1016/j.mcpro.2023.100665 |