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Thielert, M.* ; Itang, E.C.* ; Ammar, C.* ; Rosenberger, F.A.* ; Bludau, I.* ; Schweizer, L.* ; Nordmann, T.M.* ; Skowronek, P.* ; Wahle, M.* ; Zeng, W.F.* ; Zhou, X.X.* ; Brunner, A.D.* ; Richter, S. ; Levesque, M.P.* ; Theis, F.J. ; Steger, M.* ; Mann, M.*

Robust dimethyl-based multiplex-DIA doubles single-cell proteome depth via a reference channel.

Mol. Syst. Biol. 19:e11503 (2023)

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Open Access Gold as soon as Publ. Version/Full Text is submitted to ZB.

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Single-cell proteomics aims to characterize biological function and heterogeneity at the level of proteins in an unbiased manner. It is currently limited in proteomic depth, throughput, and robustness, which we address here by a streamlined multiplexed workflow using data-independent acquisition (mDIA). We demonstrate automated and complete dimethyl labeling of bulk or single-cell samples, without losing proteomic depth. Lys-N digestion enables five-plex quantification at MS1 and MS2 level. Because the multiplexed channels are quantitatively isolated from each other, mDIA accommodates a reference channel that does not interfere with the target channels. Our algorithm RefQuant takes advantage of this and confidently quantifies twice as many proteins per single cell compared to our previous work (Brunner et al, PMID 35226415), while our workflow currently allows routine analysis of 80 single cells per day. Finally, we combined mDIA with spatial proteomics to increase the throughput of Deep Visual Proteomics seven-fold for microdissection and four-fold for MS analysis. Applying this to primary cutaneous melanoma, we discovered proteomic signatures of cells within distinct tumor microenvironments, showcasing its potential for precision oncology.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

Keywords Dia ; Dimethyl Labeling ; Multiplexing ; Single Cells ; Spatial Proteomics; Quantification; Extraction

ISSN (print) / ISBN 1744-4292

e-ISSN 1744-4292

Journal Molecular Systems Biology

Quellenangaben Volume: 19, Issue: 9, Article Number: e11503

Publisher EMBO Press

Publishing Place 111 River St, Hoboken 07030-5774, Nj Usa

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Institute of Computational Biology (ICB)

Grants Projekt DEAL
Imaging Core Facility at the Max Planck Institute of Biochemistry
Helmholtz Association
International Max Planck Research School for Life Sciences - IMPRS-LS
Bavarian State Ministry of Health and Care through the research project DigiMed Bayern
Deutsche Forschungsgemeinschaft project
European Union
Max Planck Society for Advancement of Science