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 fiveplex
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.
[doi:10.25345/C5T14TW0D]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: data independent acquisition ; dimethyl labeling ; mass spectrometry ; multiplexing ; single cells ; spatial proteomics
Principal Investigators: (in alphabetical order) |
Prof. Dr. Matthias Mann, Dept. Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Munich, Germany, N/A |
Submitting User: | oroshi |
Thielert M, Itang EC, Ammar C, Rosenberger FA, Bludau I, Schweizer L, Nordmann TM, Skowronek P, Wahle M, Zeng WF, Zhou XX, Brunner AD, Richter S, Levesque MP, Theis FJ, Steger M, Mann M.
Robust dimethyl-based multiplex-DIA doubles single-cell proteome depth via a reference channel.
Mol Syst Biol. Epub 2023 Aug 21.
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