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Real-time search assisted acquisition on a tribrid mass spectrometer improves coverage in multiplexed single-cell proteomics

bioRxiv, ISSN: 2692-8205
2021
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  • Citations
    4
    • Citation Indexes
      4
      • CrossRef
        4

Article Description

In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed single-cell proteomics requires high ion injection times and high-resolution spectra to quantify the single-cell signal, however the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and present a new acquisition strategy termed RETICLE (RTS Enhanced Quant of Single Cell Spectra) that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. We show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at a MS2 injection time of 750ms using a 2h gradient.

Bibliographic Details

Benjamin Furtwängler; Nil Üresin; Bo T. Porse; Erwin M. Schoof; Khatereh Motamedchaboki; Romain Huguet; Daniel Lopez-Ferrer; Vlad Zabrouskov

Cold Spring Harbor Laboratory

Biochemistry, Genetics and Molecular Biology; Agricultural and Biological Sciences; Immunology and Microbiology; Neuroscience; Pharmacology, Toxicology and Pharmaceutics

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