Real-Time Search Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics
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Real-Time Search Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics. / Furtwängler, Benjamin; Üresin, Nil; Motamedchaboki, Khatereh; Huguet, Romain; Lopez-Ferrer, Daniel; Zabrouskov, Vlad; Porse, Bo T; Schoof, Erwin M.
I: Molecular and Cellular Proteomics, Bind 21, Nr. 4, 100219, 2022.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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T1 - Real-Time Search Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics
AU - Furtwängler, Benjamin
AU - Üresin, Nil
AU - Motamedchaboki, Khatereh
AU - Huguet, Romain
AU - Lopez-Ferrer, Daniel
AU - Zabrouskov, Vlad
AU - Porse, Bo T
AU - Schoof, Erwin M
N1 - Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
U2 - 10.1016/j.mcpro.2022.100219
DO - 10.1016/j.mcpro.2022.100219
M3 - Journal article
C2 - 35219906
VL - 21
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
SN - 1535-9476
IS - 4
M1 - 100219
ER -
ID: 301281725