Real-Time Search-Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics |
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Authors: | Benjamin Furtwängler Nil Üresin Khatereh Motamedchaboki Romain Huguet Daniel Lopez-Ferrer Vlad Zabrouskov Bo T. Porse Erwin M. Schoof |
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Affiliation: | 1. The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark;2. Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark;3. Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark;4. ThermoFisher Scientific, San Jose, California, USA;5. Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark |
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Abstract: | 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 scMS 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 an MS2 injection time of 750 ms using a 2 h gradient. |
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Keywords: | single-cell proteomics isobaric tag quantification multiplexing SPS-MS3 TMT real-time search AGC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0045" }," $$" :[{" #name" :" text" ," _" :" automatic gain control AML" },{" #name" :" keyword" ," $" :{" id" :" kwrd0055" }," $$" :[{" #name" :" text" ," _" :" acute myeloid leukemia CID" },{" #name" :" keyword" ," $" :{" id" :" kwrd0065" }," $$" :[{" #name" :" text" ," _" :" collision-induced dissociation DE" },{" #name" :" keyword" ," $" :{" id" :" kwrd0075" }," $$" :[{" #name" :" text" ," _" :" differential expression HCD" },{" #name" :" keyword" ," $" :{" id" :" kwrd0085" }," $$" :[{" #name" :" text" ," _" :" higher-energy C-trap dissociation LC-MS" },{" #name" :" keyword" ," $" :{" id" :" kwrd0095" }," $$" :[{" #name" :" text" ," _" :" liquid chromatography coupled mass spectrometry LIT" },{" #name" :" keyword" ," $" :{" id" :" kwrd0105" }," $$" :[{" #name" :" text" ," _" :" linear ion trap LSC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0115" }," $$" :[{" #name" :" text" ," _" :" leukemic stem cells OT" },{" #name" :" keyword" ," $" :{" id" :" kwrd0125" }," $$" :[{" #name" :" text" ," _" :" orbitrap PROG" },{" #name" :" keyword" ," $" :{" id" :" kwrd0135" }," $$" :[{" #name" :" text" ," _" :" progenitor RTS" },{" #name" :" keyword" ," $" :{" id" :" kwrd0145" }," $$" :[{" #name" :" text" ," _" :" real-time search S/N" },{" #name" :" keyword" ," $" :{" id" :" kwrd0155" }," $$" :[{" #name" :" text" ," _" :" signal-to-noise ratio scMS" },{" #name" :" keyword" ," $" :{" id" :" kwrd0165" }," $$" :[{" #name" :" text" ," _" :" single-cell proteomics using mass spectrometry SPS" },{" #name" :" keyword" ," $" :{" id" :" kwrd0175" }," $$" :[{" #name" :" text" ," _" :" synchronous precursor selection |
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