Real-Time Search-Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics.

Autor:	Furtwängler B; The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark; Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address: benjamin.furtwangler@bric.ku.dk., Üresin N; The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark., Motamedchaboki K; ThermoFisher Scientific, San Jose, California, USA., Huguet R; ThermoFisher Scientific, San Jose, California, USA., Lopez-Ferrer D; ThermoFisher Scientific, San Jose, California, USA., Zabrouskov V; ThermoFisher Scientific, San Jose, California, USA., Porse BT; The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark; Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark., Schoof EM; Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark. Electronic address: erws@dtu.dk.
Jazyk:	angličtina
Zdroj:	Molecular & cellular proteomics : MCP [Mol Cell Proteomics] 2022 Apr; Vol. 21 (4), pp. 100219. Date of Electronic Publication: 2022 Feb 25.
DOI:	10.1016/j.mcpro.2022.100219
Abstrakt:	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. Competing Interests: Conflict of interest K. M., R. H., D. L. F., and V. Z. are or were employees at Thermo Fisher Scientific. All other authors declare no competing interests. (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu