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

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

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

112 Downloads (Pure)


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.
Original languageEnglish
Article number100219
JournalMolecular and Cellular Proteomics
Issue number4
Number of pages12
Publication statusPublished - 2022


  • Single-cell proteomics
  • Isobaric tag quantification
  • Multiplexing
  • SPS-MS3
  • TMT
  • Real-time-search


Dive into the research topics of 'Real-Time Search Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics'. Together they form a unique fingerprint.

Cite this