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.
Highlights
Recent developments in liquid chromatography coupled mass spectrometry (LC-MS) based proteomics revealed the high potential for its application on single cells[1,2,3,4,5,6,7,8,9,10]
In the real-time search (RTS)-MS3 method, precursors detected in the MS1 scan are subjected to an MS2 scan in the linear ion trap (LIT), which is Journal Pre-proof subsequently searched against a user-defined database
The same procedure is applied in RETICLE, with the difference that RTS is used as a trigger only and precursors identified as a peptide of interest are subsequently subjected to a quantitative OT-MS2 scan
Summary
Recent developments in liquid chromatography coupled mass spectrometry (LC-MS) based proteomics revealed the high potential for its application on single cells[1,2,3,4,5,6,7,8,9,10]. Since the high resolution scan in the Orbitrap (OT) is only needed to measure accurate precursor masses in the MS1 scan and to resolve the reporter ions for quantification in the MS3 scan, peptide identification via MS2 can be performed in the more sensitive LIT20. This results in fast and sensitive MS2 acquisition, as the Tribrid design enables parallelization of OT and LIT scans. With the introduction of real-time search (RTS)[21,22] on the Orbitrap Eclipse
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