Home » Abstracts » Technological & Instrument advances in MS » TWISTT-PRM: a novel real-time acquisition method enabling large-scale targeted PRM proteomics on Thermo Orbitrap instruments

TWISTT-PRM: a novel real-time acquisition method enabling large-scale targeted PRM proteomics on Thermo Orbitrap instruments

Technological & Instrument advances in MS

Abstract #300 Posted on28 March 20259 May 2025 9 May 2025 9h51

Abstract

DDA and DIA methods are used today to identify and quantify thousands of proteins in complex biological samples. Targeted approaches like SRM and PRM are however still needed to detect low-abundance species, but the few numbers of peptides they can monitor restricts their use to certain applications.

To mitigate this limitation, we propose TWISTT-PRM (TWo Injections with Smart Triggering of Targets for Parallel Reaction Monitoring), a real-time controlled method, which is tailored for Orbitrap instruments and operates in two stages: it first collects high-confidence peptide signals from the MS survey of a reference run, and then use them in real-time during a PRM run to adjust the scheduling of targets. TWISTT-PRM was created using MSReact, a software framework we developed to facilitate the building of real-time MS acquisition methods.

Here, we benchmarked this new PRM method using dedicated sample mixtures analyzed in DIA and PRM on a Thermo Exploris 480. We assessed its accuracy and sensitivity using 708 synthetic peptides spiked in an E. coli background and evaluated the scalability on an E. coli proteome spiked into an HeLa lysate.
TWISTT-PRM outperformed DIA in sensitivity, detecting significantly more peptides at low concentrations (462 vs 62 in DIA), reducing missing values, and maintaining comparable accuracy. It enabled the use of 20-second PRM windows, allowing the targeted quantification of ~2000 E. coli peptides (E. coli/HeLa mixture) on an Orbitrap instrument.

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To mitigate this limitation, we propose TWISTT-PRM (TWo Injections with Smart Triggering of Targets for Parallel Reaction Monitoring), a real-time controlled method, which is tailored for Orbitrap instruments and operates in two stages: it first collects high-confidence peptide signals from the MS survey of a reference run, and then use them in real-time during a PRM run to adjust the scheduling of targets. TWISTT-PRM was created using MSReact, a software framework we developed to facilitate the building of real-time MS acquisition methods.

Here, we benchmarked this new PRM method using dedicated sample mixtures analyzed in DIA and PRM on a Thermo Exploris 480. We assessed its accuracy and sensitivity using 708 synthetic peptides spiked in an E. coli background and evaluated the scalability on an E. coli proteome spiked into an HeLa lysate.
TWISTT-PRM outperformed DIA in sensitivity, detecting significantly more peptides at low concentrations (462 vs 62 in DIA), reducing missing values, and maintaining comparable accuracy. It enabled the use of 20-second PRM windows, allowing the targeted quantification of ~2000 E. coli peptides (E. coli/HeLa mixture) on an Orbitrap instrument.

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