Enhancing the resolving power on an Orbitrap Astral Zoom mass spectrometer for TMTPro 35plex applications

Significance of the Topic

Multiplexed isobaric labeling using TMTpro reagents enables simultaneous quantitation of up to 32 or more samples in a single LC-MS/MS run. Resolving small mass differences of reporter ions, such as 3 mDa in expanded TMTpro sets, demands ultra-high mass resolution to ensure accurate quantitation and minimal interference.

Aims and Study Overview

This study presents a novel acquisition scheme on the Thermo Scientific Orbitrap Astral Zoom mass spectrometer, termed TMT HR mode, which separates identification (ID) scans and high-resolution quantitation (QUAN) scans. The approach allows independent optimization of parameters for peptide identification and reporter ion quantitation in TMTpro 32plex workflows, improving spectral quality and throughput.

Methodology and Instrumentation

Peptide mixtures from HeLa cell digest labeled with TMTpro 32plex were analyzed by data-dependent acquisition. ID scans employed standard Astral resolution with a wider isolation window to maximize spectral depth, while QUAN scans used extended multi-pass Astral resolution, narrower isolation windows, and tailored normalized collision energies for reporter cleavage. Parameters such as isolation width, AGC target, collision energy, and sample load were systematically optimized using a triple-knock-out yeast (TKO) standard.

Used Instrumentation

• Orbitrap Astral Zoom mass spectrometer (Thermo Scientific)
• Vanquish Neo UHPLC with direct injection (Thermo Scientific)
• Aurora Frontier XT column, 75 µm × 60 cm (Ionopticks)
• TMTpro 32plex reagents and TKO standard (Thermo Scientific)
• Proteome Discoverer 3.3 software with SEQUEST and INFERYS rescoring

Main Results and Discussion

The TMT HR mode achieved comparable identification rates to conventional MS2 methods with minimal loss of quantified proteins and peptides. Reporter ion signal-to-noise in QUAN scans was about 50% of MS2 but recovered via increased sample loads. Optimal QUAN conditions were 0.7 Th isolation width, 250% AGC target, and 55% HCD energy; ID scans used 32% HCD. Resolution exceeded 100,000 FWHM at m/z 131, fully resolving the 3 mDa reporter differences. Analysis of a 1:4 HeLa TMTpro 32plex sample yielded 4,299 protein groups and 22,146 peptides at a throughput of 440 samples per day, with >94% of proteins and 78% of peptides quantitatively complete.

Benefits and Practical Applications

This separated ID/QUAN acquisition enhances flexibility in method design, delivering high-precision quantitation in large-scale proteomic studies, quality control assays, and biomarker discovery. The workflow supports high throughput with reliable coverage in complex multiplexed experiments.

Future Trends and Opportunities

Further developments may extend this strategy to TMTpro 35plex, integrate real-time parameter adjustment through machine learning, and combine with data-independent acquisition for deeper proteome coverage. Emerging ion mobility and AI-driven analytics will likely enhance resolution and quantitation fidelity.

Conclusion

The novel TMT HR acquisition on the Orbitrap Astral Zoom mass spectrometer offers a robust solution for high-plex isobaric quantitation, achieving ultra-high resolution for reporter ions while maintaining strong identification performance and throughput, thereby advancing multiplexed proteomics capabilities.

Reference

• Zuniga et al Achieving a 35-Plex Tandem Mass Tag Reagent Set through Deuterium Incorporation Journal of Proteome Research 2024 23(11):5153-5165 DOI:10.1021/acs.jproteome.4c00668