Label-free proteomics performance with the Orbitrap Exploris 480 mass spectrometer with single-cell sensitivity

Importance of the Topic

Label-free mass spectrometry-based proteomics is essential for comprehensive protein identification and quantification in complex samples. Robust instrumentation that delivers high sensitivity, reproducibility, and ease of use is critical for transitioning from discovery to targeted quantitation, especially when analyzing large sample cohorts or single cells.

Objectives and Study Overview

This study evaluates the performance of the Thermo Scientific Orbitrap Exploris 480 mass spectrometer coupled with the FAIMS Pro interface in data-dependent acquisition mode. The goals include assessing sensitivity from single HeLa cells (~0.2 ng) to 1 µg of HeLa digest, determining cross-laboratory reproducibility, and quantifying gains in proteome coverage and dynamic range.

Methodology and Used Instrumentation

Single HeLa cells were isolated and processed via the nanoPOTS platform, while bulk HeLa Protein Digest Standard was prepared in loading buffer. LC separations employed EASY-nLC 1200 and UltiMate 3000 RSLCnano systems, Aurora and Acclaim PepMap C18 columns, and flow rates from 20 nL/min to 300 nL/min. The Orbitrap Exploris 480 mass spectrometer with FAIMS Pro was operated in TopN DDA modes across 30–120 min gradients. Data analysis utilized Proteome Discoverer 2.4 with SEQUEST, Percolator, MaxQuant for match-between-runs, and MSPepSearch for spectral library searches.

Main Results and Discussion

Across three sites and operators, the platform identified ~6700 protein groups from 200 ng HeLa digest with a coefficient of variation <1% and ~7000 proteins from 200 ng bulk digest. Incorporating FAIMS Pro increased peptide and protein identifications by ~14% and extended dynamic range to 5.5 orders of magnitude. Mass accuracy remained below ±3 ppm for ~90% of PSMs over two weeks. At low sample loads, ~2000 proteins were identified from 1 ng digest and ~800 proteins from a single HeLa cell in a 2 h gradient.

Benefits and Practical Applications

The combined Orbitrap Exploris 480 and FAIMS Pro setup offers high-throughput label-free quantitation with single-cell sensitivity, minimal sample loss, and consistent performance across laboratories. It eliminates the need for extensive peptide fractionation and supports large-scale studies in pharmaceutical, clinical, and biological research.

Future Trends and Opportunities

Advancements may include integration with multiplexed single-cell platforms, enhanced ion mobility separation, improved quantitation algorithms, and deeper coverage of post-translational modifications. These developments will further enable high-resolution studies of cellular heterogeneity and biomarker discovery.

Conclusion

The Orbitrap Exploris 480 with FAIMS Pro sets a new standard for label-free proteomics, delivering reliable, sensitive, and reproducible protein identification from bulk samples to single cells. Its performance supports scalable workflows without compromising coverage or throughput.

Reference

1. Kelly R.T. et al. Anal. Chem. 92(3):2665–2671 (2020)
2. Pfammatter S. et al. Mol. Cell Proteomics (2018)