Label-free DIA-based workflow for single-cell proteomic analysis on an Orbitrap Exploris 480 mass spectrometer

Significance of the topic

The ability to perform comprehensive proteome profiling at single‐cell resolution has the potential to uncover cellular heterogeneity, reveal unique functional states, and inform studies in basic biology, diagnostics, and therapeutic development. Label‐free data‐independent acquisition (DIA) on high‐resolution Orbitrap platforms addresses key challenges of sensitivity, coverage, dynamic range, and throughput for low‐input and single‐cell proteomics.

Aims and overview of the study

This work aims to develop and benchmark a robust, label‐free DIA workflow on the Thermo Scientific Orbitrap Exploris 480 mass spectrometer, coupled to the Vanquish Neo UHPLC system, for high‐sensitivity single‐cell proteomic analysis. Two gradient schemes—48 samples per day (SPD) and 82 SPD—were optimized using a HeLa peptide dilution series as a proxy for single‐cell input, followed by application to real single HeLa cells prepared with CellenONE.

Methodology and instrumentation

• Sample preparation: CellenONE platform for single‐cell isolation, reduction, alkylation, and trypsin digestion in nanoliter volumes.
• UHPLC separation: 25 cm Aurora Ultimate TS column on a Vanquish Neo UHPLC configured for direct injection; gradients of 14 min (11.2 min active) for 48 SPD and 24 min (19.5 min active) for 82 SPD, with flow rates adjusted to maintain comparable peak widths.
• Mass spectrometry: Orbitrap Exploris 480 with FAIMS Pro interface; Full MS at 120 k resolution and DIA at 60 k resolution; m/z range 400–800; AGC targets and collision energies optimized (NCE 26–28%); false discovery rate set to 1% at experiment level without the need for spectral libraries.
• Data analysis: Spectronaut 18 with CHIMERYS intelligent search and Thermo Scientific Proteome Discoverer 3.1.

Main results and discussion

A HeLa peptide dilution series (50 pg to 10 ng) demonstrated high sensitivity: at 250 pg, both 48 SPD and 82 SPD workflows identified over 2,500 protein groups and more than 10,000 peptides across triplicates. The faster 48 SPD method yielded up to 25% more protein identifications at higher loads. Quantitative accuracy was excellent, with log2 abundance ratios closely matching expected values and coefficients of variation below 10% for most conditions, rising to ~15% only at the lowest 50 pg load. Application to real single HeLa cells resulted in identification of >1,500 protein groups and >5,000 peptide groups per cell, with high overlap and reproducibility across biological replicates.

Benefits and practical applications

• Exceptional sensitivity enables proteome coverage from sub‐nanogram inputs and single cells.
• Two throughput options allow users to tailor experiments between maximum depth (48 SPD) and higher sample throughput (82 SPD).
• Label‐free DIA streamlines workflow by eliminating the need for pre‐built spectral libraries.
• Direct injection from 96‐well plates minimizes sample loss, supporting end‐to‐end single‐cell workflows in biological and clinical research.

Future trends and potential applications

Ongoing advances in ion mobility separation, multiplexed DIA techniques, and shorter gradients will further increase throughput and depth. Integration of machine learning for data processing, spatial proteomics, and comprehensive profiling of rare cell populations or clinical specimens represents promising directions. Coupling single‐cell proteomics with multi‐omics and automation will expand its impact in personalized medicine and high‐content screening.

Conclusion

This label‐free DIA workflow on the Orbitrap Exploris 480, paired with optimized 48 SPD and 82 SPD methods, provides a versatile platform for sensitive, reproducible, and high‐throughput single‐cell proteomic analysis. The method demonstrates robust performance across dilution series and real cell samples, delivering deep proteome coverage and accurate quantitation without spectral libraries.

References

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