Deeper proteome coverage and faster throughput for low-input and single cell samples on the Orbitrap Ascend Tribrid mass spectrometer

Deeper Proteome Coverage and Enhanced Throughput for Low-Input and Single-Cell Proteomics

Importance of the Topic

Proteomic profiling at the single cell level is essential to understand cellular heterogeneity and complex biological systems. High sensitivity and rapid analysis enable deeper insights from limited sample amounts and accelerate discovery workflows.

Objectives and Study Overview

This study assessed sensitivity, proteome coverage, and throughput using a library free data independent acquisition method on the Orbitrap Ascend Tribrid mass spectrometer. HeLa digest loads from 50 pg to 10 ng and isolated single cells were analyzed to benchmark performance.

Methodology and Instrumentation

• Sample preparation: Pierce HeLa protein digest standard diluted to loads between 50 pg and 10 ng; single cells sorted by cellenONE and prepared in 384 well plates.
• Chromatography: Vanquish Neo UHPLC at 0.2 µL per minute with Aurora Ultimate TS column, 22 minute gradient in a 25 minute cycle.
• Mass spectrometry: Orbitrap Ascend Tribrid with FAIMS Pro interface, full MS at 120000 resolution and DIA scans at 60000 resolution with adaptive isolation windows.
• Data analysis: Library free processing in Spectronaut 18 and Proteome Discoverer 3.1 with CHIMERYS, 1 percent false discovery rate; spectral libraries generated from higher load data when applied.

Main Results and Discussion

• Library free DIA yielded over 1200 protein groups from 50 pg and over 3200 from 250 pg, scaling to more than 5500 proteins at 10 ng.
• Building a DIA library from 5 and 10 ng runs increased identifications to over 4800 proteins at 250 pg input.
• Quantitative precision was high, with median coefficients of variation below 8 percent for library free and below 12 percent for library based workflows across all loads.
• Single cell analyses identified on average 3762 protein groups, increasing with pooled 5 or 10 cell samples; additional strategies incorporating low cell raw files or small libraries further boosted coverage.

Benefits and Practical Applications

• Enables deep proteome mapping from picogram to single cell inputs with high reproducibility.
• High throughput of up to fifty samples per day supports large scale proteomic studies.
• FAIMS Pro enhances signal to noise and reduces chemical noise for minute samples.

Future Trends and Applications

Ongoing developments in machine learning guided DIA analysis, miniaturized chromatography, and integrated multiomic workflows will further enhance sensitivity and throughput. Advances in ion mobility and fragmentation strategies promise deeper coverage from single cells.

Conclusion

The Orbitrap Ascend Tribrid mass spectrometer with FAIMS Pro and Vanquish Neo UHPLC delivers sensitive, rapid, and reproducible deep proteome coverage for low input and single cell samples using both library free and library based DIA strategies, advancing high resolution cellular profiling.

References

• Salvato F, Delanghe B, Kraegenbring J, Hartlmayr D, Seth A, Hakimi A, Pekar Hart T. Technical note 002879: Deeper proteome coverage and faster throughput for low input and single cell samples on the Orbitrap Ascend Tribrid mass spectrometer. Thermo Fisher Scientific, 2024.
• Spectronaut 18 User Guide. Biognosys AG.
• Proteome Discoverer 3.1 with CHIMERYS Intelligent Search Algorithm. Thermo Fisher Scientific.