Enhanced sensitivity of the Orbitrap Astral Zoom mass spectrometer for deeper proteome coverage in single-cell proteomics

Importance of the Topic

Single-cell proteomics offers detailed insights into cellular heterogeneity but is limited by small sample amounts. High-sensitivity instruments and robust workflows are essential to detect and quantify proteins at low inputs. Advances in mass spectrometry and data independent acquisition with minimal sample loss are critical to expand biological and clinical studies at the single-cell level.

Study Objectives and Overview

This study evaluates the performance of the Thermo Scientific Orbitrap Astral Zoom mass spectrometer with a library-free DIA method in Low Input mode. It aims to determine proteome coverage, quantitative precision, accuracy, and sample throughput from sub-nanogram to single-cell samples. Comparisons were made to the previous Orbitrap Astral MS to quantify improvements in depth and speed.

Methodology and Instrumentation

The workflow combined automated low-volume sample preparation, high-efficiency LC separation, and advanced mass spectrometry:

• Sample Preparation: HEK293F single cells sorted into 384-well plates, lysed in TEAB TFE buffer, digested with trypsin; HeLa and three-proteome mixes prepared at defined ratios down to 50 pg.
• LC System: Thermo Vanquish Neo UHPLC with IonOpticks Aurora Ultimate 25 × 75 micrometer C18 column, 0.2 μL min flow, 50 C column temperature, enabling 50 samples per day throughput.
• Mass Spectrometry: Orbitrap Astral Zoom MS with FAIMS Pro Duo interface and EASY-Spray NG source operated at up to 270 Hz acquisition. Low Input mode enhanced single ion detection.
• Data Analysis: Spectronaut 19.5 directDIA+ workflow against UniProt databases, with strict FDR settings and optional match between runs for improved identifications.

Key Results and Discussion

• Depth of Coverage: At 250 pg HeLa digest, the Astral Zoom MS identified on average 6,674 protein groups (+6.8) and 59,231 peptides (+20.8) compared to the previous generation.
• Low-Input Performance: From 50 pg to 2,000 pg, identified protein groups increased from 5,681 to 7,992 and peptides from 41,724 to 80,916 per sample. Match between runs further boosted identifications.
• Quantitative Precision: Over 86 of proteins had CVs below 20 and up to 71 below 10 across triplicates, with median CVs under 6.1 across sample loads.
• Accuracy with Complex Mixes: In three-proteome mixes, measured fold changes for yeast and E coli closely matched expected ratios, with relative errors below 7.5 across loads up to 5 ng.
• Single-Cell Analysis: Individual HEK293F cells yielded on average 5,842 protein groups and 34,538 peptides, representing the deepest coverage reported to date for this cell line.

Benefits and Practical Applications

The combined workflow delivers high sensitivity, precision, and throughput for low-input and single-cell proteomics. It supports detailed studies of cellular heterogeneity, biomarker discovery, and clinical applications where sample is limiting. The automation and speed of the LC MS platform enable routine analysis of large cell cohorts.

Future Trends and Possibilities

Further improvements in ion optics and trapping promise deeper coverage at even lower inputs. Integration of AI-driven data processing will accelerate results and enhance quantitation. Expanding this approach to tissue sections and clinical biopsies will broaden its impact in translational research and precision medicine.

Conclusion

The Orbitrap Astral Zoom MS in Low Input mode, coupled with optimized DIA workflows and high-efficiency LC, achieves robust and reproducible deep proteome coverage from single-cell and sub-nanogram samples. It outperforms earlier systems in sensitivity and throughput, offering a transformative solution for high-resolution proteomics in diverse research and clinical settings.

Reference

• Arrey TN et al TN 002780 Enhanced proteome coverage and throughput for single-cell samples on the Orbitrap Astral Thermo Fisher Scientific 2024
• Bubis JA et al Challenging the Astral mass analyzer to quantify up to 5300 proteins per single cell Nat Methods 2025 22 510–519
• Tan E et al HEK293 cell line as a platform to produce recombinant proteins and viral vectors Front Bioeng Biotechnol 2021 9 796991