High-throughput quantitative proteomics with Evosep Eno and the Orbitrap Astral Zoom Mass Spectrometer

Significance of the topic

High-throughput quantitative proteomics is increasingly essential for large clinical cohorts, systems biology, and high-content screening where deep, reproducible protein quantitation across thousands of samples is required. Combining robust, highly reproducible liquid chromatography with fast, sensitive mass spectrometry enables routine narrow-window data-independent acquisition (nDIA) on short gradients to deliver both throughput and quantitative fidelity.

Objectives and overview of the study

This technical note evaluates the performance of the Evosep Eno high-throughput LC system coupled to the Thermo Scientific Orbitrap Astral Zoom Mass Spectrometer for nDIA workflows across the Evosep standard methods (30, 60, 100, 200, 300, 500 samples per day). Goals were to optimize nDIA acquisition parameters for different throughputs, measure proteome depth and quantitative precision, and demonstrate chromatographic robustness and linearity at extreme throughput (500 SPD).

Methodology

• Sample: Thermo Pierce HeLa Digest with spiked PRTC peptides; typical injection load 200 ng HeLa digest. Evotips used for sample handling.
• Chromatography: Evosep Eno using standardized methods for 30–500 SPD with dedicated Evosep Performance columns (different lengths for each SPD) and Evosep Pod column oven at 40 °C.
• Acquisition: Narrow-window DIA (nDIA) on the Orbitrap Astral Zoom. Global Orbitrap full-scan resolution set to 240,000; DIA isolation-window schemes and maximum injection times were optimized per SPD. Typical precursor mass range tested 380–980 or 400–800 m/z depending on method.
• Optimization approach: Systematic testing of isolation window widths (2–6 Th) and maximum fill times (ms) to balance precursor selectivity, data points per peak (DPPP), cycle time and sensitivity.
• Data analysis: Biognosys Spectronaut (directDIA) for identifications and quantitation; Skyline for PRTC peak metrics and targeted evaluation.

Instrumentation used

• Evosep Eno LC system and Evotip sample cartridges
• Evosep Performance analytical columns (EV1182, EV1109, EV1137) and Evosep Pod column oven
• Thermo Scientific Orbitrap Astral Zoom Mass Spectrometer
• Thermo EASY-Spray Ion Source (with Evosep Pod)
• Software: Spectronaut 19.7 (directDIA) and Skyline-Daily v25.1
• Consumables: LC-MS grade solvents, Pierce HeLa Digest/PRTC standards

Main results and discussion

• Optimization at 500 SPD: Testing showed that very narrow isolation windows (2 Th) increase identifications but reduce DPPP and worsen quantitative precision. A 4 Th isolation width with 3 ms maximum Astral injection time provided the best compromise for 500 SPD, delivering high proteome depth and robust quantitation (examples below).
• Proteome depth vs throughput: Using optimized parameters, the platform identified up to ~6.5–6.8k protein groups and ~71–74k modified peptides from 200 ng HeLa at 500 SPD (short active gradient ~2.3 min, injection-to-injection ≈2.9 min). Longer gradients improved depth: the 30 SPD method yielded up to ~10.6k protein groups and ~190.7k modified peptides (five replicates).
• Quantitative precision: Across all standard Evosep methods median protein-group CVs remained low (≤5.2%) and peptide CVs were also maintained at acceptable levels (≤13%). For the 500 SPD optimized method median protein CV was ~4.4% and peptide CV ~9.6% in benchmark experiments.
• Chromatographic robustness: In a 94-run consecutive evaluation at 500 SPD, 11 isotopic PRTC peptides showed exceptional retention-time stability (standard deviations <0.4 s), median FWHM ≈1.2 s, high peak symmetry and an average of ~6 DPPP—supporting reliable quantitation even with ultrashort peaks.
• Sensitivity and linearity: Spike-in experiments spanning ~66.7 amol to 6.7 fmol per peptide (1–100 fmol PRTC in 200 ng HeLa) demonstrated strong linearity across the active gradient (R² > 0.98) and robust detection down to low-attomole ranges within the high-throughput workflow.
• Dynamic range preservation: Rank-intensity analyses showed the Astral detector maintained dynamic range and high-abundance signal fidelity across 30–500 SPD, indicating predictable trade-offs between depth and throughput without compression of high-abundance signals.

Benefits and practical applications

• Scalable throughput: The Evosep Eno + Astral Zoom combination allows users to choose standardized workflows (30–500 SPD) to match study scale and depth requirements while keeping data quality consistent across methods.
• High throughput with quantitation fidelity: The setup supports ultrafast nDIA without major sacrifices in precision or dynamic range, enabling large-cohort label-free studies, drug screens, and routine proteomic pipelines.
• Operational robustness: Short gradients and Evotip-based sample handling reduce LC variability and manual intervention, increasing reproducibility across many consecutive injections.
• Data completeness: nDIA with optimized isolation windows reduces chimeric spectra and improves identification confidence compared to broad-window DIA on very short gradients.

Future trends and potential applications

• Large-cohort clinical proteomics: The demonstrated combination of speed, sensitivity, and precision positions this workflow for biomarker discovery and validation studies requiring thousands of samples.
• High-content compound screening: Rapid, reproducible proteome readouts at hundreds of samples per day enable proteomics-informed phenotypic screening and mechanism-of-action studies.
• Automation and standardization: Integration with automated sample preparation and cloud-based data pipelines can further increase throughput and inter-laboratory comparability.
• Method refinements: Continued optimizations in isolation-window placement, intelligent acquisition strategies, and software algorithms for nDIA will further improve depth and quantitative accuracy at extreme throughput.

Conclusion

The Evosep Eno LC system coupled to the Orbitrap Astral Zoom Mass Spectrometer provides a robust, high-throughput platform for nDIA-based label-free proteomics. Optimized acquisition parameters allow routine operation at up to 500 SPD while preserving proteome depth, quantitative precision, chromatographic stability, and sensitivity across attomole-to-femtomole ranges. This makes the workflow well suited for large-scale studies that require both speed and high-quality quantitative data.

Reference

1. Evosep ApS. The six standard methods: Built for standardization. Evosep application note describing standardized Evosep Eno methods. (Evosep documentation).
2. Evosep ApS. Reproducible, inter-laboratory, scalable, and routine LC-MS-based proteomics with the Evosep Eno. Multi-lab study application note (Evosep documentation).
3. Evosep ApS. Loading protocol for Evotip Pure. Evotip sample loading and handling instructions (Evosep documentation).
4. Evosep ApS. Evosep Pod user manual. Evosep Pod operating and installation manual (Evosep documentation).