A complete and automated end-to-end sample preparation strategy for high-throughput and standardized proteomics with high sensitivity

Importance of the topic

High-throughput proteomics has become a cornerstone in biomarker discovery, drug development and clinical diagnostics. Standardized and fully automated sample preparation protocols are critical to reduce variability, increase throughput and enable sensitive detection of low-abundance proteins.

Study objectives and overview

This work presents an end-to-end, automated sample preparation workflow combining protein aggregation capture, robotic digestion and direct loading on Evotip devices. The strategy is designed to process 1–96 samples with flexible digestion times (0.5–16 h) and variable peptide loading (20–100 %) while maintaining high sensitivity and reproducibility.

Methodology and instrumentation

Protein samples (1 µg down to 5 ng HeLa lysate) undergo aggregation capture on magnetic hydroxyl microparticles, followed by a single isopropanol wash. Digestion uses a Lys-C:trypsin mix (1:100 and 1:25 ratios) at 37 °C on an Agilent AssayMAP Bravo liquid handler equipped with 96 syringes. Resulting peptides are directly loaded onto Evotip Pure using positive air pressure in ~5 min per 96 tips. LC-MS analysis is performed on Evosep One coupled to timsTOF Pro 2 (dia-PASEF, Whisper Zoom 40/80 SPD) and a QQQ 6495C for targeted assays.

Main results and discussion

Digestion efficiency increased from 67 % at 30 min to 89 % overnight, with reproducible CVs below 15 % across all conditions. Using the Whisper Zoom 40 SPD method, over 90 000 precursors and >8 000 proteins were identified from 1 µg input; at 80 SPD, >7 200 proteins were detected. Protein group CVs remained <7 % at high input and <20 % at 10 ng. A dilution series confirmed linearity (R²>0.96) across four orders of magnitude. The workflow processed 480 samples in 24 h (500 SPD) with <7 % CV for digestion efficiency.

Benefits and practical applications

• Fully automated, reducing manual errors and plate-to-plate variability
• Scalable throughput from single to hundreds of samples per day
• High sensitivity down to low nanogram inputs
• Nearly 100 % sample utilization with minimized enzyme consumption
• Flexible method parameters to balance depth and speed

Future trends and opportunities

Integration with machine-learning algorithms could optimize digestion parameters in real time. Miniaturization of workflows and multiplexing strategies may further increase throughput. Adoption in clinical labs and integration with emerging high-speed MS platforms will expand applications in precision medicine and large-scale biomarker studies.

Conclusion

The described automated sample preparation pipeline delivers high sensitivity, robustness and flexibility for large-scale proteomic studies. By combining efficient PAC capture, rapid digestion, automated Evotip loading and fast LC-MS acquisition, this workflow addresses key bottlenecks in throughput and reproducibility.

Reference

Huusfeldt M., Cabalo Zisco H., Bracamontes G., Bekker-Jensen D.B., Bache N. A complete and automated end-to-end sample preparation strategy for high-throughput proteomics with high sensitivity. Evosep Biosystems, Denmark.