Evotip Pure brings innovative sample storage and introduction to high-sensitivity and scalable LC-MS workflows

Importance of Topic

The integration of front-end sample preparation with high-sensitivity LC-MS workflows is essential for proteomics applications ranging from bulk analyses to single-cell proteomics. Minimizing sample losses, reducing carryover, and enabling reliable storage of low-input samples directly impact the reproducibility and depth of proteomic profiling.

Objectives and Study Overview

This application note evaluates Evotip Pure, a disposable sample trap designed to concentrate dilute peptide samples, streamline handling steps, and support scalable workflows. Key goals are to assess sample capture efficiency, carryover rates, robustness over hundreds of injections, and storage stability, including the impact of adding n-dodecyl-β-D-maltoside (DDM) during loading.

Methodology and Instrumentation

Sample Preparation and Digestion:

• HeLa and human plasma digests prepared by protein aggregation capture-assisted digestion.
• Peptide desalting on SPE cartridges and concentration estimation by UV spectrophotometry.
• Commercial HeLa digest used for most experiments; 250 pg HeLa digest applied in variable volumes for concentration tests.
• Assessment of DDM (0.015%) effect on low-input sample loading.

Mass Spectrometry:

• timsTOF Pro 2 mass spectrometer operating in dia-PASEF mode.
• Aurora Rapid75 analytical column at 50 °C for peptide separation.
• DIA methods (Whisper Zoom 80 SPD and 120 SPD) covering m/z 400–1000 Da with multiple ion mobility ramps.
• Data analysis via DIA-NN in library-free mode against the reviewed human proteome, with match-between-runs enabled.

Main Results and Discussion

Sample Concentration

• Loading volumes from 1 to 160 µL (250 pg HeLa) yielded consistent summed precursor intensities, demonstrating efficient concentration of very dilute samples without signal loss.

Carryover and Quantitative Precision

• Sequential analysis of 250 pg HeLa, 50 ng HeLa, 50 ng plasma, and blanks showed median CVs < 7% for 50 ng samples.
• Carryover was ≤ 0.6% for single-cell equivalents and ≤ 0.2% in blank runs due to one-time use of trap cartridges.

Robustness Across Hundreds of Runs

• Pressure profiles remained stable over 340 injections using the Whisper Zoom 120 SPD method.
• Retention time deviation for five monitored HeLa peptides averaged ~2 s, indicating high reproducibility.

Sample Storage Stability

• Over 90% of peptides remained detectable after 48 h storage on Evotip; ~75% after 72 h.
• Inclusion of DDM enhanced precursor identifications and quantitative precision at low sample loads without compromising storage performance.

Benefits and Practical Applications

Evotip Pure offers:

• Efficient capture and concentration of low-input peptide samples.
• Minimal carryover and reduced sample handling steps.
• Robust performance for high-throughput and long-term experiments.
• Compatibility with automated workflows for scalable proteomics.

Future Trends and Opportunities

As single-cell proteomics continues to demand higher sensitivity and throughput, disposable trap technologies like Evotip Pure will play a key role in enabling large-scale studies. Integration with advanced surfactants, further miniaturization, and coupling with emerging MS platforms could push detection limits and reproducibility even further.

Conclusion

Evotip Pure streamlines the interface between sample preparation and LC-MS, delivering low carryover, high recovery, and stable storage for low-input proteomic analyses. Its robustness and adaptability support both routine workflows and cutting-edge single-cell applications, paving the way for reliable, high-throughput proteomic studies.

Reference

1. Batth TS, Tollenaere M, Rüther P, et al. Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation. Mol Cell Proteomics. 2019;18(5):1027–1035.