WhisperTM Zoom drives scalable workflows for high-sensitivity applications through standardization and ease-of-use

Significance of the topic

High-sensitivity proteomics is critical for advancing biomedical research, enabling insights at single-cell resolution and complex post-translational modifications. Optimizing LC-MS workflows to maximize sensitivity, throughput, and robustness democratizes access to cutting-edge analytical performance across laboratories.

Aims and overview of the study

This application note evaluates four standardized Whisper Zoom methods (20, 40, 80, and 120 samples per day) on the Evosep One platform coupled to a timsTOF Pro 2 mass spectrometer. The objective is to demonstrate scalable, high-sensitivity workflows with consistent performance for single-cell proteomics (SCP), deep visual proteomics (DVP), and phosphoproteomics.

Methodology and Instrumentation

The study used HeLa peptide digests loaded onto disposable Evotip trap columns and analyzed in quadruplicate for each method. Chromatographic separations employed Aurora Rapid75 and Aurora Elite columns at 50 °C. Data acquisition on the timsTOF Pro 2 utilized dia-PASEF over 400–1000 Da and 0.64–1.37 1/K0 ion mobility. Library-free protein identification was performed with DIA-NN v1.8.1 against the UniProt human proteome with match-between-runs enabled.

Main results and discussion

Key findings include:

• Chromatographic peaks as narrow as 1.6 s FWHM (120 SPD) and ~2.1 s FWHM (40 SPD) support high analyte concentration and sensitivity.
• Precursor identification saturation occurs at 10 ng input for 120 SPD, 50 ng for 40/80 SPD, and ≥200 ng for 20 SPD.
• Single-cell equivalent loads (250 pg) yield comparable precursor counts across methods, with 40 SPD slightly higher.
• Robustness is evidenced by stable pump pressures over 340 runs and <2 s retention time deviation for monitored peptides.
• Inter- and intra-instrument reproducibility is maintained below 5 s and 16 s retention time deviation, respectively, across five internal and three external Evosep One systems.

Benefits and practical applications

The Whisper Zoom methods enable:

• High-throughput proteomics (up to 120 samples/day) without sacrificing depth.
• Minimal carryover and extended column life via disposable Evotip technology.
• Accessible workflows for non-expert labs in SCP, DVP, and phosphoproteomics.

Future trends and opportunities

Scaling high-sensitivity proteomics will support multi-laboratory studies, large-scale clinical cohorts, and integration with advanced sample preparation techniques. Continued innovation in microfluidic flow paths, ion mobility separations, and data-independent acquisition algorithms will further enhance depth, throughput, and reproducibility.

Conclusion

Whisper Zoom methods on the Evosep One/timsTOF Pro 2 platform deliver a balanced combination of sensitivity, throughput, and robustness. Their standardized protocols lower the barrier for adopting high-end LC-MS workflows, fostering collaboration and accelerating discovery in proteomics.

Instrument Used

• Evosep One liquid chromatography system
• Evotip disposable trap columns (Evotip Pure)
• Aurora Rapid75 and Aurora Elite columns (IonOpticks)
• timsTOF Pro 2 mass spectrometer (Bruker)
• DIA-NN software v1.8.1 for library-free data processing