Accelerating SWATH® Acquisition for Protein Quantitation – Up to 100 Samples per Day

Significance of the Topic

The integration of microflow liquid chromatography with SWATH data independent acquisition on high-speed mass spectrometers offers a robust, high-throughput solution for quantitative proteomics. By compressing analysis times to a few minutes while maintaining deep protein coverage, this approach addresses the growing demand for large-scale studies in research, industrial quality control and clinical applications.

Objectives and Study Overview

This study evaluates the impact of gradient length on protein and peptide quantitation using SWATH acquisition on the TripleTOF 6600 system. Gradients of 5, 10, 20 and 45 minutes were tested across different complex matrices (HEK, K562, yeast) and multiple instruments. The goal was to optimize acquisition parameters to enable up to 100 samples per day without compromising data quality.

Methodology and Instrumentation

• Sample preparation: Trypsin digests of HEK, K562 and yeast cell lysates, 1 µg protein load per injection.
• Chromatography: NanoLC 425 System in trap/elute mode at 5 µL/min microflow, column temperature 30 °C; gradients of 5–45 min with potential for further reduction.
• Mass spectrometry: TripleTOF 6600 with Turbo V source; SWATH acquisition with variable Q1 windows (60–100 windows) and optimized collision energy settings; MS survey scan 150 ms, MS/MS accumulation 15–25 ms.

Main Results and Discussion

• Optimization of cycle times and number of SWATH windows enabled acquisition rates up to 66 Hz, crucial for sharp peaks in short gradients.
• Using the Pan Human Library increased quantified proteins by over 30% and peptides by over 70% across all gradient lengths compared to a smaller library.
• Short gradients (5 min) yielded >1300 proteins (1 µg HEK digest) and up to 2100 proteins with the larger library; extended gradients (45 min) quantified ~2600 proteins.
• Across three instruments and matrices, peptide identifications were consistent, with only ~50% loss when moving from 45 min to 5 min gradients, demonstrating robustness.

Benefits and Practical Applications

• High-throughput quantitation of up to 100 samples per day, ideal for large cohort studies and industrial workflows.
• Substantial time savings with minimal compromise in proteome depth, supporting rapid decision-making.
• Enhanced specificity and data quality through variable window SWATH and rapid MS/MS acquisition.

Future Trends and Opportunities

• Further reductions in system delay volumes and gradient overhead to push sample rates beyond 100 per day.
• Development of deeper and more comprehensive ion libraries, combined with AI-assisted data processing for improved coverage.
• Expansion of microflow SWATH workflows into clinical and environmental proteomics for high-throughput biomarker discovery.

Conclusion

Microflow LC coupled with SWATH acquisition on the TripleTOF 6600 system delivers a powerful platform for fast, reproducible and high-quality proteome quantitation. Optimized acquisition parameters and larger spectral libraries enable researchers to balance throughput and depth, meeting the needs of modern large-scale studies.

Reference

1. Microflow SWATH Acquisition for Industrialized Quantitative Proteomics, SCIEX Technical Notes RUO-MKT-02-3637-A
2. Fast Protein Identification Experiments with Microflow LC – Up to 100 Samples per Day, SCIEX Technical Notes RUO-MKT-02-8312-A
3. Evolution of SWATH Acquisition Provides Large Gains in Quantified Proteins, SCIEX Technical Notes RUO-MKT-02-5772-A
4. SWATH Performance Kit SOP, SCIEX
5. Optimized Collision Energy Curves for TripleTOF Systems, SCIEX Community Discussion
6. SWATH Replicates Template, SCIEX Software Downloads
7. Extending Depth of Coverage with SWATH Acquisition Using Deeper Ion Libraries, SCIEX Technical Notes RUO-MKT-02-3247-A
8. Rosenberger G et al. Scientific Data 2014;1:140031