Microflow SWATH® Acquisition for Industrialized Quantitative Proteomics

Significance of Topic

Data independent acquisition (DIA) methods such as SWATH have transformed quantitative proteomics by enabling comprehensive, reproducible analysis of thousands of proteins in complex samples. Integrating microflow liquid chromatography enhances sample throughput and chromatographic robustness with only a modest sensitivity trade-off compared to nanoflow systems, addressing the demands of large-scale biomarker discovery and industrialized workflows.

Study Objectives and Overview

This work investigates the combination of microflow LC with SWATH acquisition on the NanoLC™ 400/425 system and TripleTOF® 6600 mass spectrometer. Key aims include evaluating proteome coverage, quantitative reproducibility, retention time stability, and sample throughput relative to established nanoflow strategies.

Methodology and Instrumentation

Chromatography was performed on a NanoLC™ 425 System operating at 5 µL/min using a 0.3×150 mm ChromXP™ column with a 43 min gradient (4–32% acetonitrile, 0.1% formic acid). Total protein loads ranged from 1 to 8 µg of HEK cell lysate digest.

Mass spectrometry employed a TripleTOF® 6600 with Turbo V™ source and 25 µm hybrid electrodes. Variable window SWATH methods (60, 80, 100 windows) were generated in Analyst® TF 1.7, using high sensitivity (HS, >15 000 resolution) or high resolution (HR, >30 000) MS/MS modes with extraction widths of 75 ppm and 40 ppm, respectively.

Data were processed in PeakView® 2.2 with SWATH® 2.0 using the Pan Human SWATH Ion Library. Peptides with shared or modified sequences were excluded. Quantitation metrics were set at <1% false discovery rate (FDR) and <20% coefficient of variation (CV) across five replicates.

Main Results and Discussion

Retention time reproducibility across 48 plasma injections over 2.5 days showed deviations of 4–10 seconds, enabling narrow extraction windows. High resolution MS/MS increased quantified protein numbers by 5–15% compared to HS mode. Increasing sample load to 8 µg yielded 4 500–5 000 proteins quantified per 1 h run at <1% FDR and <20% CV. Optimizing to 100 variable windows (25 ms accumulation) in HR mode produced 4 963 proteins and 30 685 peptides, with ~90% of peptides under 20% CV and ~4 orders of dynamic range.

Benefits and Practical Applications

• Throughput: 20–24 samples per day (~150 proteomes/week).
• Robustness: simplified flow path, rapid loading and re-equilibration, stable retention times.
• Quantitative quality: high specificity from variable windows and HR MS/MS.
• Flexibility: rapid switching between nanoflow and microflow on NanoLC™ systems.

Future Trends and Opportunities

Microflow SWATH is poised for wider adoption in clinical and industrial proteomics due to its balance of throughput and data quality. Future developments may include further automation, advanced window design driven by real-time density mapping, integration with AI-based data analysis pipelines, and deployment in regulated environments for biomarker validation.

Conclusion

SWATH acquisition on microflow LC platforms delivers high-quality quantitative proteomics with significantly enhanced throughput and robustness. This optimized workflow enables large-scale studies without substantial sacrifices in sensitivity, offering a versatile solution for academic and industrial research applications.

References

• SCIEX Technical note RUO-MKT-02-2879-A.
• SCIEX Technical note RUO-MKT-02-5772-A.
• SCIEX Technical note RUO-MKT-02-3252-A.
• Rosenberger G et al. Scientific Data. 2014;1:140031.
• Yansheng Liu et al., ETH Zurich (HEK cell lysate source).
• SCIEX Technical note RUO-MKT-02-8432-A.