Evolution of SWATH® Acquisition Provides Large Gains in Quantified Proteins

Significance of the Topic

SWATH® Acquisition is a data-independent acquisition (DIA) method that delivers comprehensive and reproducible proteome quantitation. Since its introduction in 2012, ongoing improvements have driven robust biomarker discovery and large-scale protein profiling by enhancing specificity, depth of coverage and throughput.

Objectives and Study Overview

This work quantifies the gains achieved in protein and peptide identification by evolving SWATH acquisition on TripleTOF® systems. Four SWATH strategies—original fixed windows (34×25 Da), intermediate fixed windows (20×20 Da), 60 variable windows and 100 variable windows—were compared under constant chromatographic, instrumental and sample conditions. Three ion libraries of increasing complexity (1D HEK, 2D K562, Pan-Human) were used to assess library-dependent gains.

Methodology and Instrumentation

Chromatography and Sample Preparation

• Trypsin digested human cell lysate separated on a NanoLC™ 425 system in trap-elute mode at 5 µL/min.
• ChromXP™ 0.3×150 mm column with a 43 min gradient from 4 to 32% acetonitrile in 0.1% formic acid; total run time 57 min; 5 µg digest injected.

Mass Spectrometry

• TripleTOF 6600 and 5600 systems equipped with Turbo V™ sources and 25 µm hybrid electrodes.
• SWATH methods built in Analyst TF 1.7 software, using 34 fixed, 20 fixed (narrow m/z), 60 variable and 100 variable Q1 windows.

Data Processing

• PeakView™ SWATH 2.0 with three libraries at 1% FDR, excluding modified and shared peptides.
• Quantification based on five replicates per method; peptide and protein identifications filtered at <1% FDR and <20% CV.

Main Results and Discussion

Variable Q1 window strategies markedly improved signal-to-noise and specificity. Compared with the original 34×25 Da approach:

• 60 variable windows delivered a 70–100% increase in peptide quantitation and similar protein gains using the Pan-Human library.
• 100 variable windows yielded up to 150% more peptides and ~120% more proteins quantified.

Gains scaled with library size: large ion libraries maximized the benefit of narrower windows. A narrow-range 20×20 Da method underperformed relative to variable windows due to limited mass coverage. Results were consistent across both 6600 and 5600 platforms.

Benefits and Practical Applications

These innovations enable:

• Enhanced specificity by adapting window widths to precursor density.
• Deeper proteome coverage through high-resolution MS/MS at fast cycle times.
• Scalable workflows using microflow chromatography for higher robustness and throughput.
• Broad applicability in biomarker discovery, QA/QC and industrial proteomics.

Future Trends and Opportunities

Emerging directions include:

• Expanded ion libraries via multidimensional fractionation and in silico prediction.
• Real-time acquisition optimization guided by machine learning.
• Integration of microflow and nanoflow platforms for diverse sample types.
• Standardized DIA workflows for regulatory and clinical applications.

Conclusion

Evolving SWATH acquisition from fixed to optimized variable windows, alongside larger ion libraries and advanced instrumentation, has driven multi-fold gains in peptide and protein quantitation. The 100 variable window strategy with a comprehensive library offers a best-practice workflow for complex proteome analysis.

References

1. Gillet L.C. et al. Mol. Cell Proteomics 11(6), 2012.
2. SCIEX Technical Note RUO-MKT-02-2879-B: Variable Q1 Window Widths in SWATH Acquisition.
3. SCIEX Technical Note RUO-MKT-02-3245-A: High Sample Loads and Smaller Q1 Windows.
4. SCIEX Technical Note RUO-MKT-02-3247-A: Deeper Ion Libraries in SWATH Acquisition.
5. Rosenberger G. et al. Scientific Data 1, 140031 (2014).
6. SCIEX Technical Note RUO-MKT-02-3637-A: Microflow SWATH for Industrial Proteomics.
7. SWATH Variable Window Calculator, SCIEX download tool.