Large Scale Targeted Protein Quantification Using WiSIM-DIA Workflow on a Orbitrap Fusion Tribrid Mass Spectrometer

Significance of the Topic

Data-independent acquisition (DIA) approaches are increasingly important for high-throughput and reproducible quantification of proteins in complex samples. The WiSIM-DIA workflow combines high-resolution accurate-mass selective ion monitoring (SIM) for quantitation with parallel CID MS/MS for sequence confirmation. Implemented on an Orbitrap Fusion Tribrid mass spectrometer, this strategy enables large-scale targeted protein quantification with high selectivity and dynamic range.

Objectives and Study Overview

This study evaluates the quantitative performance of the WiSIM-DIA workflow on the Orbitrap Fusion mass spectrometer. Two application scenarios were investigated: relative quantification of over a thousand E. coli proteins across different sample loads, and targeted analysis of signaling proteins (EGFR, AKT isoforms, PTEN) enriched by immunoprecipitation from A431 cell lysate.

Methodology and Instrumentation

Sample Preparation

• E. coli digest at 250, 500, and 1000 ng/μL.
• Immunoprecipitated A431 lysate targeting EGFR, AKT1/2, and PTEN using biotinylated antibodies and streptavidin beads.

Chromatography

• Thermo Scientific EASY-nLC 1000 with EASY-Spray PepMap C18 column (75 μm × 50 cm, 2 μm).
• 300 nL/min gradient from 5% to 35% acetonitrile over 120 minutes.

Mass Spectrometry

• Thermo Scientific Orbitrap Fusion Tribrid MS with EASY-Spray source.
• Three HRAM SIM scans (240,000 resolution, 200 amu windows, 400–1000 m/z) each followed by 17 ion trap CID MS/MS scans (12 amu isolation).

Data Analysis

• Spectral libraries from DDA data using Proteome Discoverer and SEQUEST® HT (FDR ≤1%).
• Targeted extraction with Thermo Scientific Pinpoint software: SIM quantification (±5 ppm), CID-based confirmation (spectral matching, p < 0.1).

Main Results and Discussion

• Spectral library identified 1100 E. coli proteins with ≥2 peptides at high confidence.
• WiSIM-DIA quantified 1090 proteins (98% success) across 250 vs 500 ng loads, analyzing 5,923 peptides (p < 0.1, CV ≤ 25%).
• 97% of proteins showed expected twofold change with high accuracy; 85% of peptides achieved CV < 15%.
• Immunoprecipitation workflow yielded >20-fold enrichment for EGFR, AKT1, and AKT2, confirmed and quantified by WiSIM-DIA.

Benefits and Practical Applications

• High throughput targeted quantification of thousands of proteins in a single DIA run.
• Robust selectivity and sensitivity due to high-resolution SIM quantification.
• Simultaneous sequence confirmation increases confidence in peptide identification.
• Applicable to studies of complex biological matrices and immunoenrichment workflows.

Future Trends and Applications

• Integration with advanced spectral libraries and machine learning for deeper proteome coverage.
• Automation and scaling for clinical and biomarker validation studies.
• Multiplexed analyses using isotopic labeling or novel fragmentation schemes.
• Extension to post-translational modification–focused workflows.

Conclusion

The WiSIM-DIA workflow on the Orbitrap Fusion Tribrid mass spectrometer delivers reproducible and accurate large-scale targeted protein quantification. By combining high-resolution SIM scans with parallel CID MS/MS confirmation, this approach achieves low detection limits, high selectivity, and robust quantitative accuracy suitable for diverse proteomic applications.

References

• Kiyonami R, Senko M, Zabrouskov V, Egertson J, Ting S, MacCoss MJ, Hühmer AF. Thermo Scientific Application Note AN64026.
• Prakash A, Tomazela DM, Frewen B, Maclean B, Merrihew G, Peterman SM, Maccoss MJ. J Proteome Res. 2009;8:2733–2739.
• Patel B, Meier S, Opperman K, Haney P, Kaboord B, Rogers J. ASMS Poster 514; 2014.