diaPASEF: parallel accumulation-serial fragmentation combined with data-independent acquisition

Area of Science:

• Proteomics
• Analytical Chemistry
• Mass Spectrometry

Background:

• Data-independent acquisition (DIA) in mass spectrometry typically isolates only a small fraction of ions.
• This limits comprehensive proteome analysis and quantitative accuracy.

Purpose of the Study:

• To develop a novel scan mode that maximizes peptide precursor ion capture.
• To enhance proteome coverage, reproducibility, and quantitative accuracy in mass spectrometry.

Main Methods:

• Utilized trapped ion mobility spectrometry (TIMS) coupled with a timsTOF Pro instrument.
• Developed a new scan mode leveraging ion mobility and m/z correlations to sample up to 100% of precursor ions.
• Extended existing targeted data extraction workflows to incorporate ion mobility data for improved identification specificity.

Main Results:

• Achieved deep proteome coverage from whole proteome digests and mixed organism samples.
• Demonstrated high reproducibility and quantitative accuracy, even with low sample inputs (10 ng).
• The inclusion of ion mobility significantly increased the specificity of precursor identification.

Conclusions:

• The novel scan mode effectively captures a near-complete ion current, overcoming limitations of traditional DIA.
• This approach significantly advances deep and quantitative proteome analysis, especially for challenging low-input samples.
• Ion mobility-enhanced mass spectrometry offers a powerful tool for comprehensive biological sample analysis.