Label-free quantification in ion mobility-enhanced data-independent acquisition proteomics

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Data-independent acquisition (DIA) is increasingly used in quantitative proteomics.
• Ion mobility separation (IMS) enhances liquid chromatography-mass spectrometry (LC-MS) depth.
• IMS-DIA offers an additional separation dimension for improved proteome analysis.

Purpose of the Study:

• To provide a detailed protocol for label-free quantitative proteomics using ion mobility-enhanced DIA.
• To synchronize precursor ion drift times with collision energies for efficient fragmentation.
• To enable highly reproducible and accurate protein quantification in complex samples.

Main Methods:

• Filter-aided sample preparation.
• Liquid chromatography-ion mobility-mass spectrometry (LC-IMS-MS) analysis.
• Optimized DIA data acquisition and label-free quantification.

Main Results:

• A reproducible protocol for proteome analysis handling samples of any complexity.
• Accurate label-free quantification of up to 5,600 proteins in cellular lysates.
• Successful synchronization of ion drift times and collision energies for enhanced fragmentation.

Conclusions:

• Ion mobility-enhanced DIA provides a robust workflow for deep and quantitative proteome profiling.
• The presented protocol facilitates accurate and reproducible label-free quantification.
• This method is applicable to diverse and complex proteome samples.