Additional precursor purification in isobaric mass tagging experiments by traveling wave ion mobility separation...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Data-dependent acquisition (DDA) is a prevalent LC-MS proteomics method.
• Precursor ion co-selection is a known limitation in DDA, impacting quantification accuracy.
• Isobaric mass tagging is a powerful DDA-based quantification strategy.

Purpose of the Study:

• To investigate the efficacy of traveling wave ion mobility separation (TWIMS) in reducing precursor contamination in DDA.
• To assess the impact of TWIMS on signal-to-noise ratios and reporter ion contamination in isobaric mass tagging experiments.
• To explore challenges and benefits of integrating TWIMS with DDA for proteomics.

Main Methods:

• Utilized a mixture of Saccharomyces cerevisiae and HeLa proteomes.
• Employed traveling wave ion mobility separation (TWIMS) for peptide purification prior to LC-MS.
• Analyzed data using Orbitrap instruments and isobaric mass tagging.
• Investigated the effect of a narrowed quadrupole isolation window in conjunction with TWIMS.

Main Results:

• TWIMS improved signal-to-noise ratios for sequence ions by an average of 2.85 times.
• TWIMS reduced reporter ion contamination by approximately one-third (to 14-15%).
• Combining TWIMS with a narrowed isolation window further reduced contamination to 6-9%.

Conclusions:

• TWIMS offers a significant improvement in reducing precursor contamination for DDA-based proteomics.
• TWIMS enhances the accuracy of isobaric mass tagging by mitigating co-selection interferences.
• Understanding ion mobility characteristics is crucial for optimizing TWIMS in mass spectrometry-based proteomics.