Harnessing the Ion Mobility Dimension to Improve Quantification in dia-PASEF

The aim of multiple data-independent acquisition (DIA) experiments in proteomics is to find differentially abundant proteins. Improvements in candidate discovery involve improving both identification and quantification. The latter poses a challenge due to the need to balance precision and accuracy because both can impact candidate discovery.

One approach to boost quantitative sensitivity is ion separation in the ion-mobility (IM) dimension for DIA. Here, we extended the interference removal algorithm of Spectronaut^® from the retention time (RT) dimension to the IM dimension. Additionally, we improved peak picking in the IM dimension to boost the signal to noise of a precursor. Finally, we introduced the approach of dynamic background noise estimation and removal. Testing on controlled quantitative experiments (CQE) demonstrated significantly improved quantification accuracy for dia-PASEF data without compromising candidate discovery and with only slight drops in precision.