Effects of electron-transfer/higher-energy collisional dissociation (EThcD) on phosphopeptide analysis by data-independent acquisition

In quantitative proteomics the use of data-independent acquisition (DIA) methods is getting increasingly popular and is applied to a growing number of sample types ranging from peptidomics to the analysis of post-translational modifications. Several of these sample types have been previously shown to profit from electron-based fragmentation methods such as ETD or EThcD. These fragmentation methods have so far not been implemented in DIA analysis. Here, we show the feasibility of combining DIA with EThcD fragmentation and provide insights into its performance. We show how EThcD can be used to increase peptide coverage, reaching similar success rates during targeted data extraction as standard HCD-based DIA. Furthermore, we illustrate how robust MS1 and LC results can be exploited to circumvent detrimental effects of less efficient fragmentation during targeted data extraction. Ultimately, our data demonstrates how EThcD-based MS/MS spectra can be connected to standard HCD-based DIA analyses, enabling the future use of decision-tree based spectral libraries to query large DIA data sets.