Improving Phosphoproteomics Profiling Using Data-Independent Mass Spectrometry

Mass spectrometry-based profiling of the phosphoproteome is a powerful method of identifying phosphorylation events at a systems level. Most phosphoproteomics studies have used data-dependent acquisition (DDA) mass spectrometry as their method of choice. In this Perspective, we review some recent studies benchmarking DDA and DIA methods for phosphoproteomics and discuss data analysis options for DIA phosphoproteomics. In order to evaluate the impact of data-dependent and data-independent acquisition (DIA) on identification and quantification, we analyze a previously published phosphopeptide-enriched data set consisting of 10 replicates acquired by DDA and DIA each. We find that though more unique identifications are made in DDA data, phosphopeptides are identified more consistently across replicates in DIA. We further discuss the challenges of identifying chromatographically coeluting phosphopeptide isomers and investigate the impact on reproducibility of identifying high-confidence site-localized phosphopeptides in replicates.

Automated summary

Mass spectrometry-based profiling of the phosphoproteome is a powerful method for identifying phosphorylation events. This Perspective reviews recent studies comparing data-dependent acquisition (DDA) and data-independent acquisition (DIA) methods for phosphoproteomics and discusses data analysis options. The analysis of a phosphopeptide-enriched data set shows that DDA identifies more unique phosphopeptides, while DIA provides more consistent identifications across replicates. Challenges include identifying coeluting phosphopeptide isomers and reproducibly locating high-confidence phosphorylation sites.