Significance
Protein phosphorylation is a ubiquitous post-translational modification that regulates protein function. Mass spectrometry-based approaches have revolutionised its analysis on a large-scale but phosphorylation sites are often identified by single phosphopeptides and therefore require more rigorous data analysis to unsure that sites are identified with high confidence for follow-up experiments to investigate their biological significance. The coverage and confidence of phosphoproteomic experiments can be enhanced by the use of multiple complementary fragmentation methods. Here we have benchmarked a data analysis pipeline for analysis of phosphoproteomic data generated using CID and ETD fragmentation and used it to demonstrate the utility of a data-dependent neutral loss triggered ETD fragmentation strategy for high confidence phosphopeptide identification and phosphorylation site localisation.