Evaluation of the utility of neutral‐loss‐dependent MS3 strategies in large‐scale phosphorylation analysis

Phosphopeptide identification and site determination are major challenges in biomedical MS. Both are affected by frequent and often overwhelming losses of phosphoric acid in ion trap CID fragmentation spectra. These losses are thought to translate into reduced intensities of sequence informative ions and a general decline in the quality of MS/MS spectra. To address this issue, several methods have been proposed, which rely on extended fragmentation schemes including collecting MS3 scans from neutral loss‐containing ions and multi‐stage activation to further fragment these same ions. Here, we have evaluated the utility of these methods in the context of a large‐scale phosphopeptide analysis strategy with current instrumentation capable of accurate precursor mass determination. Remarkably, we found that MS3‐based schemes did not increase the overall number of confidently identified peptides and had only limited value in site localization. We conclude that the collection of MS3 or pseudo‐MS3 scans in large‐scale proteomics studies is not worthwhile when high‐mass accuracy instrumentation is used.