Electron Transfer/Higher Energy Collisional Dissociation of Doubly Charged Peptide Ions: Identification of Labile Protein Phosphorylations

In recent years, labile phosphorylation sites on arginine, histidine, cysteine, and lysine as well as pyrophosphorylation of serine and threonine have gained more attention in phosphoproteomic studies. However, the analysis of these delicate posttranslational modifications via tandem mass spectrometry remains a challenge. Common fragmentation techniques such as collision-induced dissociation (CID) and higher energy collisional dissociation (HCD) are limited due to extensive phosphate-related neutral loss. Electron transfer dissociation (ETD) has shown to preserve labile modifications, but is restricted to higher charge states, missing the most prevalent doubly charged peptides. Here, we report the ability of electron transfer/higher energy collisional dissociation (EThcD) to fragment doubly charged phosphorylated peptides without losing the labile modifications. Using synthetic peptides that contain phosphorylated arginine, histidine, cysteine, and lysine as well as pyrophosphorylated serine residues, we evaluated the optimal fragmentation conditions, demonstrating that EThcD is the method of choice for unambiguous assignment of tryptic, labile phosphorylated peptides. Graphical