Phosphopeptide Fragmentation and Site Localization by Mass Spectrometry: An Update.

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Pioneering work by among others the groups of Hunt and Mann at the start of this century opened up the era of mass spectrometry based phosphoproteomics1-3. Through advances in sample preparation, phosphopeptide enrichment, mass spectrometric detection, peptide sequencing and dedicated database searches this field has matured and it is nowadays possible to monitor thousands of protein phosphorylation events qualitatively and quantitatively4-8. Such phosphoproteomics studies are important as protein phosphorylation is regulating nearly all biological processes and the deregulation of phosphorylation has been associated with the onset of various diseases9-15. Notwithstanding this maturation and broad acceptance of mass spectrometry based phosphoproteomics by the research community, the mass spectrometric (MS) analysis of phosphorylation remains demanding, mainly due to the often severe substoichiometric levels of phosphorylation and the intrinsic lability of the phosphate group, hampering both enrichment and unambiguous sequencing analysis of phosphopeptides. Here we review the increasing knowledge gathered about the mechanisms behind the fragmentation of phosphopeptide ions and describe additionally recent advances made to improve and facilitate the identification and site localization of phosphorylated peptides. Since our earlier related review in 200916 several fragmentation methods have been introduced and successfully applied to phosphopeptides, such as electron capture (ECD), electron transfer induced dissociation(ETD), and hybrid fragmentation techniques such as EThcD (a combination of ETD and HCD) and AI-ETD (a combination of ETD with infrared photo-activation). A plethora of data has been gathered on endogenous Ser, Thr, Tyr phosphorylation, but also on large synthetic phosphopeptide libraries, providing new insight into the mechanisms behind phosphopeptide fragmentation. Moreover, data on phosphopeptides harboring phosphorylated Histidine, Arginine or Lysine have become available, which exhibit some distinct fragmentation behaviors. Through all this acquired knowledge on phosphopeptide fragmentation and site localization mass spectrometry based phosphoproteomics has matured into a respectable tool for the broader life science research community.