Gas-phase rearrangements do not affect site localization reliability in phosphoproteomics data sets

Area of Science:

• Proteomics and Mass Spectrometry
• Post-Translational Modifications

Background:

• Intramolecular phosphate transfer during collision-induced dissociation (CID) in mass spectrometry has been reported.
• This phenomenon could potentially confound site localization in large-scale phosphorylation studies.
• Phosphorylation site assignment relies on specific fragment ions in MS/MS spectra.

Purpose of the Study:

• To investigate the impact of intramolecular phosphate transfer on phosphorylation site localization.
• To compare the reliability of CID versus electron-transfer dissociation (ETD) for site localization.
• To assess the overall validity of existing and future phosphorylation data sets.

Main Methods:

• Utilized synthetic phosphopeptide libraries, specifically doubly charged species, to study phosphate transfer.
• Compared site localization accuracy between CID and ETD using synthetic libraries.
• Evaluated site localization in a large-scale yeast phosphoproteome experiment.

Main Results:

• Intramolecular phosphate transfer was observed in synthetic phosphopeptides but was insufficient to cause mislocalization of phosphorylation sites.
• Site localization agreement between CID and ETD was high (>99.5% for synthetic libraries, 93% for yeast phosphoproteome).
• ETD did not demonstrate superior reliability over CID for phosphorylation site localization.

Conclusions:

• Intramolecular phosphate transfer does not compromise the accuracy of phosphorylation site localization.
• The reliability of current and past large-scale phosphorylation data sets is not affected by this transfer reaction.
• Both CID and ETD are reliable methods for phosphorylation site determination.