Post-translational
modifications (PTMs) alter the function and
fate of proteins and cells in almost every conceivable way. Protein
modifications can occur as a result of specific regulating actions
of enzymes, such as tyrosine kinases phosphorylating tyrosine residues
or by nonenzymatic reactions, such as oxidation related to oxidative
stress and diseases. While many studies have addressed the multisite,
dynamic, and network-like properties of PTMs, only little is known
of the interplay of the same site modifications. In this work, we
studied the enzymatic phosphorylation of oxidized tyrosine (l-DOPA) residues using synthetic insulin receptor peptides, in which
tyrosine residues were replaced withl-DOPA. The phosphorylated
peptides were identified by liquid chromatography-high-resolution
mass spectrometry and the site of phosphorylation by tandem mass spectrometry.
The results clearly show that the oxidized tyrosine residues are phosphorylated,
displaying a specific immonium ion peak in the MS2spectra.
Furthermore, we detected this modification in our reanalysis (MassIVE
ID: MSV000090106) of published bottom-up phosphoproteomics data. The
modification, where both oxidation and phosphorylation take place
at the same amino acid, has not yet been published in PTM databases.
Our data indicate that there can be multiple PTMs that do not exclude
each other at the same modification site.