Title : Determination of the sites of tyrosine O-sulfation in peptides and proteins
Abstract :
Tyrosine O-sulfation is a key post-translational modification that regulates protein-protein interactions in extracellular space
We describe a subtractive strategy to determine the sites of tyrosine O-sulfation in proteins
Hydroxyl groups on unsulfated tyrosines are blocked by stoichiometric acetylation in a one-step reaction using sulfosuccinimidyl acetate (S-NHSAc) in the presence of imidazole at pH 7.0
The presence of sulfotyrosine is indicated by the detection of free tyrosine after tandem mass spectrometry (MS/MS) analysis under conditions in which the sulfuryl group of sulfotyrosine is labile
Since phosphorylation and sulfation of tyrosine are isobaric, we used alkaline phosphatase treatment to distinguish these two modifications
Using this methodology we identified the sites and the order of sulfation of several peptides mediated by purified human tyrosylprotein sulfotransferases (TPSTs), and unambiguously determined the tyrosine sulfation sites in mouse lumican and human vitronectin