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.
authors.affiliation: Genome Center, Department of Chemistry, University of California, Davis, California 95616, USA.
authors.forename: Yonghao
authors.initials: Y
authors.lastname: Yu
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Hoffhines
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Moore
authors.forename: Julie A
authors.initials: JA
authors.lastname: Leary
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry*; D015238:Sulfotransferases / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry
chemical_list: D010455:Peptides; D011506:Proteins; C005662:tyrosine O-sulfate; D014443:Tyrosine; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase