Nature Methods
- 4, 583 - 588 (2007)
Published online: 10 June 2007; | doi:10.1038/nmeth1056
Determination of the sites of tyrosine O-sulfation in peptides and proteinsYonghao Yu1, 2, Adam J Hoffhines3, Kevin L Moore3, 4, 5, 6 & Julie A Leary11
Genome Center, Departments of Chemistry and Molecular Cell Biology, University of California, Davis, California 95616, USA. 2
Department of Chemistry, University of California, Berkeley, California 94720, USA. 3
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA
4
Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA
5
Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA. 6
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.
Correspondence should be addressed to Julie A Leary jaleary@ucdavis.edu 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.
MORE ARTICLES LIKE THIS These links to content published by NPG are automatically generated.
|
