Authors
Yonghao Yu, Adam J. Hoffhines, Kevin L. Moore, and Julie A. Leary
Summary
Tyrosine O-sulfation is one of many posttranslational modifications described in nature that can impart critical functional properties to proteins that are independent of the genes encoding them1, 2. It plays a key role in regulating protein-protein interactions in the extracellular space3 and is catalyzed by two closely related Golgi enzymes called tyrosylprotein sulfotransferases (TPST-1 and TPST–2)4, 5. In this protocol, we describe a novel subtractive strategy to determine the sites of tyrosine sulfation in peptides and proteins6. Hydroxyl groups on unsulfated tyrosines are stoichiometrically acetylated by a one-step reaction using sulfosuccinimidyl acetate in the presence of imidazole at pH 7.0. The presence of sulfotyrosine is indicated by the detection of free tyrosine after tandem mass spectrometric analysis during which the sulfuryl group of sulfotyrosine is decomposed. Since phosphorylation and sulfation of tyrosine are isobaric, we used alkaline phosphatase treatment to distinguish these two modifications. This strategy combined with the subtractive methodology is used to unambiguously determine the tyrosine sulfation sites in proteins. Application of this methodology will substantially facilitate identification of additional tyrosine-sulfated proteins and determination of the sites of sulfation, and thus, provide the foundation for a broader understanding of the role of tyrosine sulfation in protein function.Further details
The protocol was published on Protocol Exchange in 2007. To see the entire protocol, click on the source link.Advertisement
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