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Determination of the sites of tyrosine O-sulfation in peptides and proteins

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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.

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Figure 1: Overall subtractive strategy for site determination of protein tyrosine sulfation.
Figure 2: Determination of the site of sulfation in Tyr9-CCK-8.
Figure 3: RP-HPLC analysis of the sulfated CCR8 12–20 peptides generated in vitro by TPST-1 and TPST-2.
Figure 4: Identification of the sites of sulfation in lumican.
Figure 5: Identification of the sites of sulfation in lumican peptide Met29–Tyr52.

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Acknowledgements

This work was supported in part by the US National Institutes of Health, grants GM 63581 to J.A.L. and HL 074015 to K.L.M. Y.Y. acknowledges M. Leavell, E. Damoc, C. Damoc and X. Li for helpful discussions.

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Authors and Affiliations

  1. Departments of Chemistry and Molecular Cell Biology, Genome Center, University of California, Davis, 95616, California, USA

    Yonghao Yu & Julie A Leary

  2. Department of Chemistry, University of California, Berkeley, 94720, California, USA

    Yonghao Yu

  3. Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, 73104, Oklahoma, USA

    Adam J Hoffhines & Kevin L Moore

  4. Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, 73104, Oklahoma, USA

    Kevin L Moore

  5. Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, 73104, Oklahoma, USA

    Kevin L Moore

  6. Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, 73104, Oklahoma, USA

    Kevin L Moore

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  1. Yonghao Yu
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  2. Adam J Hoffhines
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  4. Julie A Leary
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Contributions

Y.Y. and J.A.L. designed the research, Y.Y. acquired the data, A.J.H. and K.L.M. expressed and purified tyrosylprotein sulfotransferases, and Y.Y., K.L.M. and J.A.L. wrote the paper.

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Correspondence to Julie A Leary.

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The authors declare no competing financial interests.

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Yu, Y., Hoffhines, A., Moore, K. et al. Determination of the sites of tyrosine O-sulfation in peptides and proteins. Nat Methods 4, 583–588 (2007). https://doi.org/10.1038/nmeth1056

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