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

Yonghao Yu, Adam J. Hoffhines, Kevin L. Moore, Julie A. Leary

Research output: Contribution to journalArticle

54 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)583-588
Number of pages6
JournalNature Methods
Volume4
Issue number7
DOIs
StatePublished - Jul 2007

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Tyrosine
Peptides
Proteins
Vitronectin
Acetylation
Phosphorylation
Extracellular Space
Post Translational Protein Processing
Tandem Mass Spectrometry
Hydroxyl Radical
Mass spectrometry
Alkaline Phosphatase
tyrosine O-sulfate

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology
  • Cell Biology

Cite this

Determination of the sites of tyrosine O-sulfation in peptides and proteins. / Yu, Yonghao; Hoffhines, Adam J.; Moore, Kevin L.; Leary, Julie A.

In: Nature Methods, Vol. 4, No. 7, 07.2007, p. 583-588.

Research output: Contribution to journalArticle

Yu, Yonghao ; Hoffhines, Adam J. ; Moore, Kevin L. ; Leary, Julie A. / Determination of the sites of tyrosine O-sulfation in peptides and proteins. In: Nature Methods. 2007 ; Vol. 4, No. 7. pp. 583-588.
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