Updated project metadata. Tyrosine sulfation is a common posttranslational modification in mammals. To date, it has been thought to be limited to secreted and transmembrane proteins, but little is known about tyrosine sulfation on nuclear proteins.To search for tyrosine sulfation on nuclear proteins, we purified the nucleus of HepG2 cells and searched for Ysulf using HPLC–MS/MS.We noted a peptide that is highly indicative of sulfation on the tyrosine 99 residue of histone H3(H3Y99sulf).We synthesized the peptides with the same primary sequence and modifications as were identified in the HPLC–MS/MS analysis: The synthesized H3Y99sulf peptides and their in vivo counterparts showed similar chromatographic and mass spectrum profiles in the same HPLC–MS/MS system . These results demonstrate that H3Y99sulf is an undocumented histone modification