biostudies-literatureDaly LABiotechnology and Biological Sciences Research Council3754-3772https://www.ebi.ac.uk/biostudies/studies/S-EPMC10696596biostudies-literatureUnknown22(12)Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalyzed by Golgi-resident tyrosyl protein sulfo transferases (TPSTs). Information on sY in humans is currently limited to ∼50 proteins, with only a handful having verified sites of sulfation. As such, the contribution of sulfation to the regulation of biological processes remains poorly defined. Mass spectrometry (MS)-based proteomics is the method of choice for PTM analysis but has yet to be applied for systematic investigation of the "sulfome", primarily due to issues associated with discrimination of sY-containing from phosphotyrosine (pY)-containing peptides. In this study, we developed an MS-based workflow for sY-peptide characterization, incorporating optimized Zr⁴⁺ immobilized metal-ion affinity Journal of proteome researchCustom Workflow for the Confident Identification of Sulfotyrosine-Containing Peptides and Their Discrimination from Phosphopeptides.PMC10696596BB/R000182/1BB/X002780/1BB/M012557/1BB/S018514/1BB/S017054/1Perkins SEyers CEEyers PAJones ARDaly LAMcDonnell EByrne DPBrownridge PJfalseCustom Workflow for the Confident Identification of Sulfotyrosine-Containing Peptides and Their Discrimination from Phosphopeptides.Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalyzed by Golgi-resident tyrosyl protein sulfo transferases (TPSTs). Information on sY in humans is currently limited to ∼50 proteins, with only a handful having verified sites of sulfation. As such, the contribution of sulfation to the regulation of biological processes remains poorly defined. Mass spectrometry (MS)-based proteomics is the method of choice for PTM analysis but has yet to be applied for systematic investigation of the "sulfome", primarily due to issues associated with discrimination of sY-containing from phosphotyrosine (pY)-containing peptides. In this study, we developed an MS-based workflow for sY-peptide characterization, incorporating optimized Zr⁴⁺ immobilized metal-ion affinity 2023-01-01T00:00:00Z2023 Dec2026-05-28T23:55:17.228Z2025-04-05T22:24:53.907ZS-EPMC106965963793928210.1021/acs.jproteome.3c00425