Established Sulfopeptide Tandem Mass Spectrometry Behavior and Sulfotransferase Assays Refute Tyrosine Sulfation as a Histone Mark

Abstract

Tyrosylprotein sulfotransferases (TPST1 and TPST2) are only known to exist in the Golgi1. Thus, Yu et al.’s findings that histone H3 sulfation occurs in the cytosol are surprising. Cytosolic sulfotransferase SULT1B1, which sulfates phenolic small molecules, was proposed to catalyze H3Y99 sulfation with 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as SO3 donor. Bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analyses were key to this conclusion. Such experiments appear to have been mainly performed following standard protocols, including reduction (reagent unknown), alkylation with iodoacetamide (IAA), and proteolytic digestion prior to nanoflow LC (nanoLC) separation of proteolytic peptides and data-dependent higher energy collision dissociation (HCD) to generate sequence-informative peptide fragments. However, the published HCD MS/MS spectra do not match established sulfopeptide fragmentation behavior. We present reannotation of these spectra and additional experiments that found a lack of evidence to support H3Y99 sulfation.