Tyrosine O-sulfation, i.e., transfer of a sulfonate group to tyrosine amino acid residues in proteins, is a widespread posttranslational modification (PTM) in eukaryotic cells with a variety of known functions in health and disease, including receptor binding, viral replication, inflammation, and retinal function. The enzymes that catalyze tyrosine sulfation are located in the Golgi apparatus. The function of this organelle is to ensure that correct protein modification occurs and to package proteins into vesicles for export to the cell surface, or the extracellular environment. Because proteins must typically enter the Golgi to become sulfated, most known sulfoproteins are secreted proteins or membrane proteins. Mass spectrometry (MS) is a powerful tool for global PTM analysis in cells and tissues; however, large scale analysis of tyrosine O-sulfation has not been feasible, due in part to its labile nature in the gas-phase environment of a mass spectrometer, and in part due to the lack of appropriate data analysis strategies. In MS experiments, proteins are typically digested into smaller peptides, which are ionized, detected, and fragmented to deduce sequence information. When measuring protein phosphorylation, another rather labile PTM known to regulate Golgi disassembly and reassembly during cell division, in interphase vs. mitotic Golgi, we found that tyrosine O-sulfation was co-enriched. This discovery is not surprising because the chemical properties of sulfation (O-SO3) are similar to phosphorylation (O-PO3H). However; high mass accuracy measurements are required to deduce the small mass difference of 0.0095 Da between these two PTMs. Even as such high performance measurements are becoming more routine, standard database search tools typically do not identify protein sulfation because this PTM is completely lost during analysis. We found that open database searching was able to overcome this problem and, thus, we were able to accomplish identification of a number of novel sulfoproteins in rat liver Golgi. While an exciting advance, the exact location of O-sulfation within proteolytic peptides could not be directly measured.
In Aim 1 of this proposal, we seek to develop improved methods for detection of intact sulfopeptides by MS, including elimination of competing phosphorylation, determination of peptide sequence effects, implementation of stabilizing adducts, and conditions that selectively dissociate sulfopeptides. To further allow sulfate site localization, in Aim 2, we seek to develop technologies for fragmenting sulfopeptides while retaining sulfate in fragment ions. These approaches include negative ion mode free radical initiated peptide sequencing, which allows sulfopeptides to enter the mass spectrometer as more stable anions, and the development of ?smart? data acquisition strategies for improved electron transfer dissociation.
The final Aim 3 seeks to apply these improved approaches for comprehensive analysis of the Golgi sulfoproteome in cells and animal tissue, particularly under perturbed Golgi conditions, which are expected to alter sulfation. These types of measurements will provide transformative information regarding the regulatory roles of tyrosine sulfation and its impact on cellular function.
Protein tyrosine sulfation plays important roles in, e.g., protein-protein interactions, inflammation, and viral infections. Due to the extreme lability of this posttranslational modification (PTM), global liquid chromatography/mass spectrometry analysis has not been feasible to date; however we discovered a novel workflow that partially enables such measurements. Here, we propose new strategies to further enhance detection and allow site determination of this widespread PTM in cells and tissues.