Protein tyrosine sulfation is a key post-translational modification (PTM) tied to many biological processes such as inflammation, hemostasis, visual functions, and viral infection. Advancement in the understanding of sulfation within the proteome is largely hindered due to the complexity in identification and characterization of the proteome for this modification <i>via</i> mass spectrometry (MS). We propose two potential methods for improving the detection of tyrosine sulfation by MS: carboxylate capping and aniline enrichment strategies. Based on known sulfotyrosine peptide sequences, ~5-10 acidic residues (Asp and/or Glu) are anticipated to be proximal to the modified tyrosine. We sought to determine the impact these residues have on ionization efficiency of sulfotyrosine-containing tryptic peptides in the more sensitive positive ion mode MS by utilizing an acetohydrazide reaction to effectively “cap” the negative residues. Additionally, the complexity of a tryptic peptide sample of the whole human proteome is too vast for analysis in a single MS preparation workflow, so enrichment strategies that decrease the sample complexity while enriching for peptides of interest are almost universally employed. We designed a tyrosine-specific biotin enrichment probe utilizing a diazonium reaction, which has been shown to be specific to tyrosine at pH 9-10. A proteomic workflow will be developed by the combination of the capping reaction and the enrichment strategy leading to enhancing the detection of tyrosine sulfation which will allow us to identify and characterize previously undetermined tyrosine sulfated peptides within the proteome.

Development of improved detection methods for proteomic analysis of tyrosine sulfation

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