Comprehensive proteomic and structural mapping of post-translational modifications of human thyroglobulin

Human thyroglobulin (hTg) is the precursor of thyroid hormones (TH) and a major iodine reservoir in the thyroid gland. Its maturation and function critically depend on multiple post-translational modifications (PTMs). To date, their global distribution, structural organization and physiological roles remain incompletely defined. Here we combined multiple proteolytic digestions with high-resolution nano-liquid chromatography-tandem mass spectrometry to produce a comprehensive map of hTg PTMs and interpreted the results in the context of its three-dimensional cryo-electron microscopy structure. Our approach enabled a near-complete (99.3%) sequence coverage of hTg, and the identification and quantification of numerous PTMs, including glycosylation, oxidation, phosphorylation, acetylation, succinylation, sulfonation, and iodination. In particular, we identified more probable N-glycosylation sites than previously reported, together with numerous methionine and tryptophan oxidation sites. Strikingly, nearly all non-hormonogenic tyrosine residues, as well as several histidine residues, were found to be iodinated. The iodination of those residues strongly correlated with residue accessibility, suggesting a role for hTg in iodine storage. The other identified PTMs were interpreted in the context of TH biosynthesis. Further functional studies are required to fully elucidate the biological significance of hTg PTMs. Together, these findings provide a structural and proteomic reference map of hTg for future studies and refine current understanding of its molecular organization and contribution to thyroid hormonogenesis.