Revisiting iodination sites in thyroglobulin with an organ-oriented shotgun strategy.
Thyroglobulin (Tg) is secreted by thyroid epithelial cells. It is essential for thyroid hormonogenesis and iodine storage. Although studied for many years, only indirect and partial surveys of its post-translational modifications were reported. Here, we present a direct proteomic approach, used to study the degree of iodination of mouse Tg without any preliminary purification. A comprehensive coverage of Tg was obtained using a combination of different proteases, MS/MS fragmentation procedures with inclusion lists and a hybrid mass high-resolution LTQ-Orbitrap XL mass spectrometer. Although only 16 iodinated sites are currently known for human Tg, we uncovered 37 iodinated tyrosine residues, most of them being mono- or diiodinated. We report the specific isotopic pattern of thyroxine modification, not recognized as a normal peptide pattern. Four hormonogenic sites were detected. Two donor sites were identified through the detection of a pyruvic acid residue in place of the initial tyrosine. Evidence for polypeptide cleavages sites due to the action of cathepsins and dipeptidyl proteases in the thyroid were also detected. This work shows that semi-quantitation of Tg iodination states is feasible for human biopsies and should be of significant medical interest for further characterization of human thyroid pathologies.
Commissariat à l'Energie Atomique, DSV, iBEB, Laboratoire des Transporters en Imagerie et Radiothérapie en Oncologie, Bagnols-sur-Cèze F-30207, France. alain.dedieu@cea.fr
Alain
A
Dedieu
Jean-Charles
JC
Gaillard
Thierry
T
Pourcher
Elisabeth
E
Darrouzet
Jean
J
Armengaud
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002403:Cathepsins / Q000032:analysis; D054879:Halogenation; D006728:Hormones / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D013954:Thyroglobulin / Q000737:chemistry* / Q000378:metabolism*; D013961:Thyroid Gland / Q000378:metabolism*; D014020:Tissue Extracts
D006728:Hormones; D014020:Tissue Extracts; D013954:Thyroglobulin; D002403:Cathepsins