Revisiting iodination sites in thyroglobulin with an organ-oriented shotgun strategy

Abstract

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.

FIGURE 1.

Schematic illustration of analytical flow. Proteomic analysis consists in direct extraction of thyroid lobes and high-throughput nano-LC ESI-MS/MS. In the dissected mouse shown on the left, the thyroid lobes are indicated with black arrows. The SDS-PAGE gel stained with Coomassie Blue Safe stain is shown on the right. The molecular masses in the first well corresponds to 188, 98, 62, 38, 28, 17, 14, and 6 kDa. The numbering of the gel slices (1–21) is also indicated.

FIGURE 2.

Unambiguous assignment of four different iodination patterns on Tyr²⁵⁷². Representative ESI CID spectra obtained for Tg peptide(2555–2577) (ILAAAVWYYSLEHSTDDYAFSR) are presented. Monoiodotyrosine (panel A), diiodotyrosine (panel B), triiodothyronine (panel C), and thyroxine (panel D) modifications on Tyr²⁵⁷² were observed. In these spectra, information from the y and b series was complete enough to assign the modifications to Tyr²⁵⁷² unambiguously. Note the presence of y₅ and y₆ ions (red labels) in all spectra.

FIGURE 3.

Specific ion pattern for peptide containing the thyroxine modification. This figure was constructed using Protein Prospector software with default resolution set to 10,000. This parameter has no effect on the ratios of the two isotopic patterns shown here. The relative height is shown for the first isotopes of an “Averagine peptide” (black line) with a molecular mass of 1087.5869 and the iodinated peptide (red line) (ILAAVWYYSLEHSTDDYAFSR) containing a thyroxine modification at Tyr²⁵⁷² with a molecular mass of 1087.6808. The elemental composition and characteristics of these two chemical entities are given below, as well as the calculated values for isotopic distribution. The isotopic distribution of iodinated peptide is different from that of noniodinated peptide with a similar mass. The effect of 4 iodine atoms should not be ignored, otherwise this type of iodinated pattern would not be recognized as a peptide pattern.

FIGURE 4.

HCD fragmentation spectrum showing unambiguous attribution of peptide(990–996) with a triiodothyronine modification on Tyr⁹⁹³. HCD fragmentation provides information from m/z = 100 Thomson. For such a small peptide, peptide(990–996), we obtained an almost complete y series. The y₃ and y₄ ions with a mass difference of 632.780 Da (for a theoretical mass difference of 632.892) indicated the presence of the modification. The triiodothyronine immonium ion, which is clearly present on the spectra (605.792), is a residue-specific marker ion that confirms the complete y series.

FIGURE 5.

CID fragmentation spectra of peptide(245–258) (ELATGELLLDEI). The almost complete b and y series obtained from a non-noisy spectrum (35 fragment ions using 42 most intense peaks) gave a Mascot ion score of 101. This spectrum assignment indicates the break on the peptide bond after an isoleucine, which cannot be explained enzymatically but by the transformation of a monoiodo (or diiodo) tyrosine into a pyruvic acid residue.