Towards the Application of Proteomics to Human Thyroid Tissue

Abstract

In this paper we describe for the first time a systematic approach to proteome analysis of human thyroid tissue. We report different methods to decrease the complexity of the human thyroid tissue proteome by applying different solubilization strategies and correcting for thyroglobulin protein abundance; to increase the protein resolution by prefractionation and by the use of narrow-range pH gradients; to detect proteins using sensitive and quantitative stains; and to identify soluble and membrane-bound thyroid tissue proteins by mass spectrometry analysis. MAIN OUTCOME/RESULTS: We found that buffers containing high contents of urea and detergents allow the best solubilization of human thyroid tissue proteins; highly variable abundance of thyroglobulin is a major pitfall of human thyroid proteome analysis, which in contrast to centrifugal ultrafiltration, size-exclusion chromatography and microdissection, can be countered best by adapting the protein amount to the thyroglobulin content per sample; prefractionation leads to a significant enrichment of proteins and allows subcellar localization of thyroid proteins; application of narrow-range immobilized pH gradient (IPG) strips allows further improvement of spot detection and separation; and protein detection with the fluorescent stain ruthenium II Tris bathophenanthroline disulfonate (RuBPs) is a highly sensitive and reliable tool for quantitative proteome analysis. Finally, in a pilot study of four patients with benign nodular thyroid disease we found that the described procedures allow a highly reproducible detection and identification of alterations in protein expression between nodular and corresponding normal thyroid tissues. Application of the described methods provides the basis for a highly sensitive and reproducible proteome analysis of the human thyroid, providing an additional novel tool to elucidate complex proteins changes in human thyroid biology as well as pathophysiology of human thyroid disease.