Proteomics provides an avenue to understand the interaction between the functional pathways within a cell and its environmental milieu, independent of any changes at the RNA level. There are only a few studies in thyroid diseases using proteomics; variability in specimen content in fibrous versus follicular tissue plus the presence of other types of cells (blood, lymphatic) makes difficult to apply global proteomic to surgery pieces; commercial cell-lines of thyroid cancer were not carefully maintained to preserve thyroid phenotype. We have developed a thyroid-tumour bank of thyrocytes in culture from surgery pieces obtained in our Hospital (CHUS) with more than 40 primary thyroid cultures (BANTTIC, Bravo, Oncogene 2003; Bravo, Clin Cancer Res 2005): from normal thyroid (NT), follicular adenomas, (T-FA), and papillary carcinomas (T-PC); they maintain expression of thyroid markers such as thyroglobulin or TSH-R.
In this study, we want to standardize a method to study proteins expressed differentially between normal samples or benign pathologies (NT, T-FA) and differentiated papillary carcinomas (T-PC2 and T-PC3) from the BANTTIC.
Soluble proteins of the cultures were suspended in Prot Buffer (7 M urea, 2 M thiourea, 5% CHAPS). Fifty micrograms of proteins were passively rehydrated into 11 cm pH gradient strips (IPG 4-7L) and focused for 9000 V. Second-dimension separation was performed using 10% acrilamide-bisacrilamide gels and detection with silver stain. Spots were picked and digested with trypsin. For matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-ToF Bruker RIV) analysis, the peptide digest mixture was directly spotted on the target plate together with a-cyane.
We have indentified common proteins in all cultures to use as controls: some belong to the cytoskeleton (beta-actin, tubulin, vimentin), others are metabolic enzymes (ATP sintase, casein-a-s1). But the pattern of staining shows differences between the NT, and T-PC2 or T-PC3. We are performing an exhaustive analysis, in order to identify specific and consistent spots/proteins useful as markers of thyroid cancer.
03 - 07 May 2008
European Society of Endocrinology