Endocrine Abstracts

Up to now there are few studies about differential proteomics in thyroid. Our group has developed a method for standard human primary thyroid culture maintaining the phenotype through passages. Thus, we believe of great interest the search for biomarkers in these cultures by 2D-PAGE coupled to MALDI-TOF/TOF-MS.

Aims: i) To standardize the conditions for sample extraction and controls. ii) To compare and identify the differential proteomic pattern between normal or benign thyroid (Pendred’s Sd. Hyperplasia) pathologies against thyroid papillary carcinomas, combining (2D) gel electrophoresis, image analysis (PDQuest) and mass spectrometry (MALDI-TOF/TOF-MS).

Results: Soluble proteins of the human thyroid culture samples (NT, T-PS, T-PC), were suspended in a lysis buffer containing 7 M Urea, 2 M Thiourea, 4% CHAPS and 40 mM DTT. 50 μg proteins were rehydrated in 7 M Urea, 2 M Thiourea, 5% CHAPS, 1.2% DeStreak, 0.5% of ampholytes 3–10, loaded in 11 cm pH gradient strips (IPG 3–10 l, Amersham) and focused for 6000 V. Second-dimension separation was performed in 15% SDS-PAGE. Spots were visualized by silver staining. Three replicate gels per sample were scanned with a GS800.

Spot detection, pattern evaluation and normalization were performed using the PDQuest software (vs 8.0.1, Bio-Rad).

The individual protein spot quantity was normalized to the total quantity of all valid spots and is expressed as parts per million (PPM).

Individual protein quantities were evaluated by the Student’s t-test within the PDQuest analysis in order to compare the two groups and identify sets of proteins that showed a statistically significant difference with a confidence level of 0.05.

Twenty two spots were statistically different in tumoral cultures (T-PC) vs normal thyrocytes (NT) and benign pathologies (T-PS). Of them, 12 were specifically increased and eight decreased in T-PC.

MALDI-TOF/TOF-MS identified some of the last spots. One of the most interesting is a low molecular weight variant protein-spot 8002.

We are performing several functional studies in order to validate this protein.

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Funding: This work was supported, however funding details unavailable.