Objectives: 

In future to use pituitary stem cells in human therapy they should be characterized to know what markers in the differentiation process are lost. Thus,we believe of great interest the search for markers in these cells by 2D-PAGE coupled to MALDI-TOF/TOF-MS. AIMS:¹ To standardize the conditions for sample extraction and controls. ² To compare and identify the differential proteomic pattern between Total pituitary, gfra2-cells against gfra2+ cells, combining (2D) gel electrophoresis, image analysis(PDQuest) and mass spectrometry(MALDI-TOF/TOF-MS).

Materials: 

The samples were taken from 16 pituitary Sprague Dawley rats. Sample from total pituitary was taken and the other part was purified to get GPScells (GRFalpha2+ and GRFalpha2- cells). Soluble proteins of pituitary cells,were suspended in a lysis buffer containing 7M Urea, 2M Thiourea, 4% CHAPS and 40mM DTT. 50 mg proteins were rehydrated in 7MUrea, 2MThiourea, 5% CHAPS, 1,2% DeStreak,0.5% of ampholytes 3-10, loaded in 11cm pHgradient strips and focused for 6000V. Second-dimension separation was performed in 15% SDS-PAGE. Spots were visualized by silver staining. 3 replicate gels per sample were scanned with a GS800. Spot detection, pattern evaluation and normalization were performed using the PDQuest software. Individual protein quantities were evaluated by the Student's t-test within the PDQuest analysis in order to compare the three groups and identify sets of proteins that showed a statistically significant difference with a confidence level of 0,05.

Results: 

137 spots were identified. Of them, 45 were statistically different between total pituitary and gfra2- cells versus gfra2+ cells. 2 spots are unique for gfra2+ cells.

Conclusions: 

We've standardized conditions for sample extraction and controls. Spots statistically different will be identified by MALDI-TOF/TOF-MS and validated.