om skin and FACS sorting, being CD105, CD73, CD90, lacking CD14 and CD34 as surface markers, being able to growth under plastic and differentiate into osteoblastic cells by osteodifferentiation induced assay and Alizarin Red stainig after 14 and 21 days. These cells were also cap able of chondro, osteo and adipogenesis, validated through histochemistry and gene expression assays, as described in the literature. Materials The protease and phosphatase inhibitor cock tail, were purchased from Roche. Modified porcine trypsin was purchased from Promega. DTT, ammonium bi carbonate, sodium cyanoborohydride, iodoacetamide, triethylammonium bicarbonate and glycolic acid, were from Sigma. CD2O,13CD2O, and sodium cyanoborodeuteride were from Isotec. Formaldehyde and ammonia solution was purchased from Merck.
Poros Oligo R3 reversed phase material was from PerSeptive Biosystems. Entinostat TiO2 beads were obtained from GL Science. EmporeTM C8 extraction disk was from 3 M Bioanalytical Technologies. The water used in all experiments was obtained from a Milli Q purification system. All other chemicals were pur chased from commercial sources and were of analysis grade. Total protein extract from murine derived mesenchymal stem cells induced with rhBMP2 Cell extracts from mesenchymal stem cells were made as previously described, with some modifications. Briefly, murine skin derived mesenchymal stem cells obtained in our laboratory, were seeded onto 100 mm diameter culture plate in Dulbeccos modified Eagles Medium containing Glutamax I, 1% penicillin streptomycin and 10% fetal bovine serum at 37 C until they reached 90% con fluence.
The medium was then changed in each experi mental group for DMEM supplemented with 200 ng ml of rhBMP2 and 10% fetal bovine serum. After the induc tion period, the cultures were washed twice with ice cold PBS buffer. After washing, cells were harvested and the cell suspension was then centrifuged at 1,000 g for 5 min. The cell pellet was ressuspended in 100 ul of lysis buffer, 2 M thiourea, 1% N octyl glycoside, 40 mM Tris containing phosphatase and proteinase inhibitors and 300 units of Benzonase. The cells were then sonicated at 40% output with intervals of 3 �� 15 s on ice to disrupt the cells and then incubated at ?80 C for 30 min. After incubation, 20 mM DTT was added, and samples were incubated at room temperature for 35 min.
Iodoacetamide was then added, followed by incubation for 35 min at room temperature in the dark. For protein precipitation, 14 ml of ice cold acet one was added to the solution, followed by incubation at ?20 C for 20 min. The proteins were pelleted by centrifugation at 6,000 g for 10 min at 4 C, and the pellet was stored at ?20 C until further use. The BCA method was used to determine the protein concentra tion of each sample. Tryptic digestion of total protein extracts Precipitated proteins from msMSC cells were solubilized in 100 mM TEAB, and 50 ug of total protein extract, quantified by the bicinchoninic acid assay