Isobaric tags for relative and absolute quantitation-based quantitative protein expression profiling of NEMO-binding domain peptide promotes osteoblast differentiation impaired by tumor necrosis factor alpha

Objective To screen for the differentially expressed proteins of NEMO- binding domain peptide promotes osteoblast differentiation impaired by tumor necrosis factor alpha. Methods The myoblast C2C12 cells as cellular model were cultured with serum- free DMEM and divided into three groups cultured with different stimulus. Cells were induced with bone morphogenetic protein(BMP-2)as the control group(group B), cells were induced with BMP- 2 and treated with BMP- 2 and tumor necrosis factor-α(TNF-α)as the experimental group(group BT), cells were induced with BMP- 2 and treated with BMP- 2, TNF-αand NBD peptide as the treatment group (group BTP). The total protein at 7 d during the differentiation was extracted and analyzed by isobaric tags for relative and absolute quantitation(iTRAQ)technology coupled with mass spectrometric analysis. Results A total of 76 significant protein spots were found by mass spectrometric analysis, among which,59 spots were up- regulated and 17 spots were down- regulated between group B and group BT. And 43 significant protein spots were found by mass spectrometric analysis, among which, 25 spots were up- regulated and 18 spots were down- regulated between group BT and group BTP.Among three groups, protein expression of periostin protein(Postn), sarcoplasmic reticulum Ca observed in the ATP share 2+ enzyme(Atp2a3),SR dependent protein(Scaf1), alphaactinin 2(Actn2), ATP synthase subunit D(Atp5d), 5 extended protein 2(Elp2), Atp5l and Niemann- Pick type C1(Npc1) were dynamic changed,implying a close linkage between above protein and osteoblast differentiation impaired by inflammation, there are other drug target to promotes osteoblast differentiation inhibited by inflammation. Conclusion iTRAQ is a useful technologic method in proteomic study of cell differentiation. Postn, Atp2a3, Scaf1,Actn2,Atp5d,Elp2,Atp5l,Npc1 could serve as potential target molecules for the mechanism study on steoblast differentiation inhibited by inflammation. Key words: Osteoblast differentiation; Bone morphogenetic protein- 2; Tumor necrosis factor-α; NEMO- binding domain peptide; Isobaric tags for relative and absolute quantitation