Detection and Identification of Differentially Expressed Proteins of HaCaT Cells Responding to 2£¬4 Dinitrobenzene Sulfonic Acid Stimulation Using 2D DIGE Coupled to HPLC-nESI MS/MS

KeyWord:2,4 dinitrobenzene sulfonic acid  occupational exposure  2 dimensional difference in gel electrophoresis  HPLC-nESI MS/MS  HaCaT

Abstract:

      Protein fluorescent dyes,Cy2,Cy3 and Cy5,were used to label protein samples.The protein integrity expression profile of keratinocytes cell HaCaT stimulated by 2,4 dinitrobenzene sulfonic acid£¨DNBS) was probed using two dimensional difference in gel electrophoresis£¨2D-DIGE).And the differentially expressed protein spots were compared and selected.Triplicate samples of protein extracts were used for both the control HaCaT cell and the DNBS treated HaCaT cell.Following the 2D-DIGE,the master DIGE gel was post stained by using Deep Purple protein fluorescent dye.The results indicated that over 1200 protein spots were averagely resolved on each DIGE gel by DeCyder software analysis.846 protein spots were matched for all three gels.And seven protein spots were found differentially expressed with the level above 50% due to the DNBS stimulation with statistically significance£¨P<0ª±05).A proteomic approach,high performance liquid chromatography combined to nano electro spray ionization tandem mass spectrometry£¨HPLC-nESI-MS/MS) was used for protein identification.The five up regulated protein spots were identified as chromosome X open reading frame 26£¨Cxorf26),human Co chaperone P23£¨PTGES3),calmodulin£¨Calm3),smooth muscle and non muscle myosin alkali light chain 6£¨MYL6),and breakpoint cluster region protein 1£¨BANF1).The two down regulated protein spots were identified as transcription elongation factor B polypeptide 2£¨TCEB2) and RPL23 protein,respectively.Only myosin has been reported to be asso ciated with certain skin disease.Those seven protein candidates identified by current work might provide some useful clues for the risk assessment of skin diseases originated from occupational exposures to chemical carcinogens such as DNBS.