2D-DIGE-HPLC-nESIMS/MS对2,4-二硝基苯磺酸损伤HaCaT细胞差异蛋白质的鉴定

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

采用Cy2、Cy3和Cy5荧光染料标记蛋白,建立了人角质形成细胞HaCaT受2,4-二硝基苯磺酸(DNBS)刺激前后的双向胶内差异凝胶电泳(2D-DIGE)图谱,每组平行样本数为3。凝胶采用蛋白荧光染料Deep Purple进行后染色(Post-stain)。DeCyder定量分析软件在每块凝胶上平均检测到1 200个以上蛋白斑点,每块胶上都匹配得到的相同蛋白质斑点有846个。其中有7个斑点丰度变化在50%以上,统计学意义显著(P值小于0.05)。利用高效液相色谱-电喷雾串联质谱(HPLC-nESI MS/MS)成功鉴定5个表达上调的斑点分别为X染色体开放阅读框26(Cxorf26)、人辅分子伴侣23(PTGES3)、钙调蛋白(CALM3)、肌球蛋白轻链6(MYL6)和断裂点丛集区蛋白1(BANF1);2个表达下调蛋白斑点被鉴定为转录延伸因子B肽链2(TCEB2)和核糖体蛋白L23(RPL23)。除MYL6被报道与皮肤疾病相关外,其它蛋白与皮肤病变的关系有待研究。该研究得到的7个差异表达蛋白为DNBS类化学致癌物职业接触者皮肤病变研究提供了有价值的线索。

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 〈 0.05 ）. 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 associated 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.