胶内差异双向电泳-电喷雾串联质谱筛选UVB损伤皮肤细胞潜在的蛋白质靶标  

Identification of Potential Protein Biomarkers for Skin Cell Damaged by UVB Irradiation Using 2D-DIGE Coupled to HPLC-nESI MS/MS

作　　者：孙明忠[1] 郑体花[2] 徐康[2] 张春艳[1] 杨帆[2] 田余祥[2] 刘淑清[2] 

机构地区：[1]大连医科大学生物技术系,辽宁大连116044 [2]大连医科大学生物化学教研室,辽宁大连116044

出　　处：《分析测试学报》2011年第4期356-361,共6页Journal of Instrumental Analysis

基　　金：教育部留学回国人员科研启动基金资助项目;国家自然科学基金资助项目(81050010);辽宁教育厅重点实验室资助项目(LS2010050)

摘　　要：联用胶内差异双向电泳(2D-DIGE)和高效液相色谱-电喷雾串联质谱(HPLC-nESI MS/MS)鉴定人角质形成细胞HaCaT应答中波紫外线(UVB)损伤的差异表达蛋白,筛选UVB影响皮肤细胞正常生理功能潜在的靶标蛋白。结果表明:UVB辐射明显影响HaCaT细胞的蛋白质表达谱,DeCyder软件在每块DIGE凝胶上平均检测到1 200多个蛋白斑点,在DIGE检测限以上有显著性表达差异的斑点有41个。HPLC-nESI MS/MS成功鉴定了9个差异表达最显著斑点,4个表达上调斑点被鉴定为26S蛋白酶体亚基p40.5(PSMD13),组织蛋白酶D(CTSD),6-磷酸葡糖酸内酯酶(PGLS),辅分子伴侣23(PTGES3);5个表达下调斑点被鉴定为层粘连蛋白受体1(RPSA),干扰素-γ诱导因子前体(IL18),钙调蛋白(CALM3),60S酸性核糖体蛋白P2类似物(RPP2)和断裂点丛集区蛋白1(BANF1)。9个差异表达蛋白中,除RPSA、CALM3和CTSD被报道与皮肤烫伤和创伤修复、角质形成细胞增殖和迁移、表皮障碍修复相关外,其他蛋白与皮肤细胞损伤和皮肤疾患研究未见报道。这些蛋白是UVB辐射直接影响皮肤细胞正常生理功能潜在的蛋白质靶标,为进一步在分子水平研究皮肤疾病有效防护提供了有价值的线索。Two-dimensional difference in gel electrophoresis （2D-DIGE） combined with high performance liquid chromatographynano-electron spray ionization tandem mass spectrometric（ HPLC -nESI MS/MS） method was developed for the identification of the differentially expressed potential protein markers of human keratinocytes cell HaCaT in response to UVB irradiation, which aims to obtain the target proteins affecting the normal physiological function of skin cells induced by UVB irradiation. The results indicated that UVB irradiation could affect the integrity protein expression profile of HaCaT cell. Over 1 200 protein spots were averagely resolved on each DIGE gel by DeCyder software analysis. 41 protein spots were found differentially expressed with statistical significance beyond DIGE detection limit. 9 protein spots differentially expressed most significantly were successfully identified by HPLC -nESI MS/MS analysis. 4 up-regulated protein spots were identified as 26S proteasome subunit p40.5 （PSMD13 ）, cathepsin D （ CTSD）, human co-chaperone P23 （ PTGES3 ） and 6-phosphogluconolactonase（PGLS）. The other 5 down-regulated protein spots were identified as laminin receptor 1 （ RPSA）, interferon-gamma inducing factor precursor（ ILl8）, calmodulin （ CALM3 ）, protein similar to 60S acidic ribosomal protein P2 （RPP2） and breakpoint cluster region protein 1 （ BANF1 ）. Only RPSA, CALM3 and CTSD proteins have been reported to be associated with skin burns and wound healing, proliferation and migration of keratinocytes and epidermal barrier repairing process. No study has been reported on the associations of the rest 6 proteins with damages from skin and skin cells. These 9 proteins are potential indicators for skin cells responding to UVB irradiation. The results from the current work provide valuable clues to effective protection for skin diseases at molecular level.

关 键 词：UVB HACAT细胞 胶内差异双向凝胶电泳 高效液相色谱-电喷雾质谱联用 

分 类 号：O657.63[理学—分析化学] O629.7[理学—化学]