一个未知的子宫雌激素反应蛋白ULF-250的鉴定

Characterization and Identification of ULF-250,an Unknown Estrogen-responsive Protein

鉴定一个未知的子宫雌激素反应蛋白ULF-250.去卵巢大鼠补充给予雌二醇造成子宫产生大量宫腔液(uterine luminal fluid,ULF),受雌激素调节的蛋白分泌其中.收集ULF分别进行SDS-PAGE和双向电泳(2-DE)分离;通过Western印迹和2D-Western确认抗ULF-250抗体识别的蛋白成分并与凝胶上的条带或斑点相对应;对目标蛋白分别用MALDI-TOF-MS和HPLC-ESI-MS/MS两种方法进行质谱分析并获得肽段序列数据;与蛋白数据库比对及文献检索鉴定未知蛋白.2D-Western显示抗ULF-250抗体特异识别的蛋白成分.从2D胶上切下对应的蛋白斑点进行MALDI-TOF-MS,结果提示:ULF-250是ebnerin/DMBT1.经SDS-PAGE分离的250 kD蛋白条带进行液-质联用分析,结果同样提示:ULF-250是ebnerin/DMBT1.与文献报道比较,ULF-250与ebnerin/DMBT1在子宫的组织定位和调节是相同的,认为ULF-250是子宫表达的ebnerin/DMBT1.通过2-DE结合质谱分析以及查阅文献,确认未知的雌激素反应蛋白ULF-250是ebnerin/DMBT1.此外,研究提示,该蛋白不仅在子宫上皮细胞表达而且分泌到子宫腔液中,其功能值得进一步研究.

To characterize an unknown estrogen-responsive protein ULF-250, which was first identified from the uterine luminal fluid （ULF） of rat in this laboratory, the ULF was collected from the estradiol-treated ovariectomized rats, and protein components of the ULF were separated by SDS-PAGE and two-dimensional electrophoresis （2-DE）. After separation, the protein bands or spots were recognized by polyclonal antiserum against ULF-250 using Western blotting and 2D-Western. The targeted protein was analyzed by mass spectrometry and compared with protein in database. The 2D-Westeru analysis showed that anti-ULF-250 antibody specifically recognized the protein spots at the molecular weight of 250 kD. The spots were excised from Coomassie blue-stained gel, and in-gel digestion with trypsin was performed. Tryptic peptides were analyzed by MALDI-TOF-MS. The peptide mass fingerprinting （PMF） and sequences obtained were matched to SWISSPROT protein database using the Mascot search algorithm （www. matrixscience, corn）. The result indicated that the ULF-250 was ebnefin/DMBT1. Additionally, the 250-kD protein band excised from SDSPAGE gel was subiected to the HPLC-ESI-MS/MS analysis. The result also suggested that the ULF-250 was ebnerin/DMBT1. The comparison of two proteins indicated that ULF-250 and ebnerin/DMBT1 were identical on localization and regulation. Therefore, we concluded that the ULF-250 was ebnerin/DMBT1 expressed in the uterus. The unknown estrogen-responsive protein ULF-250 was characterized and validated as ebnerin/DMBT1 by using 2-DE combined with MS as well as data search. In addition, our results suggested that ebnerin/ DMBT1 was not only expressed in epithelium of uterus but also secreted into the luminal fluid. The function of the protein needs further investigation.