一个未知的子宫雌激素反应蛋白ULF-250的鉴定研究

摘要 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（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 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。另外，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。因此，我们认为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，我们的研究提示，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。

Characterization and identification of the ULF-250, an unknown estrogen-responsive protein

Abstract 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 were collected from the estradiol-treated ovariectomized rats, and protein composition of the ULF were separated by using SDS-PAGE and two-dimensional electrophoresis (2-DE). After separation the protein bands or spots were recognised by Western blotting and 2D-Western using polyclonal antiserum against ULF-250. The target protein were analyzed by Mass Spectrometry and database searching. The 2D-Western analysis showed that anti-ULF-250 antibody specifically recognised the molecular weight of protein spots at 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.com). The matching results suggest that the ULF-250 is ebnerin/DMBT1. Additionally, the 250 kD protein band excised from SDS-PAGE gel was subjected for the HPLC-ESI-MS/MS analysis. The results also suggest that the ULF-250 is ebnerin/DMBT1. Comparison of two proteins indicated that both ULF-250 and ebnerin/DMBT1 are identical on localization and regulation. Therefor, we conclude that the ULF-250 is 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 searching. In addition, our results suggest 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.