人椎间盘软骨细胞外基质小分子蛋白的双向电泳和质谱鉴定

Identification of the Extracellular Matrix Small Molecular Proteins of the Intervertebral Disc by Two Dimensional Electrophoreses and Mass Spectrometry

目的初步研究双向电泳和质谱鉴定在人椎间盘的蛋白质组特性研究中的价值和作用．为进一步研究椎间盘疾病寻找有效途径。方法取人的正常椎间盘组织（包括纤维环和髓核），针对细胞外基厦分子蛋白进行蛋白萃取，通过氯化铯梯度离心去除蛋白多糖，通过固相pH梯度（IPG）等电聚焦和梯度聚丙烯酰胺双向电泳分别分离纤维环和髓核的蛋白质．分析电泳图像．使用基质辅助激光解吸附飞行时间质谱仪对蛋白质点进行分析鉴定。结果双向电泳显示。纤维环和髓核的细胞外基质小分子蛋白无明显差异．纤维环和髓核中的小分子蛋白在pH3～10、分子量16～250kd范围内能被很好地分离。对各蛋白质点行质谱仪鉴定．分别确定了萁中相同的19种蛋白质。结论双向电泳可有效地分离准间盘组织中细胞外基质小分子蛋白，通过质谱仪鉴定可以确定其组成成分，为进一步研究椎间盘软骨细胞外基质小分子蛋白的功能及在准间盘病变中的改变和所起的作用打下基础。

Objective To study the extracellular matrix small molecular proteins of the intervertebral disc by two dimensional electrophoreses and mass spectrometry, in order to find a novel effective method helpful to the intervertebral disc disease＇s prevention and therapy. Methods Proteins in both annular fibrosus and nucleus pulposus was extracted from normal subjects, the aggrecan was removed by cesium chloride gradient centrifugation, proteins was separated by two dimensional electrophoreses and identified by matrix assisted laser adsorption/ionizatlon time-of flight mass spectrometry（MALDI- TOF-MS）. Results Comparison of the two sample maps of 2-DE showed that there was no big difference between this two kind of samples in this study, the proteins were in the section of pH 3-7 and molecular weight was 16-250 kd, 19 protein bands were identified by MALDI-TOF-MS. Conclusion The extracellular matrix small molecular proteins could be separated finely by 2-DE and identified by using mass spectrometry, proteomics methods offer a new avenue for the protection and therapy of the diseases of intervertebral disc.