人椎间盘纤维环软骨细胞外基质小分子蛋白的双向电泳和质谱鉴定研究

目的初步研究双向电泳和质谱鉴定在人椎间盘纤维软骨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的治疗寻找有效途径。方法取人的正常椎间盘的纤维环组织,针对细胞外基质的小分子蛋白进行蛋白萃取,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(MALDI)对蛋白质点进行分析鉴定。结果双向电泳显示,纤维环中的胶原结合蛋白在pH3~10,相对分子质量在22000~250000范围内能够被很好地分离,对各蛋白质点进行质谱仪鉴定,确定了其中的17种蛋白质。结论双向电泳可有效地分离椎间盘纤维环组织中的细胞外基质小分子的胶原结合蛋白质,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病变中胶原结合蛋白的改变及作用打下基础。

Identification of collagen binding proteins of intervertebral disc by two dimensional electrophoresis and mass spectrometry

Objective To study the collagen binding protein of the intervertebral disc by two dimensional electrophoresis and mass spectrometry,to find a novel effective method helpful to prevent and treat the intervertebral disc disease.Methods Collagen binding proteins were extracted from fibrous rings of normal hunman samples,and separated by two dimensional electrophoresis,and identified by matrix assisted laser adsorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).Results Comparison of the two methods showed that there was no significant difference between both,the proteins were in the section of pH 3～10 and molecular weight was 22 000～250 000,17 types of proteins were identified by MALDI-TOF-MS.Conclusion The extracellular matrix small molecular proteins could be separated finely by 2-DE and identified using mass spectrometry,proteome methods offer a new avenue to protection and treatment of the intervertebral disc disease.