【摘要】 利用高效液相色谱／质谱联用技术分析了胶原蛋白Ⅱ和Ⅰ经变性和酶解处理得到的多肽混合物,比较研究了两种类型胶原蛋白降解物之间的区别,利用液相色谱质谱连用技术识别出了不同类型胶原蛋白中的特征肽段。胶原蛋白在50℃处理3h可溶解于盐溶液,凝胶过滤分析结果表明,热变性的胶原蛋白分子量在10万以上,因此在热变性过程中胶原蛋白的三螺旋结构被破坏但没有明显的随机降解。利用胰蛋白酶对胶原蛋白进行了酶切处理,在酶用量为2％,pH 8.1~8.2,温度37℃条件下,处理20h后胶原蛋白降解较完全,多肽混合物的平均分子量在1万以下。利用液质联用技术研究Ⅱ型和Ⅰ型胶原蛋白降解物中的多肽,通过多级质谱分析了胶原蛋白降解物多肽的序列。结果表明,不同类型的胶原蛋白酶解后的多肽混合物中存在特定序列的特征肽段,利用特征肽段进行胶原蛋白类型识别是可行的。更多还原

【Abstract】 Collagen type Ⅱ and Ⅰ from bovine were thermally denatured and digested with trypsin. The digest mixtures of collagen type Ⅱ and Ⅰ were analyzed with high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) . The results indicated that the heat pretreated collagens were soluble when the temperature was above 50 ℃ for 3 hours. No obvious degradation happened to the denatured collagens since the overall molecular weight was above 100kDa. The solubilized collagen Ⅱ and Ⅰ were digested with trypsin in Tris-HCl buffer (pH8.1) at 37 ℃ for 20 hours. Gel filtration ehromatography indicated that the average molecular weight of the final peptide mixture was below 10kDa. Peptides were separated with reversed-phase ehromatography and identified with MS/ MS sequencing. The results indicated that the digest mixtures of collagen Ⅱ and Ⅰ contained lots of characteristic peptides and many common peptides. Characteristic peptides were selected and may be used as index for identifying the collagen type. The research provided a possible approach for collagen identification.更多还原