基于生物质谱的胶原蛋白定量检测方法

Collagen quantitation by detection of marker peptides with HPLC-MS

摘要:

本研究旨在建立一种基于特征多肽的胶原定量检测方法，通过序列比对的方法筛选胶原蛋白特征多肽，利用胰蛋白酶将牛Ⅰ型胶原蛋白标准品进行酶解，采用液质联用技术 (HPLC-MS) 对特征多肽进行检测，建立特征多肽丰度与胶原蛋白浓度对应关系并用于实际样品分析。结果表明，牛Ⅰ型胶原蛋白中检测出6种特征多肽，其中多肽GEAGPSGPAGPTGAR由于其丰度高且二级质谱稳定适合作为定量检测的特征多肽，多肽信号强度与蛋白浓度 (0.1?3.0 mg/mL) 呈良好线性关系。将所建方法用于实际样品分析，牛跟腱胶原蛋白含量为90.2%，胶原海绵中胶原蛋白含量为93.4%，检测结果与基于氨基酸组成分析的结果一致。该研究表明基于HPLC-MS的特征多肽分析方法进行胶原蛋白定量检测具有可行性，该方法在含胶原蛋白医疗器械等生物制品质量控制方面具有应用前景。

Abstract:

A method for quantitation of collagen was established by detecting marker peptide with high performance liquid chromatography-mass spectrometry (HPLC-MS). Theoretical marker peptides were selected by sequence comparison. Bovine collagen type I was digested with trypsin. Marker peptides typical for collagen type I were identified with HPLC-MS. The relationship between the abundance of marker peptides and collagen concentration was established. The results show that GEAGPSGPAGPTGAR and the other 5 peptides showed high resolution during chromatographic separation and high signal intensity during MS analysis. Peptide signal intensity and collagen concentration showed a good linear relationship in the range from 0.1 to 3 mg/mL. Bovine tendon and collagen sponge were used as actual samples and collagen contents were determined as 90.2% and 93.4% respectively. Quantitation of marker peptides of collagen was a feasible method to identify and quantify collagens in medical device research and development.