中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (30): 7918-7925.doi: 10.12307/2026.952

• 神经组织构建 nerve tissue construction • 上一篇    下一篇

基于高效液相色谱-质谱联用的牛Ⅰ型胶原端肽含量分析

高建萍1，邢芳毓1，张  扬1，张天阳1，张  焱1，张贵锋1   

  1. 中国科学院过程工程研究所，生物药制备与递送全国重点实验室，北京市   100190

  • 收稿日期:2026-04-17 修回日期:2026-05-08 出版日期:2026-10-28 发布日期:2026-09-12
  • 通讯作者: 张贵锋，博士，研究员，中国科学院过程工程研究所，生物药制备与递送全国重点实验室，北京市 100190
  • 作者简介:高建萍，女，1987年生，山东省潍坊市人，汉族，博士，工程师，主要从事质谱分析、胶原基生物材料分析方法研究。
  • 基金资助:
    北京市自然科学基金-昌平创新联合基金资助项目(L234071)，项目名称：基于液质联用的胶原蛋白端肽定量检测方法及去除工艺研究，项目负责人：高建萍；北京市自然科学基金-昌平创新联合基金资助项目(L234021)，项目名称：胎盘多肽中促皮肤改善的成分筛选及其作用机制探索，项目负责人：张贵锋；北京市自然科学基金-昌平创新联合基金资助项目(L234070)，项目名称：液态胶原蛋白复杂体系的超高压灭菌方法和原生态表征，项目负责人：张焱

Analysis of bovine type I collagen telopeptide using high performance liquid chromatography-mass spectrometry

Gao Jianping, Xing Fangyu, Zhang Yang, Zhang Tianyang, Zhang Yan, Zhang Guifeng   

  1. Institute of Process Engineering, Chinese Academy of Sciences, State Key Laboratory of Biopharmaceutical Preparation and Delivery, Beijing 100190, China
  • Received:2026-04-17 Revised:2026-05-08 Online:2026-10-28 Published:2026-09-12
  • Contact: Zhang Guifeng, PhD, Researcher, Institute of Process Engineering, Chinese Academy of Sciences, State Key Laboratory of Biopharmaceutical Preparation and Delivery, Beijing 100190, China
  • About author:Gao Jianping, PhD, Engineer, Institute of Process Engineering, Chinese Academy of Sciences, State Key Laboratory of Biopharmaceutical Preparation and Delivery, Beijing 100190, China
  • Supported by:
    Beijing Natural Science Foundation - Changping Innovation Joint Fund, Nos. L234071 (to GJP), L234021 (to ZGF), and L234070 (to ZY)

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摘要：



文题释义：
胶原蛋白端肽：胶原蛋白两端的非三螺旋区域分别为N末端和C末端端肽。
端肽特征多肽：是指仅存在于特定类型、特定种属来源的胶原蛋白端肽中特定序列的多肽。

背景：动物来源的胶原蛋白因具有一定的免疫原性而影响其应用效果，端肽的去除率是影响免疫原性的最主要原因。因此，建立一种高精度、高灵敏度的端肽检测方法对于牛Ⅰ型胶原蛋白产品质量评价、工艺改进及免疫原性的评价具有重要意义。
目的：采用高分辨质谱识别牛Ⅰ型胶原蛋白N末端和C末端端肽特征多肽，并建立基于高效液相色谱-质谱联用的牛Ⅰ型胶原蛋白端肽特征多肽的定量检测方法。
方法：采用高效液相色谱四级杆质谱联用法，色谱柱为Zorbax C18 (2.1 mm×150 mm，5 μm)，流动相为0.1% 甲酸水-60% 乙腈水溶液(含0.1%甲酸)，梯度洗脱，流速为0.2 mL/min，柱温30 ℃；离子源为电喷雾电离，正离子扫描，多反应监测。
结果与结论：①牛Ⅰ型胶原N、C末端端肽的特征多肽分别为Q*LSYGYDE(Q*为焦谷氨酸)和LSFLPQPPQE；②N、C末端端肽特征多肽分别在0.5-20 μg/mL(r=0.999 3)、0.05-10 μg/mL(r=0.999 3)范围内线性关系良好，重复性的相对标准偏差均小于3.3%，精密度相对标准偏差均小于4.5%，加标回收率均在89%-105%之间；③N、C末端端肽特征多肽的检出限分别为12，1 ng/mL，定量限分别为25，5 ng/mL；④该方法前处理简便高效、重复性好、灵敏度高、精密度高，可用于牛Ⅰ型胶原N、C末端端肽的精确定量。
https://orcid.org/0000-0002-2470-3160 (高建萍) 


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 高效液相色谱-质谱联用, 牛Ⅰ型胶原, 端肽, 特征多肽

Abstract: BACKGROUND: Collagen derived from animal sources exhibits a degree of immunogenicity that impacts its efficacy in applications. The removal rate of terminal peptides is the primary factor influencing its immunogenicity. Consequently, to establish a highly accurate and sensitive terminal peptide detection method is significant for the quality assessment, process optimization, and immunogenicity evaluation of bovine type I collagen.
OBJECTIVE: To identify N-terminal and C-terminal marker peptides of bovine type I collagen using high-resolution mass spectrometry and to establish a quantitative detection method based on high performance liquid chromatography (HPLC)-tandem mass spectrometry (MS) for marker peptides of bovine type I collagen terminal peptides.
METHODS: The HPLC separation was performed on a Zorbax C18 colum ( 2.1 × 150 mm, 5 μm) in a gradient elute mode with water and 60% acetonitrile (both containing 0.1% formic acid). The flow rate was 0.2 mL/min. The column temperature was 30 ℃. MS detection was performed with multiple-reaction monitoring mode using positive electro spray ionization.
RESULTS AND CONCLUSION: (1) The marker peptides of N-terminal and C-terminal telopeptide from bovine type I collagen were identified and the sequences were Q*LSYGYDE (Q* was pyroglutamic acid) and LSFLPQPPQE, respectively. (2) The linear relationships of the N- and C-terminal marker peptides were in the concentration range of 0.5-20 μg/mL and 0.05-10 μg/mL, respectively. The relative standard deviations of repeatability were less than 3.3%, the relative standard deviations of precision were less than 4.5%, and the recoveries of spiked standards ranged from 89% to 105%. (3) The detection limits of N- and C-terminal telopeptide marker peptides were 12 ng/mL and 1 ng/mL, respectively, and the quantification limits were 25 ng/mL and 5 ng/mL, respectively. This method is simple, efficient, sensitive, with good reproducibility and precision, and can be used for the accurate quantification of N- and C-terminal telopeptides of bovine type I collagen. 

Key words: high performance liquid chromatography-tandem mass spectrometry, bovine type I collagen, telopeptide, marker peptide

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