Quantitation of Collagen Type V in Tissues by High-Performance Liquid Chromatography Coupled to Mass Spectrometry

A method for quantifying the bovine collagen type V (Col. V) was established based on high-performance liquid chromatography coupled to mass spectrometry by the marker peptide external standard. High-purity Col. V was extracted by the acid-enzyme hydrolysis process, and the marker peptide of Col. V was identified by LCQ mass spectrometry as GPAGPMGLTGR. A broad linear range (0.01-5.00 μg/mL) with a correlation coefficient of 0.9984 was achieved, and the limit of detection and limit of quantification were found to be 3.00 × 10^-3 and 6.25 × 10^-3 μg/mL, respectively. The method precision was 1.49%. The recovery rate was determined as 97.1-109.6% with a relative standard deviation less than 5%. The proposed method was successfully applied for the determination of Col. V contents in the bovine heart, lung, and cornea, which were 0.72 ± 0.01%, 0.23 ± 0.01%, and 2.89 ± 0.00%, respectively. The results show that the proposed method is more suitable for measuring the content of Col. V in tissue samples compared with the enzyme-linked immunosorbent assay. The marker peptide method has high accuracy and great reproducibility, and will lay a foundation for the extraction and application of Col. V. Impact statement The accurate quantitative method for collagen type V (Col. V) is particularly important in scientific research, disease diagnosis and treatment, and industrial production. In this article, we proposed a high-performance liquid chromatography coupled to mass spectrometry method based on the external standard marker peptide to quantify bovine Col. V. This method shows a higher accuracy and recovery rate than enzyme-linked immunosorbent assay (ELISA), indicating that it is more suitable for measuring the content of Col. V in tissue samples than ELISA. The establishment of this method has laid a solid foundation for the extraction and application of Col. V.