Determination of Type Ⅰ and Type Ⅲ Collagen of Liver, Lung and Kidney in Mouse by High Performance Liquid Chromatography-Tandem Mass Spectrometry

Collagen is the main component of extracellular matrix, accounting for 25%-30% of total protein. Currently, 28 types of collagen have been identified in mammals. The variation of different types of collagen in different tissues is related to age, disease and biological function. Type Ⅰ and Ⅲ collagen are the main components in liver, lung and kidney, and their content and ratio are related to inflammation and canceration. Sirius red staining and immunoassay are the traditional method for collagen detection. However, sirius red staining is a semi-quantitative method and immunoassay is not suitable for the determination of collagen content in tissues. In this study, a method of high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) was developed for the determination of type Ⅰ and Ⅲ collagen in liver, lung and kidney of mouse．The marker peptides of mouse type Ⅰ and Ⅲ collagen were identified and the sequences were GSEGPQGVR and GPSGFR, respectively. The product ions and retention time were used to identify the collagen. The peptide GLAGMK was used as the internal substance to quantitate the content of type Ⅰ and Ⅲ collagen in liver, lung and kidney. The results showed that the linear relationship of these two marker peptides was good in the concentration range of 1-500 mg/L, the relative coefficient was more than 0.99, RSD of the assay precision was less than 3.7%, and the recovery of standard addition was between 86% and 118%. The ratios of type Ⅰ collagen in liver, lung and kidney were all increased during the growth of mouse. From 0 to 1st week, the ratio of type Ⅰ collagen increased rapidly, but they increased slowly from 6th to 12th week. The ratio of type Ⅰ collagen was the highest in the kidney, and it was the lowest in the lung. The total content of type Ⅰ and Ⅲ collagen increased firstly and then decreased during the growth of mouse. The result was consistent with the trend of total collagen content in liver, lung and kidney, which was measured by hydroxyproline method. The total amount of type Ⅰ and Ⅲ collagen was the highest in the kidney, and it was the lowest in the liver. This method is simple, efficient, replicable and highly stable, and can be used for accurate quantification of type Ⅰ and Ⅲ collagen in liver, lung and kidney of mouse. This study provides an auxiliary analytical method for the diagnosis of collagen-related diseases.