High Performance Chromatography/Mass Spectrometric Analysis Of Digested Collagen

Collagen is the major insoluble protein in extra-cellular matrix (ECM). As the most important structure protein, collagen is amount to more than 30% of total proteins in mammals. Lots of researches indicated many diseases were involved with the expression, content, distribution, metabolite of different collagen type. Thus, it is necessary to develop a method to quantitatively and qualitatively determine the collagens indifferent tissues.In this research, collagens were separated from rat tails by enzymatic degradation. The crude collagen extract was thermally denatured, and then digested with trypsin. The digest mixture was subjected to analysis with high performance liquid chromatography/mass spectrometry (HPLC/MS) to determined collagen types and their relative abundance. HPLC analysis coupled with pre-column derivation was used to determine the amino acid content in crude collagen extract. The result indicated that the content of Gly was 31.5%, Hyp was more than 10%, and Pro was more than 14%. The collagens in crude extract were made soluble by thermal denaturation in 0.05 M HCl-Tris buffer (pH8.0, containing 1M NaCl) at 50℃for three hours. SDS-PAGE analysis indicated the no obvious degradation happend during thermal pretreatment. The denatured collagens were digested with trypsin. The peptides in digest mixture were identified by HPLC/MS. It was found that there were many characteristic peptides typical for collagenⅠorⅢ,which were used to determine the relative abundance of collagenⅠandⅢin rat tail. It was found that the content of collagenⅠwas obvious higher than collagenⅢ. The content of collagenⅠincreased with decease of the content of collagenⅢin whole growth stage.This research demonstrates that it is possible to determine the relative content of different collagens with HPLC/MS coupled with thermal denaturation and tryptic digestion.