Collagen Analysis Method Based On Biological Mass Spectrometry

Collagen is the most widely distributed and abundant protein in the body. This research investigated different types and content of collagen with its applications by biological mass spectrometry technology.A method for quantitative and qualitative analysis of collagen was established by detection of marker peptide using high performance liquid chromatography-mass spectrometry(HPLC-MS). Theoretical marker peptides were selected by sequence comparison. The bovine collagen type I and three different parts of bovine which are cartilage tendon and skin were digested with trypsin. Marker peptides of collagen type I were typically identified with HPLC-MS. The relationship was established between the abundance of marker peptides and collagen concentration. The method was applied for analysis animals which were by extract collagen of different weeks of BN rat skin and tail to inspecting collagen changes over weeks. The method of ELISA measured FN content and used HPLC-MS detecting hydroxylation fragments to analysis the specific binding sites of FN in collagen.The results were as follows:To the collagen type identification research, bovine tendon has type I collagen with the marker peptides of GEAGPSGPAGPTGAR and GELGPVGNP*G PAGPAGPR. Bovine cartilage has type II collagen with the marker peptides of GEAGAQ GPMGPAGPAGAR and type I collagen with the marker peptides of GAP*GP*AGPK and VGAPGPAGAR. Bovine skin has type III collagen with the marker peptides of GPP*GAGGPP*GPR and type I collagen with the marker peptides of GQAGVMGFPGPK and GQAGVMGFPGPK.To the collagen content research, the results indicated that GEAGPSGPAGPTGAR and the other five kinds of 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 of 0.1-3 mg/m L. Bovine tendon and collagen sponge were used as actual samples and collagen contents were 90.2% and 93.4%, respectively.To the binding site research, rat has type I collagen and FN in skin and tail, and both of its content increased with age. In addition, the FN content of rat skin is greater than the rat tail for the same week. The peptide GPPGPMGPPGLAGPPGESGR has four different hydroxylation modified types based on HPLC-MS, hydroxylation modification easily happened in the third GPP part in rat skins and second GPP part in rat tails, then the third GPP parts of GPP*GPMGPP*GLAGPPGESGR is the rat tails binding site and the second GPP part is the rat skins binding site.The analysis method which was based on HPLC-MS is feasible for quantitative and qualitative decetion of marker peptides of collagen, and it could be applied for for identification and quantization of collagens involved in food and medicine and other fields.