Mass spectrometric detection of marker peptides in tryptic digests of gelatin: A new method to differentiate between bovine and porcine gelatin

Gelatin is a mixture of polypeptides obtained by hydrolysis of collagen primarily from bovine and porcine skin and bones. The similarity between different gelatins makes it difficult to trace their species origin. In this work, a new method for differentiation between bovine and porcine gelatin was developed based on detection and identification of marker peptides in digested gelatins. Sequence alignment analysis indicates that bovine and porcine Type I collagen contain differential sequences. The gelatins were digested by trypsin, and the resulting peptides were analyzed by high performance liquid chromatography/tandem mass spectrometry (HPLC–MS/MS). The marker peptides specific for bovine and porcine were successfully detected in the digested bovine and porcine gelatin, respectively. Comparative analysis indicated that more marker peptides could be detected in gelatin with a higher mean molecular weight. It was found that proline hydroxylation was a key factor affecting the peptide identification. For peptides such as GPPGSAGSPGK and GPPGSAGAPGK detected in digested bovine and porcine gelatin, respectively, the sequence should be verified manually since the mass shift caused by proline hydroxylation can be confused with the mass difference between Ser and Ala residues. The results indicate that detection of marker peptides in the digested gelatin sample using HPLC–MS/MS is an effective method to differentiate between bovine and porcine gelatin.