Identification of donkey-hide gelatin and donkey-bone gelatin based on marker peptides

Donkey-hide gelatin (DHG) is a food and drug product prepared using donkey skins. However, the detection of the illegal addition of donkey bone materials to DHG remains impossible. Herein, a new method using marker peptides from bone-related specific preteins was developed to identify donkey bone gelatin (DBG) and DHG. The bone-specific proteins were identified and verified by proteomic approaches. Peptides derived from the bone-specific protein were synthesized and used to develop a liquid chromatography multiple-reaction-monitoring mass spectrometry (LC-MRM-MS) method. The capability of this method was tested using homemade adulterated gelatins and commercial DHG products. It was found that several proteins were unique to donkey bone including collagen type II, osteocalcin, and osteonectin. Three of seven marker peptides from type II collagen showed good specificity for DBG. Adulteration with DBG could be identified at a level of 50 mg/g using the LC-MRM-MS method. Among the 23 batches of commercial DHG products, three were suspected of containing donkey bone materials owing to the detection of the marker peptides. The new method established in this study could enhance the quality control of DHG, and this strategy can also be adapted to identify other foods and drugs containing protein components.