Abstract:

A method for identifying specific peptide biomarkers of animal-milk-derived components in camel milk and its products was established using proteomics. Samples were prepared by defatting, protein extraction, and trypsin hydrolysis, and proteins and peptides were identified using ultra-high performance liquid chromatography-quadrupole/electrostatic orbitrap-high resolution mass spectrometry (UHPLC-Q/Exactive-HRMS) and Protein Pilot software. Twenty two peptide biomarkers from eight species (i.e., Camelus, Bos taurus, Bubalus bubalis, Bos grunniens/Bos mutus, Capra hircus, Ovis aries, Equus asinus, Equus caballus) were identified by comparing the basic local alignment search tool (BLAST) with the Uniprot database. Verification of these marker peptides were performed quantitatively using a UHPLC-triple-quadrupole mass-spectrometry (QqQ-MS) system by multiple reaction monitoring (MRM). The pretreatment method of casein in camel milk was optimized, such as defatting, protein precipitation, and re-dissolving buffer solution. The effects of various mass-spectrometry parameters, such as atomization gas, heating- and drying-gas flow rates, and desolvation-tube (DL) and ion-source-interface temperatures on ion-response intensity were optimized. Camel milk signature peptides were detected in a mixture of milk from other seven species to ensure specificity for the selected biomarker peptides. The signature peptides of seven other species were also detected in camel milk. No mutual interference between the selected biomarker peptides of the various species was observed. Adulterated camel milk and milk powder were also quantitatively studied by adding 0, 2.5%, 5%, 10%, 25%, 50%, 75%, and 100% bovine milk or goat milk to camel milk. Similarly, the same mass proportion of bovine milk powder or goat milk powder was added to camel milk powder. A quantitative standard curve for adulteration was constructed by plotting the peak areas of characteristic cow or goat peptide segments in each mixed sample against the mass percentage of the added adulterant. The adulteration standard curves exhibited good linearity, with correlation coefficients (r²) greater than 0.99. The limits of detection and quantification (LODs and LOQs, respectively) of the method were determined as three- and ten-times the signal-to-noise ratio (S/N). The minimum adulteration LODs of bovine milk and goat milk in camel milk were determined to be 0.35% and 0.49%, respectively, and the minimum LOQs were 1.20% and 1.69%, respectively. The minimum adulteration LODs of bovine milk powder and goat milk powder in camel milk powder were determined to be 0.68% and 0.73%, respectively, and the minimum LOQs were 1.65% and 2.45%, respectively. The accuracy of the adulteration quantification method was investigated by validating the quantitative detection results for 1∶1∶1 (mass ratio) mixtures of camel milk, bovine milk, and goat milk, as well as camel-milk powder, bovine milk powder, and goat-milk powder, which revealed that this method exhibits good linearity, strong anti-interference, high sensitivity, and good repeatability for adulterated liquid-milk/solid-milk-powder samples. The adulteration results for both liquid milk and milk powder are close to the theoretical values. Finally, 11 actual commercially available samples, including five camel-milk and six camel-milk-powder samples were analyzed, which revealed that only camel signature peptides were detected in 10 samples, while camel and bovine signature peptides were both detected in one camel-milk-powder sample. The ingredient list of the latter sample revealed that it contained whole milk powder from an unidentified source; therefore, we infer that the bovine signature peptides originate from the whole milk powder. These signature peptides also demonstrate the necessity and practical significance of establishing this identification method.

Key words: liquid chromatography-tandem mass spectrometry (LC-MS/MS), camel milk, camel milk powder, adulteration quantification