Milk authenticity by ion-trap proteomics following multi-enzyme digestion

•Direct analysis of milk digests by nanoLC-ESI-IT-MS/MS.•Database post-processing of MS/MS datasets for confident sequence assignments.•Detection of milk adulteration at a level lower than 1%.•Identified cow species-specific peptide markers from β-lactoglobulin and αS1 casein.

The practice of adding adulterating substances in milk in order to raise profits is unfortunately worldwide. In addition, higher priced milk, coming from minor dairy species, is often illegally integrated with the lower priced cow milk. The presence of species-specific proteins, different from those declared in label, may be a serious problem for people with allergies. The development of proper analytical methods is therefore essential to protect consumer benefits and product authenticity.In this study, a proteomic approach for the detection of adulteration processes of specific milks in mixtures is proposed. Few microliters of milk samples have been digested with trypsin and chymotrypsin and analyzed by nanoLC-ESI-IT-MS/MS. A post-database processing was performed to obtain confident peptide sequence assignments, allowing the detection of milk adulteration at a level lower than 1%. Species-specific peptides from bovine β-lactoglobulin and αS1 casein were identified as suitable peptide markers of milk authenticity.