Eventual limits of the current EU official method for evaluating milk adulteration of water buffalo dairy products and potential proteomic solutions

•Proteomics showed limits of the EU reference method assaying bovine in water buffalo milk.•Water buffalo β-CN (f100-207) in plasminolysate generates false-positive results.•Bovine β-CN (f1-25)4P and water buffalo β-CN (f1-28)4P as novel fraud markers.•Dedicated MALDI-TOF-MS analysis as a second-generation reference method.

The European reference method (ERM) recognises the fraudulent addition of bovine (B) milk in water buffalo (WB) milk/dairy products based on concomitant isoelectric focusing (IEF) detection of B γ2- and γ3-CN fragments after corresponding plasminolysis. We here used proteomics to characterise false positive results occurring in the ERM as being due to WB β-CN(f100-209), which is also formed after plasminolysis of genuine WB milk/dairy products and comigrates in IEF with B γ2-CN. These ERM limitations were overcome by a dedicated proteomic procedure based on loading of B/WB milk/cheese CN extracts on a hydroxyapatite column, in situ trypsinolysis and elution of B β-CN(f1-25)4P and WB β-CN(f1-28)4P proteotypic peptides. Based on their similar ionisation properties and resolution in MALDI-TOF-MS, these phosphopeptides were identified as suitable markers for detection of B material in WB milk/dairy products to a detection limit of 0.8% v/v. This proteomic procedure is here proposed as integrative/alternative to the ERM.