Caira Simonetta, Nicolai Maria Adalgisa, Lilla Sergio, Calabrese Maria Grazia, Pinto Gabriella, Scaloni Andrea, Chianese Lina, Addeo Francesco
Proteomics & Mass Spectrometry Laboratory, ISPAAM, National Research Council, via Argine 1085, I-80147 Naples, Italy.
Dipartimento di Agraria, Università degli Studi di Napoli Federico II, via Università 100, Parco Gussone, I-80055 Portici, Italy.
Food Chem. 2017 Sep 1;230:482-490. doi: 10.1016/j.foodchem.2017.03.055. Epub 2017 Mar 11.
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 γ- and γ-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 γ-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.
欧洲参考方法（ERM）基于相应纤溶酶解后对牛（B）γ-酪蛋白和γ-CN片段的伴随等电聚焦（IEF）检测，识别水牛（WB）奶/乳制品中掺假添加的牛奶。我们在此使用蛋白质组学来表征ERM中出现的假阳性结果，其原因是WBβ-CN（f100-209），它也是真正的WB奶/乳制品纤溶酶解后形成的，并且在IEF中与Bγ-CN共迁移。通过基于将B/WB奶/奶酪CN提取物加载到羟基磷灰石柱上、原位胰蛋白酶消化以及洗脱Bβ-CN（f1-25）4P和WBβ-CN（f1-28）4P蛋白型肽的专用蛋白质组学程序，克服了这些ERM的局限性。基于它们在基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）中的相似电离特性和分辨率，这些磷酸肽被确定为检测WB奶/乳制品中B物质的合适标志物，检测限为0.8%（v/v）。本文提出该蛋白质组学程序作为ERM的综合/替代方法。
