Detection of plant protein in adulterated milk using nontargeted nano-high-performance liquid chromatography-tandem mass spectroscopy combined with principal component analysis

The objective of this study was to detect plant protein adulterated in fluid milk using nano-high-performance liquid chromatography (HPLC)-tandem mass spectroscopy (LC-MS/MS) combined with proteomics. Unadulterated milk and samples adulterated with soy protein, pea protein, hydrolyzed wheat protein, and hydrolyzed rice protein were prepared, with plant protein level ranged from 0.5% to 8% in total protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels clearly revealed that centrifugation at 20,000 g for 60 min would reduce band intensity of casein and albumin in milk. Results of nano-HPLC-MS/MS indicated the major proteins of soy (beta-conglycinin, glycinin), pea (vincilin, convicilin, legumin), and wheat (glutenin and gliadin) in adulterated milks, allowing detection of soy protein and hydrolyzed wheat protein at the level above 0.5% in total protein and pea protein at the level of 2 and 4%. No rice protein was identified in milk samples adulterated with hydrolyzed rice protein. Combined with principal component analysis, nano-HPLC-MS/MS could discriminate all the adulterated samples from authentic milk. This study demonstrated the feasibility of nano-HPLC-MS/MS on the detection of (hydrolyzed) plant protein adulterated in milk.