The present study aimed to identify endogenous milk peptides for species differentiation independent of heat exposure. Thus, comprehensive milk peptide profiles from five species and three types of heat treatments were analyzed by micro-flow liquid chromatography ion mobility quadrupole time-of-flight mass spectrometry (microLC-IM-QTOF) with subsequent database search leading to ≥ 3000 identified peptides. In the milks, 1154 peptides were unique for cow, 712 for sheep, 466 for goat, 197 for buffalo, and 69 for mare. Most peptides were detected in extended-shelf life (ESL) milk (2010), followed by ultra-high temperature (UHT) processed (1474) and pasteurized milk (1459 peptides), with 693 peptides present in all milk types. A blind test set of 64 samples confirmed eight species-specific, but heat-independent marker peptides in milk from cow, seven from goat, six from sheep, nine from buffalo, and three from mare. The generated peptide profiles can also be used to identify species- and heat-specific markers.
Keywords: Endogenous peptide profiling; Mammalian milk; Marker peptide; Milk heat treatment; Species specification; microLC-QTOF-MS/MS.
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