Abstract:Enzymatic hydrolysates of casein are nutritive，advanced freshening agents whose composition is associated with umami taste.In this study，porous titania microspheres with high specific surface area were synthesized by a sol-gel method and used for the first time for peptide separation and enrichment to allow liquid chromatography-tandem mass spectrometry（LC-MS/MS）analysis of a casein hydrolysate and its low abundance phosphopeptides.The casein hydrolysate was mainly composed of 16 low molecular weight peptides and free amino acids.Two of the peptides（Val-Asn-Glu-Leu-SerP*-Lys and Thr-Val-Asp-Met-Glu-SerP*-Thr-Glu-Val-Phe-Thr-Lys）were phosphopeptides.Here，a new method was demonstrated for direct analysis and identification of low abundance polypeptides and phosphopeptides in complex mixtures without monomer separation or purification.This method is expected to play an important role in protein，peptide，and nutriomics research.