Profiling the differential phosphoproteome between breast milk and infant formula through a titanium (IV)-immobilized...

Breast milk (BM) fulfills the nutritional needs of infants and sets the standard for infant formula (IF). However, profiling the differential phosphoproteome between BM and IF remains unclear. Herein, a titanium (IV) (Ti⁴⁺)-immobilized magnetic nanoplatform (Fe₃O₄@GO@PDA-Ti⁴⁺) was constructed by self-assembly polymerization of dopamine on magnetic graphene oxide, followed by immobilizing Ti⁴⁺ through chelation for phosphopeptide enrichment. Fe₃O₄@GO@PDA-Ti⁴⁺ possessed outstanding selectivity (1/1000, a molar ratio of β-casein digests to bovine serum albumin digests) and favorable sensitivity (2.5 fmol/μL), along with rapid magnetic separation. Excellent phosphopeptide capture efficiencies were obtained for BM and IF using Fe₃O₄@GO@PDA-Ti⁴⁺ as an adsorbent coupled with liquid chromatography-mass spectrometry/mass spectrometry. There were 191 and 239 phosphopeptides found in BM and IF, respectively, with 36 phosphoproteins identified in both. However, BM and IF shared only 17 phosphopeptides and 4 phosphoproteins. The variation in the phosphoproteome between BM and IF provides valuable insights into the optimization of IF humanization.