Proteomic approach-based absolute quantification of lactadherin in breast milk using full-length expressed stable isotope-labelled protein internal standard with in-gel digestion

Summary Lactadherin is a major protein in the milk fat globule membrane (MFGM) and is associated with anti-rotaviral effect. Although lactadherin level in breast milk is generally quantified using ELISA, the many contaminants in the milk samples raise concerns of low accuracy. The aim of this study was to develop a high accuracy method of absolute quantification for breast milk lactadherin using a proteomics-based technique. The developed method uses a full-length expressed stable isotope-labelled protein from the wheat germ cell-free expression system with in-gel digestion and showed much higher accuracy (97.0–109.9%,) than ELISA (27.2–31.6%). The developed method revealed lactadherin levels in Japanese breast milk (one month of postpartum 7.2 [2.2–25.7] mg/100 mL, six months of postpartum: 3.8 [1.2–22.8] mg/100 mL). This study has shown that a proteomics approach using a full-length expressed stable isotope-labelled protein with in-gel digestion achieves high accuracy quantification for breast milk lactadherin.