Quantitative determination of osteopontin in bovine, buffalo, yak, sheep and goat milk by Ultra-high performance liquid chromatography-tandem mass spectrometry and stable isotope dimethyl labeling.

Osteopontin (OPN) is a multifunctional protein present in different tissues, body fluids and milk. Different milk has different level of OPN content. To determine the amount of osteopontin in bovine, buffalo, yak, sheep and goat milk, we developed an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to detect an osteopontin signature peptide. The signature peptides selected by searching Uniprot database for trypsin digested osteopontin. The sample preparation procedure includes trypsin digestion, dimethyl labeling of tryptic peptides, purification and concentration of labeled tryptic peptide with solid phase extraction. The limit of detection and limit of quantification are 0.5 mg L-1 and 2.0 mg L-1, respectively. The method has satisfactory analytical performance with a linearity of R2 ≥ 0.998, recoveries of 103.7-111.0%, and precision of 1.8-6.2%. It is also validated and successfully applied to quantifying osteopontin content in bovine, buffalo, yak, sheep and goat milk.