10 kda ultrafilter

The methods of trypsin digestion of MFGM proteins were performed as previously described (Li et al., 2019) . Briefly, MFGM proteins (600 μg) were added to the mixture solution consisting of 4% SDS, 100 mM Tris-HCl (pH 8.0), and 100 mM dithiothreitol. The mixture was then heated at 90°C for 10 min. The samples were statically positioned and cooled to 20°C. Each sample was then loaded onto a 10-kDa ultrafilter (Millipore) containing 200 μL of urea-Tris (UT; 8 M urea and 150 mM Tris-HCl; pH 8.0) buffer. The sample mixture was centrifuged (14,000 × g, 30 min, 4°C) and washed with UT buffer. Iodoacetamide (50 mM final concentration in UT buffer) was added to the ultrafilter and mixed with the sample for 1 min. Subsequently, the sample mixture was transferred to a dark environment and incubated at 20°C for 30 min. The incubated mixture samples were centrifuged (14,000 × g, 15 min, 4°C) and washed with 100 μL of UT buffer. This step was repeated twice. The filter was then pre-washed by adding dissolution buffer (UT buffer and 25 mM NH 4 HCO 3 ). The protein was digested at 37°C for 24 h by adding 40 μL of trypsin to the filter. The digested samples were centrifuged (14,000 × g, 15 min, 4°C), and the peptide segments were collected for analysis. The absorbance of the peptides was measured at 280 nm.