The mass spectrometry of proteins specifically bound to TMAO

This is a mass spectrometry analysis of TMAO specific binding proteins. The N-hydroxylsuccinimide (NHS)-activated beads 4FF (Smart-Life Sciences Inc, SA039005) were incubated with 10 mg/ml TMAO overnight at 4°C with gentle rotation according to the instructions of manufacturer. After rinsing with deionized water, the TMAO-coupled beads were incubated with blocking buffer (0.1 M Tris-HCl, pH 8.5) for 2 h at room temperature. Then the TMAO-coupled beads were incubated with proteins extracted from mouse intestinal tissues overnight at 4°C. After washes with PBS containing 0.5% Triton X-100, the elutes were subjected to SDS-PAGE with Coomassie blue staining. The proteins specifically bound to TMAO were identified by using Q-Exactive mass spectrometer (Thermo Fisher). MS data was analyzed with Proteome Discoverer 1.3 and tandem mass spectra were searched against the MaxQuant1.6.14 algorithm. Trypsin/P is designated as a cleavage enzyme that allows up to two deletion cleavage. Precursor mass tolerance was set to 20 ppm, while fragments were detected with a tolerance of 0.1 Da.