50_100uM_DSBU_XL-MS_PHB2(V1)-Myc_HEK293

Mitochondria were isolated from HEK293 cells that stably express PHB2(V1) with an C-terminal 3×Myc tag, and were resuspended in a cross-linking buffer [1 × PBS (pH 7.2) and 50 or 100 μM disuccinimidyl didutyric urea (DSBU) for 30 min at room temperature. The cross-linked mitochondria were then lysed in RIPA buffer, and the clarified supernatants were incubated with anti c-Myc agarose beads. Proteins on the beads were digested by adding trypsin/Lys-C mix for 16 h at 37°C. The resultant peptides were divided into two halves and subjected to LC-MS/MS analysis using both Q Exactive Plus and Orbitrap Fusion Lumos spectrometers. Raw data were analyzed using Proteome Discoverer 2.2 with the XlinkX node to identify cross-linked peptides.