Sample preparation for liquid chromatography-mass spectrometry (LC-MS)

For LC-MS analysis of SPOP interactome, 293T cells were transfected with S, Flag, and Biotin-binding-protein-(streptavidin)-binding-peptide (SFB) triple-tagged backbone vector or SFB-SPOP. Twenty-four hours after transfection, control cells were treated with DMSO (Group 1) and SFB-SPOP transfected cells were treated for 24 h with DMSO (Group 2) or different drugs including mitomycin C (MMC, 1 µM) (Group 3), camptothecin (CPT, 50 nM) (Group 4), cisplatin (10 µM) (Group 5), and etoposide (10 µM) (Group 6). The cells were lysed by NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) with 50 mM β-glycerophosphate, 10 mM NaF, and 1 μg/mL pepstatin-A at 4 °C for 3 h, followed by tandem affinity purification using streptavidin beads and S tag beads and LC-MS. A total of six groups of cell lysate were analyzed (n = 6), including one group of cells transfected with an empty vector and treated with DMSO as a negative control. All the proteins identified in this group were excluded from the list of SPOP interactome. For Geminin ubiquitination site mapping, 293 T cells were transfected with Flag-Geminin, HA-Ub, and Myc-SPOP. The co-IP sample was subjected to LC-MS (n = 1/group) without additional controls. The identified Geminin ubiquitination sites were further validated by mutagenesis and co-IP assays. For identification analysis of components of the Geminin complex, 293T cells were transfected with Flag-Geminin in the presence (Group 1) or absence (Group 2) of HA-Ub and Myc-SPOP plasmids. The co-IP samples were subjected to LC-MS (n = 1/group). Group 2 was used as a negative control. All mass spectrometry experiments were performed once.