SILAC-Based Affinity Purification of Protein Complexes

A murine monocyte-macrophage cell line J774.2 (ATCC) was grown in DMEM supplemented with 10% fetal bovine serum. For metabolic labeling, the cells were transferred into arginine- and lysine-free DMEM (DMEM for SILAC, Thermo Fisher Scientific) supplemented with 10% dialyzed fetal bovine serum (Invitrogen) and heavy labeled amino acids L-arginine hydrochloride [¹³C₆ ¹⁵N₄] and L-lysine hydrochloride [¹³C₆ ¹⁵N₂] (Sigma-Aldrich) in the same concentrations as in the standard DMEM medium (“heavy” medium). After five cell divisions, the incorporation levels were checked by mass spectrometry (MS) and the labeled cells were stored as frozen stocks in DMEM with 10% DMSO at −150 °C. For the experiment, the labeled cell stock was cultivated in “heavy” medium supplemented with proline (300 mg/mL) to minimize the conversion of arginine to proline [30 (link)]. Volumes of 1 × 10⁷ of cells (heavy labeled and non-labeled) were washed in PBS, harvested in PBS completed with EDTA-free complete protease inhibitor mixture (Roche) and benzonase (Merck) and disrupted in French press by one passage at 2000 psi. Total protein concentration was determined as indicated above. The recombinant GapA-Twin-Strep tagged protein was added to freshly prepared “heavy” labeled cell lysate (6 µg of GapA to 1 mg of cell lysate), incubated for 40 min at 4 °C, and the GapA protein was purified together with its bound proteins using MagStrep “type3” XT beads, as described above. The purification was performed with the same number of lysates from non-labeled cells simultaneously (control of nonspecifically bound proteins onto the purification system). The obtained eluates were mixed and examined by LC-MS/MS analysis.