Purification and Mass Spectrometry of PrimPol

PrimPol was isolated from HEK293 cells as described previously (29 (link)). Briefly, approximately 1 × 10⁷ cells expressing Flag-tagged PrimPol were lysed in NETN buffer [150 mM NaCl, 30 mM tris (pH 7.5), 0.5% NP-40, 2.5 mM MgCl₂, and deoxyribonuclease I (100 μg/ml)] for 30 min at 4°C. Cell lysate was incubated with Anti-Flag M2 magnetic beads (Merck) for 2 hours at 4°C before beads were washed three times with wash buffer [150 mM NaCl, 30 mM tris (pH 7.5), and 0.1% NP-40]. Proteins and interacting partners were eluted with 50 μl of elution buffer [25 mM tris-HCl (pH 7.5), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 3xFLAG peptide (200 μg/ml; Merck)] and analyzed against input cell lysate by Western blot.
For mass spectrometry analysis, 10 × 10⁷ cells expressing Flag-tagged PrimPol were lysed in RIPA buffer. Protein was bound by Anti-Flag M2 magnetic beads (Merck) for 2 hours at 4°C before being washed three times in 50 mM ammonium bicarbonate. After overnight on-bead digestion at 37°C by Glu-C protease, the peptides were analyzed on an Orbitrap Exploris 480 [with FAIMS (Field asymmetric Ion mobility spectrometry)] mass spectrometer by the Proteomics Core Facility at CEITEC (Brno, Czech Republic).