Q exactive hf orbitrap lc ms ms system

1×10⁸ HepAD38 and HepBHAeΔx67 cells were induced for HBV replication for 14 days. The nuclear fractions of the cells, which contained cccDNA, were isolated using the Nuclear Extraction Kit (10009277, Cayman Chemical) and subsequently incubated with a 1:50 dilution of anti-HBc antibodies (B0586, Dako) (S3 Table) for 3 hours at 4°C under constant and gentle agitation. Then, the HBc complex pulldown by Protein G magnetic beads and intensive washing were then performed accordingly to Universal Magnetic Co-IP Kit manufacturer’s instruction (54002, Active Motif). The beads-bound proteins were reduced, alkylated, and trypsin digested directly on beads, and the subsequent peptides were identified with liquid chromatography interfaced with a hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Q Exactive HF Orbitrap LC-MS/MS system) at the Purdue University Proteomics Facility. Database search against Uniprot human database was conducted using MaxQuant to identify the probable source proteins of the peptides.

FREP followed the previously described protocol^{23 (link)}. The bait DNA fragment (rs117701653-C/5Biosg), competitor DNA fragment (rs117701653-C), and irrelevant DNA sequence were used; sequences are listed in Supplementary Table 3. Mass spectrometry was performed using a Thermo Scientific Q Exactive HF Orbitrap LC-MS/Ms system.