We then proceeded to identify the target protein(s) using affinity purification followed by mass spectrometry (MS) analysis. The purification procedure included three tandem steps after photo-cross-linking: protein of HBV (S472 strain, genotype C). Residue numbering is based on genotype D. Asterisk indicates highly conserved residues among genotypes. Epitope of mAb 2D3 was shaded in gray. (B) Effect of alterations of the critical N-terminal residues within pre-S1 region of L protein on HDV binding to PTHs. Both wild-type (WT) and mutant HDV virions carry HBV envelope proteins. Mutant HDV carries point mutation as indicated in the pre-S1 region of L protein. PTHs were incubated with HDV at 16°C for 4 hr and followed by extensive wash; bound virions were quantified by qRT-PCR for virus genome RNA copy, and the data are presented as percentage of virus binding, the binding of WT virus was set as 100%. (C) Myr-47/WT b bait peptide dosedependently inhibited HDV virion binding. The binding assay was performed similarly as panel B except that PTHs were pre-incubated with indicated peptides. (D) Inhibition of viral infection by the photoreactive peptides. Left: PTHs were pre-incubated with peptides at indicated concentrations at 37°C for 1 hr and then inoculated with HDV virus. Viral infection was examined by measuring viral RNA in infected cells with qRT-PCR 6 days post-infection (dpi). Data are presented as percentage HDV infection. Right: peptides at indicated concentrations were added to PTHs before HBV inoculation. The cell culture medium was replenished every 2 days. Secreted viral antigen HBeAg was measured by ELISA on 6 dpi, and the data are presented as percentage of that in the absence of peptides. (E) Antibody 2D3 recognizes residues 19-33 of pre-S1. Peptide NC36 (aa 4-36 of pre-S1, NLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNP) conjugated with keyhole limpet hemocyanin (KLH) was the immunogen peptide for generating mouse mAb 2D3. Binding activity of 2D3 with full-length pre-S1 protein was measured by ELISA in the presence of competition peptides at indicated concentrations. LD15 peptide compassing residues 19-33 of pre-S1 inhibited 2D3 binding in a dose-dependent manner, indicating that 2D3 recognizes an epitope within this region. HBV: hepatitis B virus; mAb: monoclonal antibody; HDV: hepatitis D virus; PTH: primary Tupaia hepatocytes; HBeAg: HBV e antigen. DOI: 10.7554/eLife.00049.003 ) or Myr-47/N9K b (N9K b ), followed by Streptavidin Dynal T1 beads precipitation and Western blot analysis using mAb 2D3. The protein cross-linked by WT b is sensitive to PNGase F treatment and shifted from ∼65 to ∼43 kDa. Right: WT b cross-linked samples were treated with 100 mM DTT and/or PNGase F as indicated and detected similarly as in the left panel. (B) Non-photoreactive Myr-47/WT peptide (WT) but not its N9K mutant competed with 200 nM of WT b peptide for cross-linking with PTHs in a dose-dependent manner. (C) The abundance of the target protein(s) in PTH cells decreased over time. PTHs on different days of in vitro culturing were photo-cross-linked with 200 nM WT b . The cross-linked samples were analyzed by Western blot. The two bands at ∼65 and ∼43 kDa were due to incomplete deglycosylation by PNGase F. (D) WT b cross-linking with primary human hepatocytes (PHH). Frozen PHH cells were thawed and plated 1 day before cross-linking. With same procedure as in panel A, 200 nM WT b but not N9K b cross-linked with a glycoprotein of molecular weight at ∼60 kDa, which shifted to ∼39 kDa upon PNGase F treatment. (E) Purification of target protein(s) for MS analysis. PTHs photo-cross-linked with 200 nM of WT b or N9K b peptide were lysed, then the peptides and their cross-linked proteins were purified in tandem with Streptavidin Dynal T1 beads, mAb 2D3 conjugated beads, and Streptavidin Dynal T1 beads in 1× RIPA buffer. Extensive wash was applied for each purification step. The samples were treated with or without PNGase F as indicated prior to the last step of Streptavidin beads precipitation. The final purified samples were subjected to SDS-PAGE followed by silver staining (left). Bracketed areas indicate the bands cut for MS analysis. Western blot analysis (right) of the same cross-linked samples were performed similarly as in panel A. The top 10 nonredundant proteins identified in the 3 samples by MS analysis are listed in Figure 2 -Source data 1. The common protein hit identified by MS analysis of the ∼65and ∼43-kDa bands cut from the WT b cross-linked sample was Tupaia NTCP (tsNTCP), and the representative MS/MS spectra and parameters of the peptide hits are shown in Figure 2 -figure supplement 5. The control band cut from N9K b cross-linked sample did not generate any hits on any of these peptides. (F) Predicted tsNTCP protein sequence. A 30-amino acid insertion unique to tsNTCP is underlined. Two peptides identified by LC-MS/MS were highlighted in green. All lysine and arginine are highlighted in red to Figure 2 . Continued on next page capturing all biotin-labeled proteins with streptavidin T1 beads, sorting out the target protein(s) with 2D3 antibody affinity beads, and then purifying with streptavidin T1 beads again to remove residual molecules that were not covalently cross-linked with the bait peptide. The purified samples were subsequently subjected to SDS-PAGE followed by silver staining. Similar to the Western blotting results with the 2D3 antibody, a ∼65-kDa protein band was visible by silver staining. The band was also shifted to ∼43 kDa upon PNGase F treatment ( Figure 2E ). Both the original 65-kDa and the shifted 43-kDa bands were subsequently excised from the gel and subjected to LTQ-Orbitrap Velos (Thermo Fisher Scientific, MA. USA) MS analysis after trypsin digestion. The tandem mass spectra were searched against a Tupaia hepatocyte protein database, which we had established by deep sequencing of the transcriptome (Figure 2-figure supplements 1-4) . Two different tryptic peptide fragments, which were identified from both the ∼65-kDa and ∼43-kDa bands ( Figure 2-figure supplement 5 ), matched to a protein homolog of human NTCP. Tupaia NTCP (tsNTCP) shares 83.9% protein sequence identity with its human counterpart and has an insertion of 30 aa near its C-terminus ( Figure 2F ). The peptide (TEETIPGTLGNSTH) containing 4 aa of this insertion (underlined) was one of the two peptides identified by the MS analysis at a high confidence level ( Figure 2-figure supplement 5) . These data suggest that NTCP is the protein specifically interacting with the WT b bait peptide.