Silver stained gel bands were cut, followed by in-gel reduction, alkylation, and trypsin digestion as previously described (Shevchenko et al., 2006) . Digested peptide mixtures containing 0.1% formic acid were loaded onto a 4 cm, 75-μm inner diameter fused silica capillary column packed with 10-μm YMC C18 material (YMC, Kyoto, Japan). After desalting, samples were separated with a Waters nano ACQUITY UltraPerformance LC (Waters, United States) and eluted to a LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, United States). The UPLC separation gradient included a 30-min gradient from 0% to 30% acetonitrile, followed by a 10-min gradient to 80% acetonitrile, then 10 min of 80% acetonitrile and back to 0% acetonitrile within 5 min. The mass spectrometer was operated in the data-dependent mode. Survey MS scans were acquired in the orbitrap with the resolution set to a value of 60,000. Each survey scan (300-2000 m/z) was followed by four data-dependent CID tandem mass (MS/MS) scans at 35% normalized collision energy and four data-dependent HCD tandem mass (MS/MS) scans at 40% normalized collision energy with 15,000 resolution in orbitrap. AGC target values were 500,000 for the survey scan, 10,000 for the ion trap MS/MS scan, and 50,000 for the orbitrap MS/MS scan. Target ions already selected for MS/MS were dynamically excluded for 30 s. Tandem mass spectra were searched against the Illumina deep sequencing-determined Tupaia hepatocyte protein database that was concatenated with reversed sequences to estimate false positives and was supplemented with the sequence of bait peptide under the Linux operating system using the ProLuCID (Xu et al., 2006) protein database search algorithm with peptide mass tolerance of ±100 ppm, fragment ion mass tolerance of ±400 ppm, half tryptic specificity, and a static modification of 57.0215 on Cys due to carboxyamidomethylation. ProLuCID search results were then filtered with DTA Select 2.0 (Tabb et al., 2002) using a cutoff of 1% for peptide false identification rate (-fp 0.01). Peptides with DeltaMass > 10 ppm (-DM 10) were rejected; the minimum number of peptides to identify a protein was set to 1 (-p 1).