DOI: 10.25345/c52r3p746
title: MassIVE MSV000093839 - MudPIT analyses of the proteins affinity-purified from HEK293T cells transiently expressing Halo-FAM60A further fractionated by size exclusion chromatography (Replicate 3)
title: Halo affinity purification for proteomic analysis | HEK293T cells (1 x 10E7) were cultured in 15-cm plates for 24 hrs, then transfected with Halo-tagged gene constructs using Lipofectamine LTX. After 48 hrs, cells were harvested and washed twice with ice-cold PBS. Cells were lysed in 300 ul of mammalian cell lysis buffer, containing 50mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1mM benzamidine HCl, 55 uM phenanthroline, 1 mM PMSF, 10uM bestatin, 5uM pepstatin A, and 20uM leupeptin. Lysed cells were passed through a 26-gauge needle 5-7 times and then centrifuged at 14,000 rpm for 30 min at 4C. The supernatant (300ul) was mixed with 700ul of Tris-buffered saline and centrifuged again at 14,000 rpm for 10 min at 4C. The clear supernatant was incubated overnight at 4C with 100ul of prewashed Magne HaloTag Beads. Post-incubation, beads were washed four times with 750ul of wash buffer and eluted with a buffer containing 50mM Tris-HCl (pH 8.0), 0.5mM EDTA, 0.005mM DTT, and 2 U of AcTEV Protease for 2 hrs at room temperature. Finally, the eluate was passed through a Micro Bio-Spin column to remove any bead residues and TCA-precipitated before proteomic analysis. Gel Filtration chromatography | Purified Fam60A protein complexes were isolated from whole cell extracts of HEK293T cells expressing Halo-Fam60A using the Halo affinity purification method. The purified Fam60A interacting proteins were then subjected to size-exclusion chromatography on a Superose 6, 10/300 GL column (Amersham Bioscience) using a buffer composed of 40mM HEPES (pH 7.9), 350mM NaCl, 5% glycerol, 0.1% Tween 20, and 1.0mM DTT. Fractions of 500ul were collected to analyze the co-fractionation profiles of Fam60A and its associated proteins. The column calibration was performed using a set of gel filtration markers, including Blue Dextran 2000, thyroglobulin (669kDa), Ferritin (440kDa), beta-amylase (200kDa), Alcohol dehydrogenase (150), and bovine serum albumin (66kDa). Multidimensional Protein Identification Technology (MudPIT) analysis | Precipitated proteins were resuspended in 30uL of 100mM Tris pH 8.5 with 8M urea to denature proteins. Cysteines were reduced and alkylated prior to digestion with recombinant LysC and modified trypsin. Reactions were quenched by the addition of formic acid to the final concentration of 5%. After digestion, peptide samples were pressure-loaded onto 100 um fused silica microcapillary columns packed first with 9 cm of reverse phase material, followed by 3 cm of 5um Strong Cation Exchange material, followed by 1 cm of 5um C18 RP. The loaded microcapillary columns were placed in-line with a 1100 Agilent Quartenary HPLC. The application of a 2.5 kV distal voltage electrosprayed the eluting peptides directly into Thermo linear ion trap (LTQ) mass spectrometers equipped with a custom-made nano-LC electrospray ionization source. Full MS spectra were recorded on the eluting peptides over a 400 to 1600 m/z range, followed by fragmentation in the ion trap on the first to fifth most intense ions selected from the full MS spectrum. Dynamic exclusion was enabled for 90 s. Mass spectrometer scan functions and HPLC solvent gradients were controlled by the XCalibur data system. Data Processing | RAW files were extracted into .ms2 file format using RawDistiller v. 1.0, in-house developed software. MS/MS spectra were first searched using ProLuCID v. 1.3.3 with a mass tolerance of 500 ppm for peptide and fragment ions. Trypsin specificity was imposed on both ends of candidate peptides during the search against a protein database containing non-redundant human proteins (NCBI 2016-06-10 release), as well as 426 common contaminants. To estimate false discovery rates (FDR), each protein sequence was randomized (keeping the same amino acid composition and length) and the resulting "shuffled" sequences were added to the database. Masses of 57.00 Da was differentially added to cysteine residues to account for alkylation by CAM and 16.00 Da were differentially added to methionine residues. DTASelect v.1.9 was used in combination with an in-house software, swallow v1.0 to select and sort peptide/spectrum matches (PSMs) to FDRs of less than 1% at the peptide and protein levels.