ABSTRACT: Flp-In 293 T-REx cells (Thermo Fisher Scientific) stably expressing FlagBirA alone or FlagBirA-LIMP-2 were generated. After selection , cells were incubated for 24 hours in complete media supplemented with tetracycline (Sigma) and biotin (BioShop). Cells were collected and pelleted, the pellets were washed twice with PBS and snap frozen. Cells were resuspended in 10 ml of modified RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% SDS, 1:500 protease inhibitor cocktail (Sigma-Aldrich), 1:1,000 benzonase nuclease (Novagen), incubated on an end-over-end rotator , sonicated then centrifuged. Supernatants were transferred to a fresh 15 ml conical tube. Streptavidin-sepharose beads (GE) were added and the mixture incubated. Beads were pelleted by centrifugation at 2,000 rpm for 2 minutes and transferred with 1 ml of lysis buffer to a fresh Eppendorf tube. Beads were washed once with 1 mL lysis buffer and twice with 1 ml of 50 mM ammonium bicarbonate (pH =8.3). Beads were transferred in ammonium bicarbonate to a fresh centrifuge tube and washed two more times with 1 ml ammonium bicarbonate buffer. Tryptic digestion was performed by incubating the beads with MS-grade TPCK trypsin (Promega, Madison, WI) dissolved ammonium bicarbonate (pH 8.3). The following morning, additional MS-grade TPCK trypsin was added, and beads were incubated 2 additional hours. Beads were pelleted by centrifugation at 2,000 x g for 2 min, and the supernatant was transferred to a fresh Eppendorf tube. Beads were washed twice with 50 mM ammonium bicarbonate, and these washes were pooled with the first eluate. The sample was lyophilized and resuspended in buffer A (0.1% formic acid). 1/5th of the sample was analyzed per MS run.
Resuspended peptides were subjected to high performance liquid chromatography (HPLC) performed on pre-columns (150mm I.D.) and analytical columns (75mm I.D.) assembled in-house using fused silica capillary tubing (InnovaQuartz, Phoenix, AZ) packed with C18 (Magic, Michrom Bioresources, Auburn, CA). A 120min reversed-phase LC gradient (0-100% organic, 0.1% HCOOH) running at 250 nl/min on a Proxeon EASY-nLC pump was applied in-line with a hybrid LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA). A parent ion scan was performed in the Orbitrap at 60,000 resolution (fwhm), with up to the 20 most intense precursors selected for MS/MS (minimum ion count of 1000 for activation), using standard collision-induced dissociation (CDI) fragmentation. Dynamic exclusion was activated such that MS/MS of the same m/z (within a range of 15 ppm; exclusion list size = 500) detected twice within 15 seconds were excluded from analysis for 30 sec. For protein identification, Thermo .RAW files were converted to the .mzXML format using Proteowizard (PMID 24939128), then searched using X!Tandem (PMID 14976030) against the human database (Human RefSeq Version 45), using a parent ion mass tolerance error of 15ppm and fragment ion mass of 0.4 Da. Oxidation (M), acetylation (protein N-terminus) and deamidation (N, Q) were included as potential modifications. Each biological replicate was analyzed using two technical replicates. Data were analyzed using the trans-proteomic pipeline PMID 16729052) via the ProHits software suite (PMID 20944583). Proteins identified with a Protein Prophet cut-off of 0.9 were analyzed with the SAINT (PMID 21131968) algorithm using 14 Flag-BirA only samples as negative controls. High confidence interactors were defined as those with a SAINT score over 0.8 (bayesian false discovery rate (BFDR) lower than 0.01).
Brian Raught 
