Chapter 3: Development of biological and chemical tools for discovery of the OGT
3.6 Methods
3.6.2 Tandem affinity purification protocol for OGT pulldown and lentiviral production 146
The conditions for tandem affinity purification were optimized using HEK293T cells transfected with lentivirus expressing rat OGT-FLAG-HA and FLAG-HA-OGT. In order to generate lentivirus, 10 cm plates were coated with 0.1% wt/vol. gelatin A for 2 hours followed by rinsing twice with sterile water and air drying. Then, HEK293FT cells were plated in COMPLETE media (10% FBS, Na pyruvate, P/S, nonessential amino acids, and HEPES in DMEM High Glucose). At 60% confluence, the media was removed, and then the cells were plated with serum-free DMEM High Glucose with GlutaMAX transfected with ViraPower™ Lentiviral Packaging Mix (pLP1, pLP2, and
pLP/VSVG), the Tet-OGT-FH or Tet-FH-OGT and Lipofectamine™ 3000 according to the manufacturer’s instructions. After 6 hours, the serum-free media was replaced with COMPLETE media and then the media was collected and then replaced every 12 hours thereafter for 72 hours total. The collected media was then filtered using a 0.45 µm sterile filter or spun 2000 x g for 5 min in order to remove or pellet cellular debris. Then, the media was concentrated using 50-ml pre-washed Amicon-15 30 kDa centrifugal concentrators. HEK293T cells were transduced with the lentivirus in order to create HEK293T cells that would express OGT-FH with the addition of doxycycline (Tet-OGT- FH HEK293T cells).
Tet-OGT-FH HEK293T cells cultured with or without 1 µg/ml doxycycline or OGT-FH mouse brains or WT mouse brains were then lysed in 1% Triton X-100 in TBS with PIC-EDTA followed by enrichment using ANTI-FLAG M2® M2 affinity gel
agarose beads with end-over-end rotation overnight at 4°C. After this, the FLAG beads were washed three times with TBS (Tris Buffered Saline with Triton X-100). Following washing, the OGT-FH was eluted using 150 ng/µL 3xFLAG peptide in TBS (19 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl) with 1% Triton X-100. Then, the eluted
proteins were added to HA beads and enriched with end-over-end rotation overnight at 4°C. The beads were washed with TBS three times followed by elution with 3M NaSCN.
Proteins in the final tandem affinity purification elution were separated on NuPAGETM NovexTM 4-12% Bis-Tris protein gels in MOPS buffer (50 mM MOPS, 50 mM Tris Base,
0.1% SDS, 1 mM EDTA, pH 7.7). Following protein gel electrophoresis, the proteins were either then prepared for mass spectrometry (see below), silver stain, or transferred onto an Immobilon-FL PVDF membrane in transfer buffer (25 mM Tris, 190 mM
glycine, 20% methanol) for western blot. For silver stain, the gel was stained using the Silver Stain Plus kit according to the manufacturer’s instructions. For western blot, the membrane was blocked for 1 h at RT with 5% bovine serum albumin (BSA) in 1x TBST (TBS with 0.1% Tween 20). The blot was then incubated for 1 h at RT or overnight at
4°C with the indicated primary antibodies (1:1,000 dilution): HA-Tag (C29F4) Rabbit mAb, ANTI-FLAG M2® mouse mAb, α-Tubulin mouse mAb, and OGT rabbit Ab. After primary incubation, the membrane was rinsed three times with 1x TBST and then
incubated with the appropriate Alexa Fluor® 680 or 800 conjugated secondary antibody (1:5,000) in 5% BSA in TBST for 1 h at RT, washed three times with TBST for 5 min, and imaged with an Odyssey scanner.
For crosslinking, the cells were placed on ice, washed once with PBS/Ca2+/Mg2+
buffer (0.1 mM CaCl2, 1 mM MgCl2 in PBS), then incubated for 2 hours in either 1 mM DTSSP (crosslinking solution) in PBS/Ca2+/Mg2+ or PBS/Ca2+/Mg2+ (non-crosslinking solution), and quenched in 20 mM Tris, pH 7.4 in PBS/Ca2+/Mg2+ buffer for 15 min.
Following crosslinking, the cells were lysed in 0.5% Triton X-100 in Buffer A (10 mM HEPES, 150 mM NaCl, 1 mM EGTA, 0.1 mM MgCl2, pH 7.4) with PIC-EDTA for 30 min with rocking. Following lysis, the cells were scraped from the plate and centrifuged at 15,000 x g for 15 min. All crosslinking and lysis conditions were performed on ice or at 4°C with ice-cold buffers. Then, the supernatant was analyzed for protein
concentration using the BCA assay according to the manufacturer’s instructions.
Afterwards, the tandem affinity purification proceeded with 10 mg of each sample in parallel as described above.
3.6.3 Mass spectrometry for OGT interactome
Following tandem affinity purification, the final HA pull down eluent was added to two prewetted 10K MWCO Amicon filter units and washed 3 times in TEAB buffer (100 mM triethylammonium bicarbonate). Then the concentrate was spun down to 50 µL
of liquid and then 10 mM DTT in 100 mM TEAB was incubated with gentle rotation for 60 min at 37°C. After reduction, the solution was washed twice with 100 mM TEAB, concentrated to 50 µL, and then alkylated with 50 mM 2-iodoacetamide (IAA) in 100 mM TEAB in the dark with rotation for 40 min at RT. The solution was washed twice with 100 mM TEAB, concentrated to 20 µL, and then digested overnight at 37°C with trypsin in 1 mM CaCl2, 100 mM TEAB with a final protease to protein ratio of 1:20 (wt/wt). The peptides were washed twice with 100 mM TEAB and then combined for a final volume of 100 µL. Then, 4 µL of either 4% (vol/vol) CH2O or CD2O (for light or intermediate labelling respectively) was added to the samples, mixed, and then spun down as previously described.46 Afterwards, 4 µL of 0.6M NaBH3CN was added to the samples and then rotated for 1 hour at RT. The solution was quenched by adding 16 µL of 1% (vol/vol) ammonia solution and then acidified with 8 µL formic acid. The sample was desalted using HPLC and then resuspended in 10 µL of 0.1% formic acid. At this point, the light- and intermediate-labelled samples were mixed and analyzed by LC- MS/MS at the Caltech Proteomics Exploration Laboratory. About 250 ng of digested peptides were analyzed with an EASY-nLC 1000 and Thermo Orbitrap Fusion™
Tribrid™ Mass Spectrometer (ThermoFisher Scientific) as previously described.47