Native IP-MS was performed with about 50 ml of packed floral tissues25 (link). Floral tissues were ground into fine powder using RETCH homogenizer and resuspended in 25 ml of IP buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 0.1% Tergitol, 0.5 mM DTT, and cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)). Clumps were broken by Dounce homogenizer. Lysate was filtered through Mirachoth and centrifuged at 20,000 g for 10 min at 4 ◦C. Supernatant were incubated with 200 ul of anti-FLAG M2 magnetic beads (Sigma) at 4 ◦C for 2 h with rotation. Beads were washed 5 times with IP buffer. Bead bound proteins were eluted with 300 ul of 250 ug/ml 3xFLAG peptide (Sigma), with vigorous mixing at 37 ◦C for 15 min each elution for a total of two elutions. The eluted proteins were subjected to trichloroacetic acid precipitation and mass spectrometric analysis.
Crosslinked IP was performed with 15 g of floral tissues and resuspended in Nuclei Isolation Buffer (50 mM HEPES, 1 M sucrose, 5 mM KCl, 5 mM MgCl2, 0.6% Triton X-100, 0.4 mM PMSF, 5 mM benzamidine, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)) to reach a final volume of 40 ml and supplemented with 1% formaldehyde for 12 min with rotation. Glycine was added immediately to stop the crosslinking. Clumps were broken by Dounce homogenizer and lysate was filtered through Miracloth and centrifuged at 1500 g for 10 min at 4 ◦C. Nuclei pellet was resuspended and washed with NRBT buffer (20 mM Tris-HCl pH 7.5, 2.5 mM MgCl2, 25% glycerol, 0.2% Triton X-100) twice and resuspended in 6 ml of RIPA buffer (1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). Resuspended nuclei were split into 3x 2 ml aliquots for sonication for 20 min (30 s on/30 s off) with Bioruptor Plus (Diagenode). Sheared lysate was centrifuged at 8000 g for 15 min at 4◦C and combined supernatant was incubated with 200 ul of FLAG-M2 magnetic beads (50% slurry, Sigma) for 2 h at 4 ◦C with rotation. Beads were washed, eluted, and precipitated as described in native IP.
The TCA precipitated samples were resuspended in 50 μl of digestion buffer (8 M urea, 100 mM Tris pH 8.5). Each sample was reduced and alkylated by adding TCEP and iodoacetamide to final concentrations of 5 mM and 10 mM, incubated at room temperature in the dark for 20 min, and then digested by 0.1 μg of Lys-C (Thermo Scientific, 90051) and 0.8 μg Trypsin (Thermo Scientific, 90057) proteases at 37 °C overnight. The digested samples were quenched by the addition of formic acid to 5% (v./v.) final concentration. Finally, each sample was desalted via C18 tips (Thermo Scientific, 87784) and reconstituted in 15 μL of 5% formic acid before analyzed by LC–MS/MS.
Digested peptides were resuspended in 5% formic acid and fractionated online using a 25 cm long, 75 uM inner diameter fused silica capillary packed in-house with bulk C18 reversed phase resin (length, 25 cm; inner diameter, 75 uM; particle size, 1.9 μm; pore size, 100 Å; Dr. Maisch GmbH). The 140-minute water–acetonitrile gradient was delivered using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific) at a flow rate of 300 nl/min (Buffer A: water with 3% DMSO and 0.1% formic acid and Buffer B: acetonitrile with 3% DMSO and 0.1% formic acid). Fractionated peptides were ionized and analyzed by tandem mass spectrometry (MS/MS) Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Data was acquired using a Data-Dependent Acquisition (DDA) method comprised of a full MS1 scan (Resolution = 120,000) followed by sequential MS2 scans (Resolution = 15,000) to utilize the remainder of the 3 second cycle time.
Data analysis including peptide and protein identification was performed using MS2 spectra were searched using the ProLuCID algorithm against Arabidopsis reference proteome followed by filtering of peptide-to-spectrum matches (PSMs) by DTASelect using a decoy database-estimated false discovery rate of <1%.
Crosslinked IP was performed with 15 g of floral tissues and resuspended in Nuclei Isolation Buffer (50 mM HEPES, 1 M sucrose, 5 mM KCl, 5 mM MgCl2, 0.6% Triton X-100, 0.4 mM PMSF, 5 mM benzamidine, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)) to reach a final volume of 40 ml and supplemented with 1% formaldehyde for 12 min with rotation. Glycine was added immediately to stop the crosslinking. Clumps were broken by Dounce homogenizer and lysate was filtered through Miracloth and centrifuged at 1500 g for 10 min at 4 ◦C. Nuclei pellet was resuspended and washed with NRBT buffer (20 mM Tris-HCl pH 7.5, 2.5 mM MgCl2, 25% glycerol, 0.2% Triton X-100) twice and resuspended in 6 ml of RIPA buffer (1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). Resuspended nuclei were split into 3x 2 ml aliquots for sonication for 20 min (30 s on/30 s off) with Bioruptor Plus (Diagenode). Sheared lysate was centrifuged at 8000 g for 15 min at 4◦C and combined supernatant was incubated with 200 ul of FLAG-M2 magnetic beads (50% slurry, Sigma) for 2 h at 4 ◦C with rotation. Beads were washed, eluted, and precipitated as described in native IP.
The TCA precipitated samples were resuspended in 50 μl of digestion buffer (8 M urea, 100 mM Tris pH 8.5). Each sample was reduced and alkylated by adding TCEP and iodoacetamide to final concentrations of 5 mM and 10 mM, incubated at room temperature in the dark for 20 min, and then digested by 0.1 μg of Lys-C (Thermo Scientific, 90051) and 0.8 μg Trypsin (Thermo Scientific, 90057) proteases at 37 °C overnight. The digested samples were quenched by the addition of formic acid to 5% (v./v.) final concentration. Finally, each sample was desalted via C18 tips (Thermo Scientific, 87784) and reconstituted in 15 μL of 5% formic acid before analyzed by LC–MS/MS.
Digested peptides were resuspended in 5% formic acid and fractionated online using a 25 cm long, 75 uM inner diameter fused silica capillary packed in-house with bulk C18 reversed phase resin (length, 25 cm; inner diameter, 75 uM; particle size, 1.9 μm; pore size, 100 Å; Dr. Maisch GmbH). The 140-minute water–acetonitrile gradient was delivered using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific) at a flow rate of 300 nl/min (Buffer A: water with 3% DMSO and 0.1% formic acid and Buffer B: acetonitrile with 3% DMSO and 0.1% formic acid). Fractionated peptides were ionized and analyzed by tandem mass spectrometry (MS/MS) Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Data was acquired using a Data-Dependent Acquisition (DDA) method comprised of a full MS1 scan (Resolution = 120,000) followed by sequential MS2 scans (Resolution = 15,000) to utilize the remainder of the 3 second cycle time.
Data analysis including peptide and protein identification was performed using MS2 spectra were searched using the ProLuCID algorithm against Arabidopsis reference proteome followed by filtering of peptide-to-spectrum matches (PSMs) by DTASelect using a decoy database-estimated false discovery rate of <1%.
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