ABSTRACT: The Organomercury-resin-assisted capture (MRC) of S-nitrosoproteins was done with modifications as described in (Doulias and Gould, 2018). Entire inflorescences pre-anthesis (corresponding to FD stages up to 12/13) of soil grown WT and hot5-2 plants were harvested, shock frozen in lN2 and ground to a fine powder. 5 mL of plant material was used to extract proteins using a 1:1 ratio of extraction buffer (250 mM Hepes pH 7.7, 1 mM DTPA, 0.1 mM neocuproine, 40mM MMTS, 1% TritonX-100). After 5 min of vortexing, samples were centrifuged at 16.000 x g at 4 C for 15 min to separate cell debris from soluble protein. Centrifugation was repeated, protein concentration measured using the BCA assay (Thermo Scientific, USA) and 10 mg of total protein per sample used for the subsequent S-nitrosoprotein enrichment. For photolysis controls 0.1 M mannitol and 5 mM MMTS were added to 10 mg of protein homogenate, vortexed briefly and placed into borosilicate glass vials. The vial was placed on top of an ice bucket and proteins were illuminated under a UV lamp for 7 min in time mode using a UV-crosslinker with a 254 nm light source (Stratalinker Model 1800, Stratagene, USA). Each sample had a corresponding UV-treated negative control analyzed under identical conditions. Proteins were obtained after acetone precipitation and centrifugation at 4500 x g for 15 min at 4 C by resuspending in blocking buffer (250 mM Hepes pH 7.7, 1 mM DTPA, 0.1 mM neocuproine, 50 mM MMTS, 2.5 % SDS). All samples were alkylated for 35 min in the dark in a 50 C water bath with frequent vortexing, followed by another round of acetone precipitation to remove any non-reacted MMTS. Proteins were resuspended to 0.5 mg/ ml using loading buffer (250 mM MES pH 6.0, 1 mM DTPA, 1 % SDS). Organo-mercury resin was synthesized as mention in (Doulias and Gould, 2018). After alkylation, proteins were loaded onto 15 ml of activated organo-mercury resin slurry (50 %) and incubated for 60 min at RT in the dark to allow binding of S-nitrosated proteins to the resin material. Following the incubation, columns were washed with 50 CV of 50 mM Tris-HCl pH 7.5, 0.3 M NaCl, 0.5 % SDS, 50 CV of 50 mM Tris-HCl pH 7.5, 0.3 M NaCl, 0.05 % SDS, 50 CV of 50 mM Tris-HCl pH 7.5, 0.3 M NaCl, 1 % Triton X-100, 50 CV of 50 mM Tris-HCl pH 7.5, 0.3 M NaCl, 0.1 % Triton X-100, 0.1 M Urea and 200 CV of H2O. For on-column trypsin digest, columns were pre-equilibrated with 5 CV of 0.1 M NH4CO3 and then loaded with 1 CV of 0.1 M NH4CO3 containing 1 ug/ml trypsin gold (Promega V5280, Promega, USA). The flow-through containing the digested peptides of S-nitrosated proteins (elution 1) was collected in glass tubes after incubation overnight at RT in the dark. The remaining S-nitrosated peptides that were still bound to the organo-mercury resin were washed with 40 CV of 1 M NH4CO3, 0.3 M NaCl, 40 CV of 1 M NH4CO3, 40 CV of 0.1 M NH4CO3 and 200 CV of H2O. Bound peptides (elution 2) were then oxidatively released by incubation with 1 CV of 1 % performic acid for 45 min at RT and then collected in glass tubes. Following lyophilization of the samples and resuspension in 300 ul of 0.1 % formic acid, sample volume was reduced to 30 ul using a vacuum concentrator (Thermo Savant SPD111V, Thermo Scientific, USA) and then centrifuged for 20 min at 16,000 x g at 4C to remove any precipitate. Peptides were desalted using C18 tips as recommended by the manufacturer (Pierce C18 Tips, Thermo Scientific, USA) and resuspended into 15 ul of 1 % formic acid.
For the LC-MS\MS run, a 5 uL injection was loaded by a Thermo Easy nLC 1000 UPLC on to a 2 cm trapping column and desalted with 12 uL mobile phase A (0.1% formic acid in water). Peptides were eluted at 300 nL/min on to a 75 um x 15 cm RSLC column (Thermo) using a linear gradient of 5-35% mobile phase B (0.1% formic acid in acetonitrile) over 90 minutes. Ions were introduced by positive ESI using a stainless steel capillary at 2.1 kV into a Thermo Orbitrap Fusion tribrid mass spectrometer. Mass spectra were acquired over m/z 350-2000 at 120,000 resolution (m/z 200) and 1 second cycle time with an AGC target of 1e6, and data-dependent acquisition selected the top speed most abundant precursor ions for tandem mass spectrometry by HCD fragmentation using an isolation width of 2 Da, and automatic gain settings. Peptides were fragmented with a normalized collision energy of 27 %, and fragment spectra acquired in the linear ion trap. Targeted mass exclusion was activated to avoid MS/MS of ions previously identified as Rubisco or keratin peptides
Elizabeth Vierling