Proteins were extracted from phloem and xylem samples and prepared for proteomic analysis using the following protocol. 200 mg of the homogenized phloem and tissue samples were added to 500 µL of extraction buffer (0.5% sodium deoxycholate, 50 mM 1,4 dithiotreitol, 1 µM Pepstatin (Thermo Fisher Scientific), 1X Complete Mini Roche (Sigma Aldrich) in 50 mM ammonium bicarbonate) was added to each sample. Mechanical extraction was then performed using a Mixer mill MM400 (Retsh) with three inox beads of two cycles of 2 min at 30 Hz, turning the tube racks 180° between the cycles. Samples were centrifugated at 10 000 × g for 15 min at 4 °C to remove pellet debris. The supernatant was then filtered using a 0.45 µm Centrifugal Filter Membrane (Millipore) at 12 000 × g. Five volumes of acetone at -20 °C was added to the filtered sample and incubated at -20 °C overnight. After centrifugation at 16 000 × g for 15 min at 4 °C, the major part of the supernatant was discarded and the remaining acetone was left evaporated under the fume hood. The pellet was then resuspended with 50µL of 50 mM ammonium bicarbonate and protein concentration was measured using Bradford assay.
For each sample, a volume corresponding to 20 µg of proteins was used for subsequent analysis. Volumes were adjusted to 30 µL using 50 mM ammonium bicarbonate and sodium deoxycholate was added to a final concentration of 1%. The samples were heated at 95 °C for 5 min for protein denaturation. Cysteine disulfide bridges were reduced and alkylated using the following procedure. 1,4 dithiothreitol was added to a final concentration of 0.2 mM and incubated at 37 °C for 30 min. This was followed by the addition of iodoacetamide to a final concentration of 0.8 mM and incubated at 37 °C for 30 min in the dark.
Enzymatic digestion of the protein samples was initiated using 400 ng of trypsin enzyme (Promega), corresponding to an enzyme:protein ratio of 1:50, followed by an incubation at 37 °C overnight. Enzymatic digestion was stopped by acidification using 30µL of 3% acetonitrile, 1% trifluoroacetic acid, 0.5% acetic acid. This step also allowed the precipitation of sodium deoxycholate. Samples were finally centrifugated at 16 000 × g for 5 min and the supernatants were collected. The peptides resulting from trypsin digestion contained in these supernatants were purified on StageTips according to [33 (link)] using C18 Empore reverse phase. The samples were finally vacuum dried and stored at -20 °C prior to mass spectrometry analysis.
For each sample, a volume corresponding to 20 µg of proteins was used for subsequent analysis. Volumes were adjusted to 30 µL using 50 mM ammonium bicarbonate and sodium deoxycholate was added to a final concentration of 1%. The samples were heated at 95 °C for 5 min for protein denaturation. Cysteine disulfide bridges were reduced and alkylated using the following procedure. 1,4 dithiothreitol was added to a final concentration of 0.2 mM and incubated at 37 °C for 30 min. This was followed by the addition of iodoacetamide to a final concentration of 0.8 mM and incubated at 37 °C for 30 min in the dark.
Enzymatic digestion of the protein samples was initiated using 400 ng of trypsin enzyme (Promega), corresponding to an enzyme:protein ratio of 1:50, followed by an incubation at 37 °C overnight. Enzymatic digestion was stopped by acidification using 30µL of 3% acetonitrile, 1% trifluoroacetic acid, 0.5% acetic acid. This step also allowed the precipitation of sodium deoxycholate. Samples were finally centrifugated at 16 000 × g for 5 min and the supernatants were collected. The peptides resulting from trypsin digestion contained in these supernatants were purified on StageTips according to [33 (link)] using C18 Empore reverse phase. The samples were finally vacuum dried and stored at -20 °C prior to mass spectrometry analysis.
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