Ptmscan succinyl lysine motif succ k kit

Total proteins were extracted from the stolons of the bermudagrass plants using the Plant Total Protein Extraction Kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The protein concentration was also quantified using the Bradford method. For trypsin digestion, 100 μg of protein was firstly reduced with 10 mM DTT for 45 min at 50 °C and alkylated with 50 mM iodoacetamide for 45 min at room temperature in the dark. The protein solution was then diluted with four volumes of digestion buffer (100 mM TEAB, pH 8.0) containing 2 μg of Trypsin Gold (Promega, Madison, USA) and incubated at 37 °C overnight. The digested peptides were desalted using a Strata-X C18 SPE column (Phenomenex, Torrance, USA) and lyophilized by vacuum centrifugation. Acetylated and succinylated peptides were enriched from the lyophilized peptides using the PTMScan® Acetyl-Lysine Motif [Ac-K] Kit (Cell Signaling Technology, Danvers, USA) and PTMScan® Succinyl-Lysine Motif [Succ-K] Kit (Cell Signaling Technology) according to the manufacturer’s instructions, respectively.

For global succinylome analysis, 1 mg of proteins of each sample was reduced with 10 mM DTT for 1 h at 34 °C, alkylated with 50 mM iodoacetamide for 1 h in the dark and then quenched by additional of 38 mM DTT. Each sample was diluted with 50 mM TEAB to a final concentration of 1 M Urea. Each sample was digested with 55 μg trypsin (1:18 w/w) for 18 h at 35 °C. The digests were cleaned up with Bond Elute C18 100 mg/mL cartridge (Agilent) and peptides were eluted with 50% ACN-0.1% TFA and dried down by a SpeedVacuum. Subsequent enrichment for succinylated peptides was conducted using a PTMScan® Succinyl-Lysine Motif [Succ-K] kit (Cat # 13764, Cell Signaling Technology, Inc., Danvers, MA, USA) following the vendor’s recommended procedures. Specifically, the peptides from each sample were reconstituted in 350 μL immunoaffinity purification (IAP) buffer and transferred to the vial containing 20 μL equilibrated Succ-K motif antibody beads, incubated on a vortex mixer at 4 °C for 2 h. After centrifugation at 2000 × g for 30 s, the beads were washed twice with 250 μL IAP buffer and three times by water. Finally, the enriched peptides were eluted three times with 55 μL of 0.15% TFA. The eluted fractions were pooled together, dried down and reconstituted in 22 µL of 0.5% formic acid (FA) for subsequent label-free quantitative analysis by nanoscale LC-MS/MS.