Quantification of Cellular Spermidine

Cells were washed with PBS and the pellet resuspended in lysis buffer (80% methanol + 5% Trifluoroacetic acid) spiked with 2.5 μM 1,7-diaminoheptane (Sigma). The cell suspension, together with acid-washed glass beads (G8772, Sigma), was transferred to a bead beater tube and homogenized in a bead beater (Precellys 24, Bertin Technologies) for four cycles (6500 Hz, 45 s) with 1 minute of ice incubation between each cycle. The homogenized samples were centrifuged at 13,000 g for 20 minutes at 4°C. The supernatant was collected and dried overnight. For chemical derivatization, 200 μL trifluoroacetic anhydride was added to the dried pellet and incubated at 60°C for 1 hour, shaking at 1200 rpm. The derivatization product was dried, re-suspended in 30 μL isopropanol and transferred to glass loading-vials. The samples were analyzed using a GCxGC-MS system as described (Yu et al., 2017 (link)). The following parameters were used for quantification of the 1D-GC-qMS data: type: area, slope: 1000/min, width: 0.04 s, drift 0/min and T. DBL: 1000 min without any smoothing methods used. Cellular spermidine amount was normalized to total protein levels determined by BCA assay for each sample first followed by comparison between samples.