Speedvac

Protein was collected from cell lines by PTS buffer (12 mM sodium deoxycholate, 12 mM N-Dodecanoyl sarcosinate, 50 mM Ammonium bicarbonate, Protease inhibitor cocktail) and purified by using 2-D Clean-Up Kit (GE Healthcare). After the sample was heated in 95° C for 5 min, 0.25 M DTT, 0.375 M IAA, and 50 mM Ammonium bicarbonate were added by steps to each sample with vortex, and then incubated in room temperature for 30 min. Trypsin was added into each protein sample and incubated at 37° C overnight. Then, samples were added 1% Trifluoroacetic Acid in ethyl acetate to each sample with vortex and centrifuged in 15,600 g for 3 min. To dry them up, Speedvac (TOMY SEIKO, Tokyo, Japan) was performed for 30 min and re-suspended by 2 M Urea and 1% TFA. After centrifuging in 20,000 g for 3 min, the supernatant was separated into 7 fractions by the tips balance adjustment. Dry up all fractionations by Speedvac and re-suspended with 20% Paraformaldehyde and buffer contained with1% TFA, 2% ACN. The protein in samples was analyzed by LC-MS/MS (Thermo Fisher Scientific) and identified proteins by Mascot search with the databank of Swiss-Prot. The pathway analysis was performed to make clear the relation among the identified proteins using Targetmine (https://targetmine.mizuguchilab.org/).

One gram of sediment sample (wet weight) was transferred to a 15-mL Falcon tube and twice extracted with 5 mL methanol. The extraction was carried out by vortexing (30 s), and sonication (10 min), followed by centrifugation (10 min, 9,000 g, 10°C). Pooled methanol extracts were transferred to a new Falcon tube (15 mL) and dried in a vacuum concentrator (40°C, TOMY Speed Vac). One mL of methanol was added to the resulting dried extract, vortexed (1 min), and sonicated (10 min). Finally, the suspension was transferred to an Eppendorf tube (2 mL) and centrifuged (10 min, 14,000 g, 10°C) to give a clear methanol solution. The clear methanol solution was decanted in an Eppendorf tube (2 mL) and dried in the vacuum concentrator (40°C). The resulting extract was finally dissolved in methanol (200 mL) and centrifuged (10 min, 14,000 g, 10°C) to give a clean methanol extract. The methanol extract was either analyzed immediately or stored at −30°C.

Lipidomic analysis was performed as previously described [63 (link)]. In details, lipids were isolated from HCC cells JHH7 transduced with shCtl or shTG2 using the chloro-form/methanol/water extraction method. The chloroform phase containing lipids was evaporated under vacuum in SpeedVac (CC-105, TOMY, Tokyo, Japan) and reconstituted in 50 μL isopropanol (Wako Industries). Lipids were mixed with 15 mg/mL DHB matrix (1:5 v/v) in 90% acetonitrile/0.1% tri-fluoroacetic acid aqueous solution, and 0.5 μL of the mixture was loaded onto a MAL-DI-TOF target plate (MTP 384 target plate ground steel, Bruker Daltonics). Mass spectrometric analysis was performed using a rapifleX MALDI Tissuetyper mass spectrometer (Bruker Daltonics) at a spatial resolution of 20 µm. Peak calibration was carried out with a mixture of 10 mg/mL DCTB and 1 mg/mL cesium triiodide (1:1 v/v). Each collected spectrum was the sum of 10 single spectra obtained by shooting the laser at random positions on the target spot. Data were analyzed using FlexImaging software (Bruker Daltonics). Peak intensity was normalized to the protein concentration.