Vn01h02

The protein extracts were processed according to the Filter Aided Sample Preparation (FASP) protocol [44 (link)] using spin filter devices with 10kDa cutoff (Sartorius, VN01H02). 150 μl lysate were diluted in 8 M Urea/100 mM Tris-HCl pH 8.5, the filters were extensively washed with the urea solution, treated with 10 mg/ml iodoacetamide in the urea solution and incubated for 30 min in the dark for the alkylation of cysteines. The proteins on the top of the filters were washed three times with 50 mM ammonium bicarbonate and finally the proteins were digested adding 1 μg trypsin/LysC mix in 80 μl 50 mM ammonium bicarbonate solution (Mass spec grade, Promega) and incubated over night at 37°C. The peptides were eluted by centrifugation, followed by speed-vac-assisted solvent removal, reconstitution in 0.1% formic acid, 2% acetonitrile in water and transferring to LC-MS glass sample vials. Peptide concentration was determined by nanodrop absorbance measurement at 280 nm.

About 100 µg proteins of each sample per tube were prepared. Then they were reduced with 12 mM (final concentration) dithiothreitol for 1 h at 37°C. Subsequently, protein alkylations were performed with 50 mM (final concentration) iodoacetamide for 1 h in dark at room temperature. Then the mixture was transferred to centrifugal units (VN01H02, Sartorius, Germany) and centrifuged at 12,000×g for 20 min, and then the filtrate was discarded. After centrifugation, 8 mM urea solution was added into the centrifugal units and centrifuged, this step was repeated twice. After that, 100 µL dilute buffer (50 mM triethylammonium bicarbonate) was added into the centrifugal units and centrifuged. Then 50 µL dilute buffer containing 2 µg modified trypsin (Promega) was added into the centrifugal units at 37°C overnight. After trypsin digestion, the resulting peptides were collected using centrifugation. Then the peptides were labeled with iTRAQ reagents (AB Sciex, USA) according to the manufacturer's instructions. For each sample of three time points (i.e., S1, S2, and S3) was iTRAQ labeled 3 times except S3. (i.e., 113-, 116-, 119- tags for S1, 3 replicates. 114-, 117-, 121- tags for S2, 3 replicates. 115-, 118- tags for S3, 2 replicates.)

PNGase F digestion was performed on a 10 kDa cutoff filter (Sartorius #VN01H02) prior to FASP digestion. Filters were first equilibrated with successive washes of 0.1% FA followed by 8M urea, 100 mM ABC pH 8.0. All washes were spun through at 14,000 × g for 15 min. Samples were then loaded onto filters and spun at 14,000 × g for 20 min before washing with 200 μl of 8M urea, 100 mM ABC. Filters were then washed with three aliquots of 100 μl 50 mM ABC pH 8.0 before adding 1000U PNGase F (NEB #P0704) in 2M urea, 100 mM ABC pH 8.0 directly to the top of the filter membrane. PNGase F digestion was allowed to proceed for 2 h at 37 °C before spinning at 14,000 × g for 15 min to remove digest volume, retaining deglycosylated protein on the membrane. Filters were then washed with 100 μl 50 mM ABC pH 8.0 to remove residual buffer. Trypsin digest was performed in 20 mM ABC pH 8.0 with 0.02% ProteaseMax (Promega #V207 A) for 16 h using a 1:100 enzyme:protein ratio. Samples were eluted and acidified in 150 μl 0.2% FA. Aliquots of digested samples containing 10 μg of protein were desalted using Pierce C18 Spin Tips (Thermo Scientific #84850) according to the manufacturer's protocol.