Ultrafiltration filter

20 μg protein lysate per sample were reduced with 25 mM DTT at 60 °C for 30 min and alkylated with 50 mM iodoacetamide in the dark for 10 min. After alkylation, the sample was loaded on an ultrafiltration filter (10 kDa cutoff, Sartorius, German) for FASP digestion. Trypsin was added at a ratio of 1 : 100 (enzyme to protein) at 37 °C for 14–16 h. The samples were spun at 20 000 g at 4 °C for 10 min. The peptides were desalted using Ziptip C18 pipette tips (Merck KGaA, Darmstadt, Germany) according to manufacturer's instructions. After drying, the peptides were resuspended in 0.1% formic acid.

For each sample, 6300 μg of total proteins were diluted in 4% SDS, 100 mM Tris–HCl (pH 8.0), and 100 mM DTT and heated at 100ºC for 5 min. Each sample was then cooled to RT(room temperature) and loaded onto an ultrafiltration filter (30 kDa cutoff, Sartorius, Germany) containing 200 μL of UA buffer (8 M urea, 150 mM Tris–HCl, pH 8.0) followed by centrifugation at 14,000×g for 30 min and an additional washing step with 200 μL of UA buffer. 100 µL of 50 mM iodoacetamide in UA buffer was subsequently added to the filter to block the reduced cysteine residues, and the samples were then incubated for 30 min at RT in the dark followed by centrifugation at 14,000×g for 30 min. The filters were washed with 100 μL of UA buffer and centrifuged at 14,000×g for 20 min, and 100 µL dissolution buffer was added to elute protein. The procedure was repeated twice. The protein suspensions were then digested with 40 μL of trypsin (Promega, Madison, WI, USA) buffer (2 μg trypsin in 40μL dissolution buffer) at 37 °C for 16–18 h. Finally, the filter unit was transferred to a new tube and centrifuged at 14,000×g for 30 min. The resulting peptides were collected as a filtrate, and the peptide concentration was determined at absorbance of 280 nm^{11 (link)}.

Protein digestion was performed using the FASP method [40 (link)]. A total of 300 µg proteins from each sample were placed on an ultrafiltration filter (30 kDa cutoff, Sartorius, Gottingen, Germany) that had 200 µL UA buffer (8 M urea, 150 mM Tris–HCl, pH 8.0). It was then centrifuged at 14,000g for 30 min and washed with 200 µL of UA buffer. About 100 µL of 50 mM iodoacetamide was added to the filter to block reduced cysteine residues. The samples were maintained at room temperature for 30 min in the dark, followed by centrifugation at a speed of 14,000g for 30 min. UA buffer (100 µL) was used to wash the filters twice. Approximately 100 µL of a dissolution buffer (Applied Biosystems, Foster City, CA, USA) was placed on the filter. This was centrifuged at 14,000g for 20 min, and then repeated twice. The protein suspensions were subjected to enzyme digestion with 40 µL of trypsin (Promega, Madison, WI, USA) buffer (4 µg trypsin in 40 µL of dissolution buffer) for 16–18 h at 37 °C. The final filter unit was transferred to a new tube that was spun at 14,000g for 30 min. The peptides were collected as a filtrate and the concentration of the peptides was measured at an optical density with a 280 nm wavelength (OD₂₈₀).

Protein digestion was conducted using the FASP procedure (Wisniewski et al., 2009 (link)). In brief, 300 mg of proteins were loaded onto an ultrafiltration filter (30 kDa cutoff; Sartorius, Goettingen, Germany) containing 200 µL of UA buffer (8 M urea, 150 mM Tris-HCl, pH 8.0), centrifuged at 14,000× g for 30 min, and then washed with 200 µL of UA buffer. Then, 100 µL of 50 mM iodoacetamide in UA buffer was subsequently added to the filter to block reduced cysteine residues. The samples were incubated for 30 min at room temperature in the dark and then centrifuged at 14,000× g for 30 min. The filters were washed three times with 100 µL of UA buffer and then centrifuged at 14,000× g for 30 min after each washing step. Next, 100 µL of dissolution buffer (Applied Biosystems, Foster City, CA, USA) was added to each filter and then centrifuged at 14,000× g for 30 min, which was repeated three times. The protein suspensions were then digested with 40 µL of trypsin (Promega, Madison, WI, USA) buffer (6 µg trypsin in 40 µL of dissolution buffer) at 37 °C for 18 h. Finally, the filter unit was transferred to a new tube, and 40 µL of dissolution buffer was added followed by centrifugation at 14,000× g for 30 min. The resulting peptides were collected as a filtrate, and the peptide concentration was analyzed at OD280.