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Trypsin digestion by filter aided sample preparation (FASP) 

YH Yi Hao
MY Ming Ye
XC Xiaona Chen
HZ Hongli Zhao
AH Ayshamgul Hasim
XG Xia Guo
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100 µg protein sample was brought to 100 mM DTT and 200 µL UA buffer (8 M urea, 150 mM Tris-HCl, pH 8.0) were added. The sample was transferred to a Microcon YM-30 filter unit and centrifuged at 14,000g for 15 min. The supernatant was discarded and 200 µL UA buffer were added, followed by centrifugation for 15 min at 14,000g. The following steps were each repeated twice: (1) 100 µL of 50 mM iodoacetamide (IAA) in UA was added and the sample was centrifuged for 10 min at 14,000g; (2) 100 µL of 50 mM NH4HCO3 buffer was added and the sample was centrifuged for 10 min at 14,000g. Finally, 40 µL trypsin buffer (2 µg trypsin in 40 µL 50 mM NH4HCO3) was added and the mixture was shaken for 1 min at 600 rpm and incubated for 16–18 h at 37 °C. The digested sample was transferred to a new collection tube. After centrifugation for 10 min at 14,000g, the filtrate was collected, desalted with C18-SD Extraction Disk Cartridge (3 M, United States) and quantified by measuring extinction at 280 nm in a UV spectrophotometer.

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