Updated PubMed. Human induced pluripotent stem cells were differentiated into NSC for 7 days. Cells on days 0, 1, 2, 3, 5 and 7 of differentiation (2.0 × 10⁶) were washed twice with DPBS (−) and then mixed with 100 μL of dissolution buffer consisting of 12 mM sodium deoxycholate, 12 mM N-lauroylsarcosine, 100 mM Tris-HCl pH 9.0, 1% O-GlcNAcase inhibitor, 1% Halt™ protease, and phosphatase inhibitor cocktail. The harvested cells were incubated at 95°C for 5 min and then sonicated to remove the viscosity. One hundred micrograms of protein was diluted to 2.5 μg/μL with dissolution buffer, and the proteins were reduced by adding 10 mM 2-mercaptoetanmol and incubating the mixture at 37℃ for 30 min, followed by another incubation with 20 mM acrylamide at 37℃ in the dark for 60 min. One microgram of mass spectrometry grade lysyl endopeptidase was added to the solution and incubated at 37℃ for 60 min. Then, 2 μg of trypsin was added to the solution and incubated at 37℃ for 6 h. Add an equal amount of ethyl acetate and 0.1% formic acid. After stirring, the supernatant was removed after centrifugation at 14,000 xg. The supernatant was desalted with an Oasis PriME HLB 1 cc Extraction Cartridge according to the manufacturer’s instructions. The protein fraction was dried using a Speed Vac concentrator, and the dried sample was reconstituted with 0.1% FA/3% ACN and analyzed by an Q-Exactive mass spectrometer coupled with a nanoLC instrument.