Quantitative proteomics for efficient differentiation of human induced pluripotent stem cells into neural stem cells using data independent acquisition mass spectrometry.

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.