Analysis of protein profile changes during cardiac differentiation of hiPSC using DIA-MS

The non-target siRNA transfected cells (Day 7) were washed twice with DPBS(-) and mixed in buffer D consisting of 12 mM sodium deoxycholate, 12 mM N-lauroylsarcosine, and 100 mM Tris-HCl pH 9.0. The cells were collected and incubated at 95 ℃ for 5 min. The samples were sonicated to remove viscous matter. Protein concentration was determined using a BCA protein assay. Next, 100 μg of protein was diluted to 1.0 μg/μL in dissolution buffer D. The proteins were reduced by incubating with 10 mM 2-mercaptoethanol at 37 ℃ for 30 min. Alkylation was performed by incubation with 20 mM acrylamide at 37 ℃ in the dark for 60 min. The protein solution was incubated with 1 μg of lysyl endopeptidase mass spectrometry grade at 37 ℃ for 60 min. Next, 50 mM Tris-HCl pH8.5 containing 1.5 mM CaCl₂ was added with 2 μg of trypsin and cells were incubated at 37 ℃ for 16 h. The reaction was terminated by adding 500 μL of ethyl acetate followed by 10 μL of formic acid. After stirring and centrifugation at 14,000 xg, the supernatant was discarded. Desalting was performed using an Oasis PRiME HLB 1 cc Extraction Cartridge following 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 to a concentration of 0.1 μg/µL proteins.