wild-type and LGI3-FH KI mouse brain TAP sample, Shotgun proteomics with label-free quantification

The proteins in the tandem affinity purification (TAP) eluates from the wild-type control and Lgi3-FH KI mouse brains were concentrated by Trichloroacetic acid/acetone precipitation and redissolved in the denaturing buffer containing 7 M guanidine hydrochloride, 0.5M Tris-HCl (pH 8.5), and 10 mM EDTA. Then, proteins were reduced with dithiothreitol and alkylated with iodoacetamide, concentrated, and digested with trypsin at 37°C overnight (in-solution digestion). The obtained peptides were separated via nano-flow liquid chromatography (EASY-nLC1000, Thermo Fisher Scientific) using a reverse-phase C18 column (0.075 Å~ 125 mm; Nikkyo Technos). The liquid chromatography eluent was coupled to a nano ion spray source attached to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). For shot-gun analysis including protein identification, label-free quantification, and volcano plot analyses, the Mascot2.6.1 (Matrix Science) and Proteome Discoverer2.2 software (Thermo Fisher Scientific) were used. Peptide and protein identifications were calculated with protein false discovery rate (FDR) < 0.01. No filtering was applied and obtained protein lists with a high FDR confidence (< 0.01) from control and LGI3-FH KI replicates were used for statistical analysis to compare protein abundances based on peak intensity.