Identification of the readthrough full-length B4GALNT1 protein derived from M4 mutant cDNA. (A) A peptide of B4GALNT1 containing the nonsense mutation at AA228 was shown. Green-coloured sequence is actually identified in MS of WT ProtA-B4GALNT1 fusion protein. This figure shows nonsense mutation at 682nd nucleotide from C to T, and possible readthrough sequences by replacing a new AA with the termination codon, TGA. (B) ProtA-B4GALNT1 fusion proteins consisting of WT or M4 mutated products were purified from culture supernatants and served for western blotting as described in Materials and Methods section. A faint band of M4 protein could be detected, while WT protein was stained with much more intensity. A CBB-stained gel and excision sites (1, 2) were shown. (C) MS analysis was performed by in-gel digestion with trypsin as described in Materials and Methods section. Cover maps of the identified fragments from WT and M4 mutant were presented. Cover rates of the individual samples were also attached. An arrow indicates the nonsense mutation site (AA228). Upper two panels are from measurement by Easy-nLC 1200^TM and Orbitrap Eclipse^TM (Thermo Scientific), and lower two are from Ion Mobility system (FAIMS Pro^TM) (Thermo Scientific) that can detect further minor components. Note that a fragment immediately following the PTC was identified. Green zones indicate individual peptide fragments identified. Detailed peptide sequences identified are presented in Supplementary Fig. S3.