Figure 2.

Liquid Chromatography-Tandem Mass Spectrometry of wtRPGR^ORF15 and coRPGR^ORF15

(A) Following overexpression in HEK293T cells, wtRPGR^ORF15 and coRPGR^ORF15 were purified from whole cell lysate by SDS-PAGE. In each lane, a single band above 180 kDa was identified as RPGR^ORF15 (arrow). This band was missing in the negative control (nc) lanes. No additional bands are visible, which would be different between WT and coRPGR^ORF15 lanes. (B) Identified RPGR^ORF15 bands were then excised for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. More than 80% of peptide sequence could be directly identified (indicated by dark arrows below amino acid sequence). Unidentified amino acids (no arrows) are largely based in the ORF15 sequence, which escapes LC-MS/MS analysis because of its repetitive, glutamic acid and glycine-rich sequence. Note that the C-terminal sequence following the ORF15 region was identified, which rules out premature termination of translation. Together with the identical migration pattern between the wtRPGR^ORF15 and coRPGR^ORF15 samples in the SDS-PAGE (A), this suggests that codon optimization does not result in alternative splicing, but in a protein product equivalent to the wtRPGR^ORF15.