Fig. 5

a Vector pBR-CMV-5ATG-63-EGFP produces FMRpolyG containing 63 CGG repeats fused to EGFP under a strong CMV enhancer and promoter. As a positive control for FMRpolyG detection by LC-MS/MS, an expression plasmid was generated with a CMV promoter driven FMRpolyG-GFP fusion construct. The human FMR1 5′ untranslated region (UTR) and Exon 1 sequence, from Transcriptional Start Site I (TSS-I) to the first three base pairs of Exon 2, were placed upstream and in frame with EGFP to create a stable fusion protein. The translational start site of FMRpolyG was modified from the native ACG to the canonical ATG to further drive FMRpolyG expression. b LC-MS/MS analysis of FMRpolyG-GFP expressed in an SK-N-MC heterologous cell expression system identifies multiple proteotypic peptides. Transiently transfected SK-N-MC cells containing the pBR-CMV-5ATG-63-EGFP plasmid were SDS solubilized and whole cell protein lysates were trypsin digested and analyzed by LC-MS/MS. GFP signal was observed in FMRpolyG and GFP expressing cells, but not null transfection controls, indicating successful transfection of the constructs. Peptide sequence coverage of FMRpolyG-GFP (48%) is indicated in yellow with modified residues in green. LC-MS/MS identified three unique FMRpolyG-GFP peptides present only in FMRpolyG-GFP expressing cells that map to native FMRpolyG protein. A representative mass spectrum of the FMRpolyG specific peptide SPPLGGGLPALAGLK is provided with y/b ions labeled