FIGURE 2.

Tryptic peptide coverage of the p53 protein expressed from the R213X expression plasmid. (A) H1299 cells were transfected with plasmid expressing the TGA termination codon at codon 213 and treated with Gentamicin as in Figure 1. Samples were processed as indicated in the methods to produce tryptic peptides from the gel slice (Supplementary Figure S1). The figure shows representative tryptic peptide coverage of p53 as the blue annotated lines with several gaps. The 213 PTC codon peptide containing an R insertion is highlighted by a red dotted line. The low recovery and/or coverage of p53 tryptic peptide sequence C-terminal to the PTC is highlighted by the green box. The overlapping tryptic peptide coverage of p53 is staggered C-terminal to the 213 PTC position (overlapping blue lines from amino acids ∼270 through to the C-terminal SBP tag. The SBP tryptic peptide sequence (brackets) is also detected at the C-terminus (425-LEHHPQGQREP-435) which also confirms the readthrough of p53. The purple line highlights the localization of the reproducibly recovered tryptic peptide (121-SVTCTYSPALNK-132) that we use to introduce a termination codon as a model readthrough peptide (See Supplementary Figures S2–S4). (B) An example of a tryptic peptide that terminates at the PTC at codon 213; 203-VEYLDDRNTF*-212 Additional parameters include; scan: 23191; −10LgP PEAKS score: 50.71; mass of the precursor peak: 1270.58, m/z 636.30 (z = 2); error in ppm: 0.9; RT 33.54 min); see Figure 4B, line 5b, columns 1-3. Additional representative MS/MS spectra of tryptic peptides 203-VEYLDDRNTFR-213, which incorporates the wild-type arginine amino aid at codon 213, and 121-SVTCTYSPALNK-132 which forms our model PTC peptide, are shown in Supplementary Figures S2A, B.