Fig. 1.

Heavy-isotope pulse-chase to identify long-lived proteins present in aged cells. (A) Schematic representation of experimental design to identify long-lived proteins in aged mother cells (details in Materials and Methods). (B) Representative plots of the ¹³C/¹²C ratio for peptides in given gel slices that correspond to specific proteins. The x axis is approximate MW of peptide (estimated from gel slice); y axis is median ratio ¹³C/¹²C for all peptides mapped to that protein in a gel slice. The size of dots reflects the relative number of peptide observations in that slice. Error bars are SD of the medians calculated from 1,000 bootstrapped samples. The expected MW of the full-length unmodified protein is plotted as a vertical dashed red line. Mrh1 is an integral membrane protein that appears across many gel slices, each with a similar ratio. Sur7, an integral membrane eisosome component, appears only in gel slices corresponding to its expected size with a high abundance of heavy label. Bgl2, a cell wall component, shows enrichment for heavy label. The full-length version of Ssa2, a small heat-shock protein, is primarily newly synthesized (very low ¹³C/¹²C), whereas the original ¹³C label is predominantly in low MW gel slices that correspond to fragmented Ssa2 protein.