Figure 3: Technical Validation of acquired turnover rates.

(a) Bar charts showing in each of the samples: the number of proteins (i) identified at 1% FDR (light blue), (ii) quantified with isotope incorporation data at ≥4 time points (blue), and (iii) quantified with derived turnover rates passing stringency filters (dark blue). (b) Peptide decay curves across a range of goodness-of-fit (R²) and standard error (s.e.) values. Panels show experimental data of the fractional abundance of the 0th peptide isotopomers (A₀) (y) over time (x), illustrating representative qualities of fitting at various R² and s.e. values passing the stringency filter. Red line: best-fit kinetic curve. Red area: upper and lower bounds of fitting. (c) Histograms of R² (top) and s.e. (bottom) for the fitted peptide data. The R² histograms include all quantified peptides; s.e. histograms include only peptides passing stringency filters. Colors of stacked histograms reflect the number of time points at which the peptide’s isotope fractional abundance was quantified. (d) (Left panel) Cut-offs at various values of s.e. (x) and R² (y) were sampled stepwise to determine their effects on the intra-protein variance of turnover rates (heatmap colors), calculated as the median of the median absolute deviations of turnover rates of peptides identified to the same proteins. Using R² as sole filter excludes a subset of well-fitted peptides (lower left). (Right panel) Density plot showing distribution of R² (x) versus log₂ turnover rate (y) for peptides passing the stringency filter. Colors of density contours denote two groups of accepted peptides (blue: R²≥0.8; red: s.e. ≤0.05). Blue line: local regression. Accepted peptides with R²<0.8 have lower turnover rates. (e) Turnover rates of 14 distinct peptides from one protein (ATP5H). (Left) The amino acid (aa) position and length (x) of the peptides along the protein sequence are plotted against log₂ turnover rates of the peptides (y), showing consistent turnover. Indices refer to peptide sequences in the middle. (Right) Overlaid decay curves for the 14 ATP5H peptides. Isotope abundance (A₀) is rescaled to fractional synthesis (y) to normalize the position of each peptide curve over time (x).