Fig. 2: Measuring peptide clearance with PPToP.
a Experimental setup. HeLa cells were grown in isotopically light (L)-labeled medium and pulsed for a specified time with heavy (H)-labeled medium (Replicates 1–3; L and H reversed for Replicate 4). After lysis and trypsin-digest, phosphorylated peptides were enriched using Fe-IMAC. Both the eluate enriched for phosphorylated peptides (phospho, in yellow) as well as the flow-through (unmodified, in blue) were measured with mass spectrometry. Boxplots show replacement of old with new labeled protein over time, y axis: median values over replicates on the protein level. Outliers have been omitted for clarity. b After correction for cell growth (see “Methods”), clearance traces for each peptide are compared to the trace of the median of all unmodified peptides (representing the entire protein pool P) to find peptides with significantly different clearance. See “Methods” for details. c Fitting of the initial slope to assign peptides to faster or slower clearance. d Example traces of phosphopeptides (yellow) whose clearance significantly deviates from the protein median (gray). Numbers over facets represent phosphosites phosphorylated on the peptides in question. e Volcano-like plot showing peptides with significantly slower or faster clearance compared to the protein median. x axis shows the difference in fit compared to the protein median signed by the difference in initial slope: sign(Δinitial slope)*√(RSS0−RSS1). f Significantly more peptides in the phospho fraction exhibit differing clearance, with the majority of hits being slower. In contrast, most hits of unmodified peptides exhibit faster clearance. g Phosphoproteins are more likely to show differing clearance in at least one of their peptides compared to all proteins. h Phosphosites exhibiting faster clearance are more likely to be conserved and functionally important as shown by lower SIFTS scores14,15. Below: sample sizes of each group (n); above: p values from a two-sided Wilcoxon signed-rank test. Boxplots in a and h consist of median line, box: upper and lower quartiles, whiskers: 1.5 times interquartile range, points: outliers. Source data are provided as a Source data file.
