Figure 3.

The use of time courses in evaluating the quantification accuracy. (A) Tracking the missed cleaved peptides over time helps to evaluate the completeness of the digestion with respect to the selected peptides. (B) In the case of unstable peptides even at the maximum, the signal intensity does not reflect the level of endogenous peptides. (C) When slow forming and unstable peptides are combined, there is no time point at which the integrated area ratio reflects the true stoichiometry. (D) When stable peptides are inspected, the time point of AQUA peptide addition does influence the observed stoichiometry, which equals the true stoichiometry. (E) Degradation reactions that have the same rate in the peptide and protein context decrease the observed stoichiometry when the AQUA peptide is added after the digestion. The true stoichiometry can be assessed when the AQUA peptide is added early in the workflow. (F) Degradation reactions that have faster rate or only occur in the peptide context lead to wrong stoichiometries irrespective of the time point of AQUA peptide addition. Notably, the addition prior or upon digestion will mask this problem since a stable stoichiometry is observed. To detect such degradation reaction, the addition after digestion is much more indicative.