Reverse response curves were generated to evaluate the detection sensitivity, linearity, and reproducibility of selected peptides from salivary proteins. These curves were then used to calibrate the assay for precise quantification over a broad dynamic range (0.01–512 fmol/µg).

Serially diluted heavy peptides were spiked into trypsin-digested samples of pooled saliva, which already contained the corresponding light peptides. The LC-MRM assay was then used to measure the concentration-dependent responses of the spiked-in heavy peptides by measuring the peak area ratio of heavy to light signals.

The aliquots (1 µg) of the digested saliva were separately mixed with the serially diluted heavy peptides (0.0078, 0.0156, 0.0312, 0.0625, 0.125, 0.25, 0.5, 2, 8, 32, 128, and 512 fmol), and the resulting samples were lyophilized and stored at -20 °C until further analysis by LC-MRM.