Figure 1. Targeted quantitative proteomics method.

Our experimental method is described. Briefly, adult female heads, antennae or THAs are pulverized, proteins extracted under denaturing conditions, clarified by centrifugation, digested with trypsin, quenched and desalted. Targeted quantitative proteomics was performed using multiple reaction monitoring (MRM) on a triple quadrupole (QTrap 5500). MRM data were acquired in-triplicate or quadruplicate from samples collected every 4 hr for 24 hr. Peak areas for each peptide corresponding to the protein were integrated and the data normalized. AUC response (area under curve) was graphed for each protein vs. time, shown here after median transformation of the data. Note every protein had 2–3 underlying peptides used for quantification/qualification. An example of the three peptides used to quantify protein levels of OBP1 over 24 hr is shown. Targeted (quantitative MS/MS) proteomics is advantageous as it is highly specific, sensitive and linear with respect to quantity. In this work we demonstrate relative protein abundance rather than absolute quantities. Horizontal bars indicate day/night (white/black). See Fig. S1 for a detailed methodology and example results.