Stable isotope dilution–parallel reaction monitoring–mass spectrometry (SID-PRM-MS) validation of rickettsial protein RC0497. A: Annotated MS/MS spectrum of stable isotope-labeled RC0497 peptide LLLSLDSTGE[K(13C6,15N2)]. B: Liquid chromatography (LC) chromatogram for transitions of stable isotope-labeled standard peptide LLLSLDSTGE[K(13C6,15N2)]. C: LC chromatogram for transitions of native peptide LLLSLDSTGEK from the serum of mice with lethal dose (LD) rickettsial infection. D: SID-PRM-MS quantification of RC0497 peptide LLLSLDSTGEK in the mouse serum. The y axis uses a log10 scale. Dot-product (dotp) values show the expected similarity in peak shape of native peptides to the library spectra. ***P < 0.001 (t-test). Ctrl, control mice; SIS, stable isotope labeled; SLD, sublethal dose.