Discovery of Mouse Spleen Signaling Responses to Anthrax using Label-Free Quantitative Phosphoproteomics via Mass

Inhalational anthrax is caused by spores of the bacterium Bacillus anthracis (B. anthracis), and is an extremely dan-gerous disease that can kill unvaccinated victims within 2 weeks. Modern antibiotic-based therapy can increase the survival rate to 50%, but only if administered presymp-tomatically (within 24–48 h of exposure). To discover host signaling responses to presymptomatic anthrax, label-free quantitative phosphoproteomics via liquid chroma-tography coupled to mass spectrometry was used to compare spleens from uninfected and spore-challenged mice over a 72 h time-course. Spleen proteins were de-natured using urea, reduced using dithiothreitol, alkylated using iodoacetamide, and digested into peptides using trypsin, and the resulting phosphopeptides were enriched using titanium dioxide solid-phase extraction and ana