Inhalational
anthrax is caused by spores of the bacterium Bacillus anthracis (B. anthracis), and is an extremely dangerous disease that can kill unvaccinated victims within 2 weeks. Modern
antibiotic-based
therapy can increase the survival rate to ∼50%, but only if administered presymptomatically (within 24-48 h of exposure). To discover host signaling responses to presymptomatic
anthrax, label-free quantitative phosphoproteomics via liquid chromatography coupled to mass spectrometry was used to compare spleens from uninfected and spore-challenged mice over a 72 h time-course. Spleen
proteins were denatured 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 analyzed by nano-liquid chromatography-Linear Trap Quadrupole-Orbitrap-MS(/MS). The fragment ion spectra were processed using DeconMSn and searched using both Mascot and SEQUEST resulting in 252,626 confident identifications of 6248
phosphopeptides (corresponding to 5782 phosphorylation sites). The precursor ion spectra were deisotoped using Decon2LS and aligned using MultiAlign resulting in the confident quantitation of 3265 of the identified
phosphopeptides. ANOVAs were used to produce a q-value ranked list of host signaling responses. Late-stage (48-72 h postchallenge) Sterne strain (lethal)
infections resulted in global alterations to the spleen phosphoproteome. In contrast, ΔSterne strain (asymptomatic; missing the
anthrax toxin)
infections resulted in 188 (5.8%) significantly altered (q<0.05)
phosphopeptides. Twenty-six highly tentative phosphorylation responses to early-stage (24 h postchallenge)
anthrax were discovered (q<0.5), and ten of these originated from eight
proteins that have known roles in the host immune response. These tentative early-
anthrax host response signaling events within mouse spleens may translate into presymptomatic diagnostic
biomarkers of human
anthrax detectable within circulating immune cells, and could aid in the identification of pathogenic mechanisms and therapeutic targets.