Disease detection in historical samples currently relies on DNA extraction and 
amplification, or immunoassays. These techniques only establish pathogen 
presence rather than active disease. We report the first use of shotgun 
proteomics to detect the protein expression profile of buccal swabs and cloth 
samples from two 500-year-old Andean mummies. The profile of one of the mummies 
is consistent with immune system response to severe pulmonary bacterial 
infection at the time of death. Presence of a probably pathogenic Mycobacterium 
sp. in one buccal swab was confirmed by DNA amplification, sequencing, and 
phylogenetic analyses. Our study provides positive evidence of active pathogenic 
infection in an ancient sample for the first time. The protocol introduced here 
is less susceptible to contamination than DNA-based or immunoassay-based 
studies. In scarce forensic samples, shotgun proteomics narrows the range of 
pathogens to detect using DNA assays, reducing cost. This analytical technique 
can be broadly applied for detecting infection in ancient samples to answer 
questions on the historical ecology of specific pathogens, as well as in 
medico-legal cases when active pathogenic infection is suspected.