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.

Funding: This work was supported in part by National Geographic Society and the Secretariat of Culture of the Province of Salta, Argentina; and by grant NIH/NCRR 1S10RR025072-1 (Orbitrap). No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2012 Corthals et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Detecting the immune reaction to the pathogen in the host provides positive evidence of active pathogenic infection [9] . Existing methods, such as antibody-binding immunoassays, are ill suited for archeological applications because they require fresh tissues, use a small number of targeted antibodies, and are prone to both false positives and false negatives [10] , [11] . Proteomics approaches can identify and quantify proteins directly, and offer three distinct advantages in archeological and forensic research [12] . First, proteins can potentially outlast DNA by thousands to millions of years [13] , [14] , pushing back the time frame for detection of responses to infection. Second, protein detection does not rely on amplification, so there is less susceptibility to contamination than in PCR [15] . Third, a broad spectrum of proteins can be characterized from small samples, resulting in a more resolved picture of immune response than from immunoassays [16] . In this paper, we present methods for obtaining proteomic-quality samples from 500-year old Andean mummies, and results documenting immune response in these ancient human samples. Our results show that shotgun proteomic applications complement results from forensic DNA analyses by providing evidence of active infection and pointing to the pathogens triggering observed immune responses.

Over the last decade, forensic techniques relying on ancient DNA extraction and PCR amplification have provided critical evidence to resolve longstanding historical questions, such as uncovering pathologies linked to the early death of Tutankhamen [1] , or identifying the presence of the pathogen Yersinia pestis in bodies excavated from medieval cemeteries [2] , [3] . Because extraneous DNA can be easily amplified during PCR, forensic applications rely on strict controls to avoid false positives [4] , [5] . When used to infer infection in historical samples, DNA techniques can confirm pathogen presence but cannot positively infer disease because a pathogen could be present without causing infection [6] , [7] , [8] . Such applications are particularly valuable in an archeological context, in which differentiating between natural and deliberate causes of death can significantly change the interpretation of a historical event [1] , [2] . Detection of a pathogen, however, is necessary but not sufficient to determine disease because the pathogen could be present without causing infection [6] , [7] , [8] .

Methods

Archaeological Context In 1999, a team of archaeologists led by Johan Reinhard and Constanza Ceruti, uncovered the site of three burials 25 m from the 6,739-m summit of Llullaillaco, a high elevation volcano in the province of Salta, Argentina. The expedition recovered the preserved bodies of two young children (a 7 year-old boy and a 6-year old girl) and one 15-year old adolescent girl known as “the Maiden”. The three children had been sacrificed to Pachamama, the earth goddess, in the ritual of Capacocha [17], [18], [19]. The outstanding condition of the mummies (fig. 1) was the result of the combination of freezing temperature, mild humidity, anaerobic environment and the presence of natural disinfectants. The bodies were buried about 50 cm underground, and the empty space within the tombs was packed with volcanic ash. The ash inhibited the growth of decomposing bacteria and fungi, and acted as a barrier to moisture, protecting the bodies from external humidity while preserving internal moisture. This atmosphere provided the conditions for the subcutaneous fat of the bodies to transform into soap in a process called adipocere [19], [20]. Finally, a layer of packed snow rendered the tombs airtight shortly after their closing. As a result, the bodies were exceptionally preserved and provided more high-quality physical evidence for their state at the time of death than comparable finds from that period anywhere in the world. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. The children of Llullallaico. a) La Doncella (the Maiden); b) El Niño (the Boy); and c) La Niña (the Girl). https://doi.org/10.1371/journal.pone.0041244.g001

Sampling All three Llullaillaco mummies are preserved at Museum of High Mountain Archaeology (MAAM) in Salta (Argentina). They are in airtight, self-contained capsules and maintained at −20°C, in a mix of liquid nitrogen vapor and 2% oxygen. Sampling took place in the cold laboratory adjacent to the mummies’ repository, at −5°C. We sampled a small, blood-soaked piece of cloth from the boy’s cloak, against which his mouth rested. We took four contact mouth swabs from the lips of the Maiden and the boy, since the lips of both presented blood and saliva deposits. The mummy of the young girl (“La Niña”) showed signs of having been struck by lightning (fig. 1) and was not sampled. All samples were placed dry in individual sterile and sealed vials to prevent contamination. They were kept dry at room temperature to avoid any oxidative or hydrolytic lesions to the DNA. The samples were shipped and maintained dry until analyses.

Proteomic Sample Preparation and Analysis Three samples were obtained from the mummies: 1) a 3 mm2 piece of fabric from the boy, 2) a cotton swab from the lips of the boy, and 3) a cotton swab from the lips of the Maiden. All were processed with the same protocol. The excised tips of the cotton swabs and the fabric were cut off and placed in separate low-protein binding 1.5 ml polypropylene microfuge tubes. All sample tubes received 50 mM NH 4 HCO 3 sufficient to cover the sample and incubated at 23°C for 10 min followed by 10 min submersion in a bath sonicator at 23°C. The samples were centrifuged for 5 min at 16,000 G and 23°C, and the supernatant transferred to fresh tubes. The moist fabric and cotton were transferred to 500-µl polypropylene tubes perforated with a 22-gauge needle hole in the bottom. The tubes were place into the original 1.5 ml tubes and the combined tubes centrifuged for 1 min at 16,000 G. The passed-through buffer was combined with the removed supernatants. The tubes containing the supernatant and pass-through were centrifuged at 16,000 G for 5 min and the resultant supernatants (∼100 µl) transferred to fresh tubes. The volume of supernatants was reduced to 20 µl using a Speed-Vac, and each tube subsequently received 20 µl of ACN. The samples were reduced by the addition of 1 µl of 0.1 M DTT and incubated 30 min at 23°C. The samples were alkylated by the addition of 1 µl of 0.2 M iodoacetamide and incubated for 30 min at 23°C in the dark. Each tube then received 10 µl of 5X Invitrosol followed by 1 µl of trypsin at 1 mg/ml. The samples were incubated overnight at 37°C. Following incubation, the samples were centrifuged at 16,000 G for 5 min, the supernatants transferred to fresh tubes, and the volumes reduced to 20 µl in a Speed-Vac. Each tube received 5 µl of 0.1% TFA and sufficient volume of 2% (v/v) acetonitrile, 0.2% formic acid to bring the total volume to ∼50 µl. Each sample was divided into 3 ∼ 15-µl aliquots. One aliquot was subjected to immediate mass spectrometry (MS) analysis, while the others were quick-frozen in liquid N 2 and stored at −80°C. Fifteen µl of the peptide mixture from each residual sample was analyzed by automated microcapillary liquid chromatography-tandem mass spectrometry on a Thermo LTQ-Orbitrap XL mass spectrometer. Fused-silica capillaries (100 µm i.d.) were pulled using a P-2000 CO 2 laser puller (Sutter Instruments, Novato, CA) to a 5-µm i.d. tip and packed with 10 cm of 5-µm Magic C18 material (Agilent, Santa Clara, CA) using a pressure bomb. This column was then placed in-line with an Eksigent 2D HPLC with autosampler. The column was equilibrated in buffer A (2% acetonitrile, 0.1% formic acid), and the peptide mixture was loaded onto the column using the autosampler. The HPLC separation at a flow rate of 300 nl/min was provided by a gradient between Buffer A and Buffer B (98% acetonitrile, 0.1% formic acid). The HPLC gradient was held constant at 100% buffer A for 5 min after peptide loading, followed by a 30-min gradient from 5% buffer B to 40% buffer B. Then, the gradient was switched from 40% to 80% buffer B over 5 min and held constant for 3 min. Finally, the gradient was changed from 80% buffer B to 100% buffer A over 1 min, and then held constant at 100% buffer A for 15 more minutes. The application of a 1.8-kV distal voltage electro-sprayed the eluted peptides directly into the mass spectrometer equipped with a custom nanoLC electrospray ionization source. Full mass spectra (MS) were recorded on the peptides over a 400–2000 m/z range at 60,000 resolution (at m/z 400), followed by five tandem mass (MS/MS) events sequentially generated in a data-dependent manner on the first, second, third, fourth and fifth most intense ions selected from the full MS spectrum (at 35% collision energy). Mass-spectrometer scan functions and HPLC solvent gradients were controlled by the Xcalibur data system (ThermoFinnigan, San Jose, CA). Tandem mass spectra were extracted from raw files with the program RawXtract (fields.scripps.edu). The spectra were searched against a human protein database containing 87,061 protein sequences downloaded as FASTA-formatted sequences from EBI-IPI (database version 3.68) [21] and 54 common contaminant proteins, for a total of 87,115 target database sequences. To calculate confidence levels and false positive rates, a decoy database containing the reverse sequences of 87,115 proteins appended to the target database [22] and the SEQUEST algorithm [23] was used to find the best matching sequences from the combined database. The peptide mass search tolerance was set to 50 ppm. A static modification on cysteines of 57.02146 Da was included. No enzymatic cleavage conditions were imposed on the database search, so the search space included all candidate peptides whose theoretical mass fell within the mass tolerance window, despite their tryptic status. DTASelect [24] was used to filter good peptide matches from the SEQUEST result. Table S1 a full list of the proteins and peptides detected.