Mass spectrometry can also be used for the analysis of ancient microbial proteins. Several studies have reported the use of matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF MS) for ancient animal proteins or peptides (17,18,33,50,51), which paved the way for a unique application in paleomicrobiology (52). In our laboratory, we have developed a simple and efficient protocol to extract proteins from ancient dental pulp specimens (Figure 3) for MALDI-TOF MS analysis. After incubating 0.1–1 g dental pulp with 1 mL 500 mM EDTA, pH 8.0, with agitation at room temperature for 24 h, the specimen is sonicated five times (1 min each), centrifuged at 17,900× g for 40 min at room temperature, and dialyzed overnight in 2 L of a solution containing 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl. The dental pulp collected from eight individuals from four burial sites dating between 700 and 8500 years ago yielded 0.13±0.3 mg protein/mg dental pulp (53). Further MALDI-TOF MS profiling of the dental pulp allowed for classification of ancient mammal individuals (53). Ancient mammals were previously identified by analyzing dried muscle and skin collected from well-preserved museum specimens, hair shafts from permafrost-preserved animals, and bones and teeth of buried animals (54-57). These tissues are directly exposed to the environment, and contamination and degradation of surface proteins have been observed (58). The analysis of ancient mycobacterial proteins in ancient human remains was recently achieved using MALDI TOF/TOF MS (52). One could also predict that MALDI-TOF MS could detect host signature specific to infection.Late serology
Late serology is based on the previous observation that IgG can be detected by dot-blotting and using a modification of this method called quantitative dot-blotting in 0.12-Myr-old equid bones from Atapuerca and 1.6-Myr-old equid and hominid fossil bones from Venta Micena, Orce, Granada (59). In late serology analysis, immunoglobulins extracted from ancient bone specimens are exposed to a pathogen-specific antigen (60). The reactivity of such ancient immunoglobulins against T. pallidum demonstrated the presence of syphilis in a 200-year-old skeletal specimen from Easter Island with anthropologic lesions indicative of treponemal infection; a parallel PCR-based approach demonstrated the presence of T. pallidum subspecies pallidum DNA (60). The authors found that extensively purified IgG reacted strongly with T. pallidum, and they concluded that late serology had the advantage of being far less prone to contamination and false-positive results than aDNA PCR; however, late serology cannot distinguish among T. pallidum subspecies (60).Analyzing ancient microbial lipids
The detection of ancient microbial lipids has been applied to the detection of M. tuberculosis in ancient skeletons (61-64). Indeed, mycolic acids and mycocerosic acids are unique components of the M. tuberculosis cell wall and can be used to detect this pathogen in ancient human skeletons (64-66). These lipids appear to be particularly robust (66) and detectable in minute quantities in ancient remains (63). High-performance liquid chromatography (HPLC) is one method for detecting ancient mycobacterial lipids (62,64) and complements the detection of M. tuberculosis aDNA (67). Likewise, detection of M. tuberculosis aDNA in the Karkur calcified pleura (AD 600) was confirmed by parallel HPLC-based detection of mycolic acids, establishing the first detection of M. tuberculosis in a nonmummified tissue other than bone (61). By parallel analysis of M. tuberculosis aDNA and mycolic acids in 1000-year-old ribs collected in an early medieval (Anglo-Saxon) cemetery in Addingham, West Yorkshire, it was shown that mycolic acids are more reliable when diagnosing ancient tuberculosis than IS6110 and that mycolic acids can be of value in tracing tuberculosis back into antiquity (64). Additionally, HPLC-based detection of mycolic acids has been used as an independent way to confirm the detection of M. tuberculosis aDNA in human ribs, arm bones (adult), and long bones (infant) at the site of Atlit-Yam, 10 km south of Haifa. Calibrated radiocarbon dates range from 9250–8160 years before the present, indicating a date during the last phase of the Pre-Pottery Neolithic C period (68). Mycocerosic acids and other specific components of the M. tuberculosis cell wall have been detected in the ribs of 49 individuals from the 19th- and 20th-century Coimbra Identified Skeletal Collection, half of those being historically documented as tuberculosis (66). This work indicated that mycocerosic acids are valuable biomarkers for the diagnosis of ancient tuberculosis (66). MALDI-TOF MS has been recently used to detect mycolic acids in five bone specimens from different archaeological periods (AD 600 to AD 1600); a diagnosis of tuberculosis was made based on positive M. tuberculosis complex DNA detection (69). Ancient mycolic acid MALDI-TOF MS profiles were compared with the spectra derived from noninfected, healthy bone samples from the processed cemeteries as negative controls (69). This report suggests that MALDI-TOF-MS is a rapid and reproducible technique for the detection and identification of ancient mycobacterial infections (69). However, recent reinterpretation of the data pointed to a lack of convincing evidence that the MALDI-TOF MS peaks were indeed mycolic acids (70). Use of MALDI-TOF MS for the detection of nonprotein ancient biomolecules remains an open field of research.