Palaeoproteomic analysis of skeletal remains from Grotta Guattari (Italy)

Excavations at Grotta Guattari (Italy; 66-65 ka) in the previously unexplored Antro del Laghetto area uncovered numerous morphologically unidentified skeletal remains from both the cave's internal (aquatic) and external (dry) sectors, providing an opportunity to investigate how contrasting depositional environments influence protein preservation. Taxonomic identifications were attempted using MALDI-ToF (n = 200) and LC-MS/MS (n = 490). LC-MS/MS provided limited taxonomic identifications for specimens from aquatic environments, whereas MALDI-ToF generated no reliable taxonomic identifications for the same environment. However, it should be noted that the proteomic analysis was performed on randomly selected bone specimens from different locations throughout the cave, so direct performance comparisons between MALDI-ToF and LC-MS/MS cannot be made. The protein aggregation capture (PAC) extraction method failed to generate taxonomic results, whereas a consecutive acid and ammonium bicarbonate (Ambic) extraction combined with LC-MS/MS yielded validated genus-level assignments for 142 specimens. Among these, four specimens were assigned to the genus Homo following additional validation, while acknowledging the limitations imposed by false discovery rate filtering and the absence of a Neanderthal proteome in the reference database. These findings highlight the importance of tailored proteomic workflows for highly degraded archaeological assemblages.