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1000 - MOLECULAR SIGNATURES OF PROTEIN AGING IN ANCIENT HUMAN BONES FROM THE ERUPTION AREA OF VESUVIUS. Georgia Ntasi (1) - Piero Pucci (1) - Gennaro Marino (2) - Leila Birolo (1) University of Naples Federico II / Chemical Sciences, University of Naples Federico II / Chemical Sciences, Naples (1) - University of Naples / Suor Orsola Benincasa, University of Naples / Suor Orsola Benincasa, Naples (2) Keywords: Paleoproteomics, Mass Spectrometry, Cultural heritage, Protein Degradation, Ancient proteins Introduction: In search of markers for protein aging, we use the singular case of collagen from human bones from the excavations sites in Pompei, Herculaneum, Oplonti, S.Paolo Belsito, and Scalandrone Bay as control reference site. Deamidation of glutamine and asparagine is considered as aging molecular marker [1]. Moreover, other chemical modifications that are a likely signature of diagenetically derived alterations were considered. Methods: Specific proteomic protocols and different sample preparation were adapted to improve protein extraction and sequence coverage yields. The analyses were performed with the use of LC-MSMS. The exploitation of bioinformatic tools such as the error tolerant search in the Mascot suite [2] or the open mass modification utility in the Protein Prospector database search [3] were used for data processing in search of unexpected chemical modifications. Results: Ancient human bones (100) have been analyzed by proteomic approach following an adapted protocol based on the already developed one reported in [4]. From the raw LC- MSMS data and the use of Mascot platform, COL1α1 and COL1α2 were detected with a protein sequence coverage up to 80% in all the samples. All the deamidation sites were listed and their occurrence and deamidation state were statistically evaluated. The statistical analysis is suggestive of the most detected positions, that were considered the best candidates for the quantification of the deamidation level as protein’s aging biomarkers in ancient human bones. Similar approach was used to evaluate also other chemical modifications. Conclusions: The analysis of the deamidation in the different samples suggests that the deamidation level in the samples from Pompei is low in respect to an extensive deamidation observed in the samples from Herculaneum and Scalandrone (our control reference). Our preliminary results seem to suggest that the low deamidation level could be linked to the peculiar burial conditions and to the “lower” temperature experienced by Pompei inhabitants in comparison to Herculaneum ones. Novel Aspect: This set of samples constitutes an extraordinary test case to evaluate deamidation and other chemical modifications as molecular signatures of protein aging. 1.Leo G, Cartechini L, Pucci P, Sgamellotti A, Marino G, Birolo L. Proteomic strategies for the identification of proteinaceous binders in paintings. Anal Bioanal Chem. 2009;395(7):2269-2280. doi:10.1007/s00216-009-3185-y. 2.Cappellini E, Jensen LJ, Szklarczyk D, et al. Proteomic Analysis of a Pleistocene Mammoth Femur Reveals More than One Hundred Ancient Bone Proteins. J Proteome Res. 2012;11(2):917-926. doi:10.1021/pr200721u. 3.Hill RC, Wither MJ, Nemkov T, et al. Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen. Mol Cell Proteomics. 2015;14(7):1946-1958. doi:10.1074/mcp.M114.047787.

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4.Vinciguerra R, De Chiaro A, Pucci P, Marino G, Birolo L. Proteomic strategies for cultural heritage: From bones to

paintings ☆. Microchem J. 2016;126:341-348. doi:10.1016/j.microc.2015.12.024.