Proteins and their modifications of the natural mummy of Cangrande della Scala (Prince of Verona, Northern Italy, 1291-1329) were studied. The nano-LC-Q-TOF analysis of samples of rib bone and muscle from the mummy showed the presence of different proteins including Types I, III, IV, V, and XI collagen, hemoglobin (subunits alpha and beta), ferritin, biglycan, vitronectin, prothrombin, and osteocalcin. The structure of Type I and Type III collagen was deeply studied to evaluate the occurrence of modifications in comparison with Type I and Type III collagen coming from tissues of recently died people. This analysis showed high percentage of asparaginyl and glutaminyl deamidation, carbamylation and carboxymethylation of lysine, as well as oxidation and dioxidation of methionine. The most common reaction during the natural mummification process was oxidation-the majority of lysine and proline of collagen Type I was hydroxylated whereas methionine was oxidated (oxidated or dioxidated). To the best of our knowledge, this is the first study which reports the protein profile of a natural mummified human tissue and the first one which describes the carbamylation and carboxymethylation of lysine in mummified tissues.

Department of Diagnostic and Public Health Unit of Forensic Medicine University of Verona Verona Italy

Institute of Physiology Academy of Sciences of the Czech Republic Prague 14220 Czech Republic

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Pataridis S, Eckhardt A, Mikulikova K, Sedlakova P, Miksik I (2008) Identification of collagen types in tissues using HPLC‐MS/MS. J Sep Sci 31:3483–3488. PubMed

Pataridis S, Eckhardt A, Mikulíková K, Sedláková P, Miksik I (2009) Determination and quantification of collagen types in tissues using HPLC‐MS/MS. Curr Anal Chem 5:316–323.

Myllyharju J, Kivirikko KI (2004) Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet 20:33–43. PubMed

Prockop DJ, Kivirikko KI (1995) Collagens: molecular biology, diseases, and potentials for therapy. Annu Rev Biochem 64:403–434. PubMed

Asara JM, Schweitzer MH, Freimark LM, Phillips M, Cantley LC (2007) Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry. Science 316:280–285. PubMed

Janko M, Zink A, Gigler AM, Heckl WM, Stark RW (2010) Nanostructure and mechanics of mummified type I collagen from the 5300‐year‐old Tyrolean Iceman. Proc R Soc B 277:2301–2309. PubMed PMC

Robinson NE, Robinson AB (2004) Molecular clocks: deamidation of asparaginyl and glutaminyl residues in peptides and proteins. Cave Junction, OR: Althouse Press.

Hurtado PP, O'Connor PB (2012) Deamidation of collagen. Anal Chem 84:3017–3025. PubMed

Hill RC, Wither MJ, Nemkov T, Barrett A, D'Alessandro A, Dzieciatkowska M, Hansen KC (2015) Preserved proteins from extinct Bison latifrons identified by tandem mass spectrometry; hydroxylysine glycosides are a common feature of ancient collagen. Mol Cell Proteomics 14:1946–1958. PubMed PMC

Miksik I, Sedlakova P, Pataridis S, Bortolotti F, Gottardo R, Tagliaro F (2014) Prince Cangrande's collagen: study of protein modification on the Mummy of the Lord of Verona, Italy (1291‐1329 AD). Chromatographia 77:1503–1510.

Wadsworth C, Buckley M (2014) Proteome degradation in fossils: investigating the longevity of protein survival in ancient bone. Rapid Commun Mass Spectrom 28:605–615. PubMed PMC

Orlando L, Ginolhac A, Zhang G, Froese D, Albrechtsen A, Stiller M, Schubert M, Cappellini E, Petersen B, Moltke I, Johnson PLF, Fumagalli M, Vilstrup JT, Raghavan M, Korneliussen T, Malaspinas A‐S, Vogt J, Szklarczyk D, Kelstrup CD, Vinther J, Dolocan A, Stenderup J, Velazquez AMV, Cahill J, Rasmussen M, Wang X, Min J, Zazula GD, Seguin‐Orlando A, Mortensen C, Magnussen K, Thompson JF, Weinstock J, Gregersen K, Roed KH, Eisenmann V, Rubin CJ, Miller DC, Antczak DF, Bertelsen MF, Brunak S, Al‐Rasheid KAS, Ryder O, Andersson L, Mundy J, Krogh A, Gilbert MTP, Kjaer K, Sicheritz‐Ponten T, Jensen LJ, Olsen JV, Hofreiter M, Nielsen R, Shapiro B, Wang J, Willerslev E. (2013) Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse. Nature 499:74–78. PubMed

Cappellini E, Jensen LJ, Szklarczyk D, Ginolhac A, da Fonseca RAR, Stafford TW, Jr , Holen SR, Collins MJ, Orlando L, Willerslev E, et al. (2012) Proteomic analysis of a Pleistocene mammoth femur reveals more than one hundred ancient bone proteins. J Proteome Res 11:917–926. PubMed

van Doorn NL, Wilson J, Hollund H, Soressi M, Collins MJ (2012) Site‐specific deamidation of glutamine: a new marker of bone collagen deterioration. Rapid Commun Mass Spectrom 26:2319–2327. PubMed

Deyl Z, Miksik I (2000) Advanced separation methods for collagen parent [alpha]‐chains, their polymers and fragments. J Chromatogr B Biomed Sci Appl 739:3–31. PubMed

Miksik I, Deyl Z (1997) Post‐translational non‐enzymatic modification of proteins II. Separation of selected protein species after glycation and other carbonyl‐mediated modifications. J Chromatogr B Biomed Sci Appl 699:311–345. PubMed

Niwa T (1997) Mass spectrometry in the search for uremic toxins. Mass Spectrom Rev 16:307–332. PubMed

Yim MB, Yim HS, Lee C, Kang SO, Chock PB, Protein glycation—creation of catalytic sites for free radical generation. In: Park SC, Hwang ES, Kim HS, Park WY, Eds. (2001) Healthy aging for functional longevity: molecular and cellular interactions in senescence, Annals of the New York Academy of Sciences, Vol. 928, pp 48–53. PubMed

Mikulikova K, Eckhardt A, Pataridis S, Miksik I (2007) Study of posttranslational non‐enzymatic modifications of collagen using capillary electrophoresis/mass spectrometry and high performance liquid chromatography/mass spectrometry. J Chromatogr A 1155:125–133. PubMed

Fu MX, Requena JR, Jenkins AJ, Lyons TJ, Baynes JW, Thorpe SR (1996) The advanced glycation end product, Nepsilon‐(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions. J Biol Chem 271:9982–9986. PubMed

Saxena AK, Saxena P, Wu X, Obrenovich M, Weiss MF, Monnier VM (1999) Protein aging by carboxymethylation of lysines generates sites for divalent metal and redox active copper binding: relevance to diseases of glycoxidative stress. Biochem Biophys Res Commun 260:332–338. PubMed

Miyata T, Inagi R, Asahi K, Yamada Y, Horie K, Sakai H, Uchida K, Kurokawa K (1998) Generation of protein carbonyls by glycoxidation and lipoxidation reactions with autoxidation products of ascorbic acid and polyunsaturated fatty acids. FEBS Lett 437:24–28. PubMed

Verbrugge FH, Tang WHW, Hazen SL (2015) Protein carbamylation and cardiovascular disease. Kidney Int 88:474–478. PubMed PMC

Trelstad RL, Lawley KR, Holmes LB (1981) Nonenzymatic hydroxylations of proline and lysine by reduced oxygen derivatives. Nature 289:310–312. PubMed

Davies MJ, Fu SL, Wang HJ, Dean RT (1999) Stable markers of oxidant damage to proteins and their application in the study of human disease. Free Rad Biol Med 27:1151–1163. PubMed

Xu G, Chance MR (2007) Hydroxyl radical‐mediated modification of proteins as probes for structural proteomics. Chem Rev 107:3514–3543. PubMed

Dean RT, Fu S, Stocker R, Davies MJ (1997) Biochemistry and pathology of radical‐mediated protein oxidation. Biochem J 324:1–18. PubMed PMC

Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro‐purification, enrichment, pre‐fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906. PubMed