Identification of Protein Remains in Archaeological Potsherds by Proteomics

Abstract

We demonstrate here the possibility of identifying proteins trapped in few milligrams of the clay matrix of It 1200-1400 AD [i'iupiat potsherd fragment from Point Barrow, Alaska, by It dedicated proteomics approach. The four main steps of a proteomics analysis, (i) protein extraction from biological samples, (ii) protein hydroly· sis using a hydrolase enzyme, (iii) nanoLC, nanoESI MS, and MS/ MS analysis of the generated peptides, and (iv) protein identification using protein databank proceeded from genomic data, hltve been optimized for a rcheological remains. llrieRy our procedure starts by grinding the potsherds, extraction with 1 % triRuoroacetic acid, digestion with excess of trypsin, nanoLC, nanoESJ Ff-ICR analysis, and data mining by homology search. The developed conditions were evaluated on protein extracts from remains obtained by heated muscle tissues an!! blubbers of different seal and whale species, these samples representing the main diet sources of the Eskimo population . Most of the proteins were identified by seque nce homology to other species due to the lac k of cetacean and pinniped proteins in the databanks. More interestingly, two proteins, myo· globin and hemoglobin, respectively, identified in muscle tissue samples and blubber samples highlight s everal specific peptides of cetacean and pinniped species; these peptides arc siJ!nificant to prove the presence of these marine species in the analyzed samplcs. Based on the developed methodology and on protein identification results obtained from the heated seaVwhale muscle tissues and blubbers, the analysis of the clay matrix of a 1200-1400 AI) Ifiupiat potsherd fragment from Point Harrow was investigated. The described method succeeds in identifying four peptides corresponding to the harbor seal myoglobin (species Phoca ritulina) with a measured mass accuracy better than 1 ppm (MS and MS/ MS experiments) including one specific peptide of the cetacean and pinniped species and one specific peptide of the seal species. These results hiJ.lhlight, fur the first time , a methodology able to identify proteins from a few milligrams of archeologi· cal potsherd buried for years; the obtained results confirm the presence of a seal muscle tissue protein in this Punuk potsherd.

Validation of a Non-Targeted LC-MS Approach for Identifying Ancient Proteins: Method Development on Bone to Improve Artifact Residue Analysis

“…Mass spectrometry approaches that provide a general scan of archaeological protein residues from cooking pottery, on the other hand, may not pinpoint original residues because exogenous proteins can overwhelm their signal (but see Cappellini et al 2010;Heaton et al 2009;Nielsen-Marsh 2005;Solazzo et al 2008). Put simply, when using mass spectrometry, a morass of exogenous protein from soil bacteria can overwhelm the signals of more archaeologically meaningful residues leaving them undetected.…”

“…Analysis of ancient bone proteins has witnessed substantial advancement during the last decade (see Buckley and Wadsworth 2014;Cappellini et al 2014;Welker et al 2015), some of which can be attributed to the peptide fingerprinting methodology, ZooMS, (Buckley et al 2009;van Doorn et al 2011) but which also relates to the general applicability of high throughput MS to bone (Cappellini et al 2012. In contrast, MS analysis of ancient protein residues from artifacts for the study of past subsistence has lagged (Barker et al 2012, Barnard 2007; but see Heaton et al 2009;Solazzo et al 2008). To address this deficiency, our paper focuses on the analysis of bone proteins using the extraction methodology of Barker et al (2012; Data, Methods & Taxonomies sive evaluation of our methodology as applied to multiple taxa to identify ancient proteins from several sites.…”

Two‐dimensional Fourier transform ion cyclotron resonance mass spectrometry: reduction of scintillation noise using Cadzow data processing

“…[8][9][10] The advances in electronics and computer technology, combined with the fact that gas-free in-cell fragmentation modes (IRMPD [11] and ECD [12,13] ) are routinely installed on commercial FTICR instruments, make it possible to develop 2D FTICR-MS as a useful analytical method for complex samples. The resolving power and mass accuracy of 1D FTICR-MS is sufficient to determine the raw chemical formulae of complex samples even in very low amount archeological samples [14] and 2D FTICR-MS has the potential to give more structural information on each compound.…”

Ötzi, the Tyrolean Iceman

“…Recent innovations in electrospray ionization mass spectrometry (ESI-MS) may allow archaeological researchers to resolve molecular structures of both fats and proteins preserved in archaeological residues that have not thus far been possible to characterize through conventional gas chromatography and mass spectrometry techniques (Mirabaud et al, 2007;Solazzo et al, 2008;Stevens et al, 2010). In electrospray ionization mass spectrometry, a small amount of the substances being analyzed is dissolved in a volatile mixture of solvents and introduced into a chamber where a charged field at the tip of a hypodermic needle disperses the mixture into a fine spray of charged droplets (Fenn et al, 1990;Pramanik et al, 2002).…”

“…Triacylglyceride distributions of ruminant carcass fats and dairy foods become indistinguishable from one another following burial for prolonged periods of time (Heron and Evershed, 1995), but Mirabaud et al (2007) were able to match triacylglyceride distributions of milk fats from modern European cattle to those of potsherd extracts from a long submerged Neolithic settlement at Clairvaux XIV in France. French and American researchers also used ESI-MS to identify protein peptide sequences of seal muscle and whale blubber in the clay matrix of an 800-year-old Inupiat potsherd from Point Barrow in northern Alaska through comparison with modern reference samples using a proteinmatching database (Solazzo et al, 2008). While electrospray ionization mass spectrometry could prove useful in identifying organic compounds in archaeological residues recovered from anaerobic burial environments or those that have remained frozen for prolonged periods, this instrumental technique may have little applicability in characterizing organic residues from the vast majority of archaeological sites.…”