Evaluation of microwave-assisted enzymatic digestion and tandem mass spectrometry for the identification of protein residues from an inorganic solid matrix: implications in archaeological research

Abstract

A method based on microwave-assisted enzymatic digestion and liquid chromatography-tandem mass spectrometry analysis is presented for the identification of proteins incorporated within solid matrices using protein standards bound to experimental cooking pottery as a validation model. The implementation of microwave irradiation allowed for a significant decrease in overall analysis time in addition to select enhancement of peptide recovery as determined by label-free relative quantitation. We envision that the reported methodology will provide new avenues for scientific discovery in areas such as archaeology and forensics. Results of this series of experiments are part of an ongoing project directed at developing a comprehensive methodology for extracting proteinaceous residues from archaeological pottery.

Archaeological Protein Residues: New Data for Conservation Science

“…In one of the most surprising recent examples, collagen, a structural protein found in bones, was recovered and identified from a fossilized hadrosaur (Brachylophosaurus canadensis) bone dated to approximately 80,000,000 years ago (Schweitzer et al 2009). This study and others that demonstrate the preservation of biomolecules in ancient and/or experimental samples suggest that the potential for Stevens et al (2010). Four different proteins (bovine serum albumin, bovine casein, bovine collagen and horse myoglobin), were cooked with ceramic at different ratios.…”

“…Specifically, it has been demonstrated that proteins bind to mineral, e.g., ceramic, matrices via the interplay of inter and intra-molecular non-covalent forces including ion exchange, water bridges, Van der Waal bonding, and hydrophobic interactions ( Figure 1). Subsequently, these residues are difficult to remove and detect without the use of corrosive acids, strong detergents, and/or highly sensitive analytical equipment (Craig and Collins 2002;Stevens et al 2010). In addition to binding proteins, such matrices may also facilitate preservation by trapping organics within small pore spaces that restrict the access of microorganisms (Brady and Weil 2002:514) and/or within complex organic conglomerates (Kleber et al 2007).…”

“…Despite some successes in the recovery and identification of archaeological protein residues, several limitations have hindered the widespread application of protein-based studies, most of which are rooted in the shortage of published methodological research (but see Barnard et al 2007;Buckley et al 2010;Brandt et al 2002;Collins 2000, 2002;Solazzo et al 2008;Stevens et al 2010). Too often, it seems that the focus of residue research has been on providing archaeologically meaningful results rather than on addressing fundamental assumptions regarding the behavior of organic chemicals over long periods of time and the suitability of the analytical techniques that have been applied for recovering and identifying archaeological proteins.…”

“…Without additional knowledge to this effect, sampling strategies are reduced to guesswork and key opportunities may be missed due to a potentially misguided assumption that proteins are not likely to preserve under certain conditions. Lastly, much current research is limited by the failure to report quantitative data (but see Collins 2000, 2002;Shanks et al 2004;Solazzo et al 2008;Stevens et al 2010). Without knowing quantitative information, such as the amount of protein that sorbs to ceramic matrices, the percentage of total protein recovered using different extraction strategies, the quantity of protein residues recovered from archaeological samples, or a probability estimate of a particular protein match, it is difficult to gauge the validity of results.…”

Archaeology, Heritage, and Moral Terrains: Two Cases from the Mesa Verde Region

“…Much of this work has been done experimentally, and the audience is referred to Barker et al (2012Barker et al ( , 2015 and Stevens et al (2010) for details. For the purposes of this paper, suffice it to say that we used experimental cooking pottery to develop our approaches.…”