Assessing the degradation of ancient milk proteins through site-specific deamidation patterns

Abstract

The origins, prevalence and nature of dairying have been long debated by archaeologists. Within the last decade, new advances in high-resolution mass spectrometry have allowed for the direct detection of milk proteins from archaeological remains, including ceramic residues, dental calculus, and preserved dairy products. Proteins recovered from archaeological remains are susceptible to post-excavation and laboratory contamination, a particular concern for ancient dairying studies as milk proteins such as beta-lactoglobulin (BLG) and caseins are potential laboratory contaminants. Here, we examine how site-specific rates of deamidation (i.e., deamidation occurring in specific positions in the protein chain) can be used to elucidate patterns of peptide degradation, and authenticate ancient milk proteins. First, we characterize site-specific deamidation patterns in modern milk products and experimental samples, confirming that deamidation occurs primarily at low half-time sites. We then compare this to previously published palaeoproteomic data from six studies reporting ancient milk peptides. We confirm that site-specific deamidation rates, on average, are more advanced in BLG recovered from ancient dental calculus and pottery residues. Nevertheless, deamidation rates displayed a high degree of variability, making it challenging to authenticate samples with relatively few milk peptides. We demonstrate that site-specific deamidation is a useful tool for identifying modern contamination but highlight the need for multiple lines of evidence to authenticate ancient protein data.

Dairying enabled Early Bronze Age Yamnaya steppe expansions

“…Additionally, within the two identified casein peptides, there is only one possible amino acid deamidation site, which renders any estimation of the antiquity of these peptides exceedingly challenging. A previously published paper 38 demonstrates the extreme variability in deamidation of amino acids in milk proteins, which further limits our ability to confirm the authenticity of this dairy finding. The calculus from the two additional Botai individuals demonstrated adequate preservation, but also lacked evidence for dairy consumption.…”

The spread of herds and horses into the Altai: How livestock and dairying drove social complexity in Mongolia

“…Peptide mass tolerance was set at 10 ppm, with fragment mass tolerance of 0.01 Da, with an allowance for monoisotopic mass shifts. As we reduced and alkylated the disulfide bonds, carbamidomethyl of cystine (C) was set as a fixed modification, and based on previous observations of modifications detection in ancient dental calculus [ 29 – 31 ], we chose deamidation of asparagine (N) and glutamine (Q) and oxidation of methionine (M) as our variable modifications.…”

Forensic proteomics

“…The role of protein modifications also plays a potential role here. For instance, deamidation of hair proteins has been measured to increase over a period of decades [292] and has also been used to estimate the post-mortem interval [355], date archaeological material and separate out archaeological protein from modern contaminating keratins [88,179,[360][361][362][363]. Deamidation is known to occur at different rates depending on the amino acid, flanking protein sequences and protein flexibility J o u r n a l P r e -p r o o f [364][365][366][367][368].…”

“…Deamidation is known to occur at different rates depending on the amino acid, flanking protein sequences and protein flexibility J o u r n a l P r e -p r o o f [364][365][366][367][368]. These complex and variable chemical and environmental factors result in a high level of variance of deamidation at different time points, indicating it is more of a preservation and exposure signal than an indicator of time [102,183,363]. These processes will be accentuated and possibly dominated by the introduction of environmental microflora that will use the protein as a supply of nutrient and introduce non-specific proteases.…”