Minimizing Laboratory-Induced Decay in Bone Proteomics

Proteomics methods are being increasingly
used to study archaeological
and palaeontological bone, assisting in species identification and
phylogenetic studies as well as improving our understanding of bone
diagenesis. More recently, there are developing interests in the study
of post-translational modifications, some of which are potentially
diagnostic of decay, but none of the previous extraction methods have
been developed in light of this. To be able to record close to natural
deamidation levels of samples, an extraction procedure should minimize
laboratory-induced decay, such as asparagine and glutamine deamidations,
which are considered most strongly related with decay and known to
occur frequently with standard laboratory procedures. Here we tested
numerous methods to identify an optimal approach of extracting proteins
from bone while minimizing artificial decay. Using a weak acid to
partially demineralize the bone sample, then subsequent incubation
of the acid insoluble fraction with guanidine hydrochloride and enzymatic
digestion in ammonium acetate, we observed an ∼50% reduction
in deamidation while also substantially decreasing the protocol length.
We propose this optimized method as appropriate for studies of archaeological,
palaeontological, as well as potentially forensic investigations using
proteomics where decay measurements could act as “molecular
timers”.