Comparing the Use of Magnetic Beads with Ultrafiltration for Ancient Dental Calculus Proteomics

Over the past two decades, proteomic
analysis has greatly developed
in application to the field of biomolecular archaeology, coinciding
with advancements in LC–MS/MS instrumentation sensitivity and
improvements in sample preparation methods. Recently, human dental
calculus has received much attention for its well-preserved proteomes
locked in mineralized dental plaque which stores information on human
diets and the oral microbiome otherwise invisible to other biomolecular
approaches. Maximizing proteome recovery in ancient dental calculus,
available only in minute quantities and irreplaceable after destructive
analysis, is of paramount importance. Here, we compare the more traditional
ultrafiltration-based and acetone precipitation approaches with the
newer paramagnetic bead approach in order to test the influence of
demineralization acid on recovered proteome complexity obtained from
specimens as well as the sequence coverages matched for significant
proteins. We found that a protocol utilizing EDTA combined with paramagnetic
beads increased proteome complexity, in some cases doubling the number
of unique peptides and number of proteins matched, compared to protocols
involving the use of HCl and either acetone precipitation or ultrafiltration.
Although the increase in the number of proteins was almost exclusively
of bacterial origin, a development that has implications for the study
of diseases within these ancient populations, an increase in the peptide
number for the dairy proteins β-lactoglobulin and casein was
also observed reflecting an increase in sequence coverage for these
dietary proteins of interest. We also consider structural explanations
for the discrepancies observed between these two key dietary proteins
preserved in archaeological dental calculus.