Human Bone Paleoproteomics Utilizing the Single-Pot, Solid-Phase-Enhanced Sample Preparation Method to Maximize Detected Proteins and Reduce Humics

Sample
preparation has become an important part of bone proteomics
and paleoproteomics and remains one of the major challenges to maximizing
the number of proteins characterized from bone extractions. Most paleoproteomic
studies have relied on in-solution digestion with the inclusion of
filter-aided sample preparation (FASP) as effective methods to detect
the proteome. However, neither of these are optimal because few proteins
have been detected utilizing only in-solution digestion and the molecular
weight cutoff of FASP may miss remaining fragments of proteins in
fossil bone. The recently developed single-pot, solid-phase-enhanced
sample preparation (SP3) overcomes these issues by not relying on
molecular weight while still controlling where the proteins are digested.
Here, historical human bones were extracted with either 500 mM tetrasodium
EDTA or 400 mM ammonium phosphate dibasic, 200 mM ammonium bicarbonate,
4 M guanidine HCl and digested with the SP3 method. Across all samples,
78 ± 7 (400-200-4) and 79 ± 17 (EDTA) protein accessions
were identified, including previously difficult to detect proteins
such as osteopontin. SP3 also effectively removed 90% or more of the
coextracting humic substances (based on reduced absorbance) from extracted
proteins. The utility of SP3 for maximizing the number of protein
detections in historical bones is promising for future paleoproteomic
studies.