Timothy Cleland

Single-pot solid-phase-enhanced sample preparation for paleoproteomics and removal of humics

Timothy P. Cleland§, Christine A.M. France§

§ Museum Conservation Institute, Smithsonian Institution, Suitland, MD, USA

Sample preparation for ancient- and paleoproteomics is essential to detect the maximum number of proteins, identify in vivo and diagenetic post-translational modifications on the proteins without inducing artifacts, and separate interfering molecules (e.g., humic substances). Recent sample preparation improvements including the usage of filter-aided sample preparation (FASP) resulted in more complete characterization of ancient proteomes compared to in solution digestion alone. However, the FASP method uses a molecular weight cutoff system and as a result may lead to the loss of smaller preserved protein fragments. The recent development of the single-pot solid-phase-enhanced sample preparation (SP3) method can overcome the loss of low molecular weight protein fragments in the FASP method while also potentially separating non-proteinaceous contaminating molecules (e.g., humic substances). Using historical human bones and multiple protein extraction methods, we applied the SP3 method to evaluate the numbers of proteins extracted, effects of protein extraction method on SP3, apparent removal of humics, and reproducibility between SP3 applications. We have found insignificant (p >> 0.05) differences in protein concentration, especially for both collagen I alpha chains, between replicates of the SP3 method suggesting it has high levels of reproducibility. Additionally, after protein capture, desalting, and drying, residual brown residue was observed that we interpret as humic substances not retained by the SP3 beads. This suggests the SP3 method effectively removes these contaminating molecules. SP3 represents a promising new method to prepare paleorproteomic samples without the limitations of molecular weight cutoffs.