Elena Schroeter

A method to concentrate and clean peptides from fossils with high humic content for mass spectrometry

Elena Schroeter§, Michael Goshe§, Mary Schweitzer§

§ North Carolina State University, Raleigh, NC 27695, USA

Humics are diagenetic products of decaying organic matter that co-extract during protein solubilization from fossil specimens. These dark substances are notoriously difficult to separate from proteins in solution, and cause a number of problems for researchers attempting protein sequencing of fossil taxa. I introduce a method that combines elements from multiple techniques into a single extraction protocol that both concentrates proteins and removes these humic substances from fossil extracts, allowing for clean samples that can be analyzed by mass spectrometry (MS) without interference. This method includes 1) a non-demineralizing extraction buffer, which eliminates the protein loss observed during the commonly applied demineralization stage of extraction; 2) filter-aided sample preparation (FASP) of peptides, which concentrates and digests proteins in one filter and allows the separation of large humics after digestion; 3) centrifugal stage-tipping, which allows the removal of small humics in a process that, unlike manual stage-tipping, is uniform and can be performed simultaneously on multiple samples. Application of this protocol to a 1000-year-old moa bone with very high humic content resulted in samples that were colorless, and which produced a wider window into the moa proteome than previous MS analyses on this same specimen using a more standard MS workflow.