LAP-MALDI MS analysis of amelogenin from teeth for biological sex estimation

The biological sex estimation of human individuals can be achieved by extracting fragments of the amelogenin protein from small areas of tooth enamel. The amelogenin gene can be found on both sex chromosomes (X and Y) with chromosome-specific differences in its sequence, and consequently the sequences of the expressed protein in teeth. Virtually all current analytical techniques used to identify the occurrence of the male Y chromosome-specific proteoform employ proteoform-specific peptide analysis by LC-ESI MS/MS, which typically results in longer analytical times due to the LC separation step, despite recent efforts of shortening the LC step. We report a rapid analytical workflow for biological sex estimation by combining minimal acid extraction of amelogenin peptides, including the Y chromosome-specific SM_oxIRPPY peptide, with LAP-MALDI (liquid atmospheric pressure matrix-assisted laser desorption/ionization) MS and MS/MS analysis but without the use of an LC system. A total of 27 peptides from amelogenin and ameloblastin were characterized by MS/MS, revealing oxidation and deamidation as chemical modifications and information on the maturation of amelogenin. The entire sample preparation and analysis time for biological sex estimation using the applied workflow is ≤ 10 minutes, of which only 1 minute is needed for the MS and MS/MS data acquisition. The sample preparation is minimally hazardous, requiring 10 % HCl for peptide extraction, and can be undertaken in non-specialized labs before being submitted to MS and MS/MS analysis. The developed workflow can also facilitate the MS/MS analysis of many other amelogenin peptides without LC separation, providing further proteomic information on protein expression and mRNA transcription. It was applied to the teeth of five males and five females, whose biological sex had been estimated using osteological techniques, from three archaeological sites.