SPEED-E: A modified version of the sample preparation by Easy extraction and Digestion(-free) protocol for enamel-based sex estimation in archaeological remains

Accurate estimation of biological sex in archaeological human remains is critical when considering demographic, resource partitioning, and various sex-based cultural issues in historic societies. Recent developments in paleoproteomics of enamel have allowed for the estimation of biological sex through sex chromosome-linked amelogenins. This method is highly advantageous when traditional osteological sex estimation is precluded by incompleteness, poor preservation, or juvenile age. Here, we have developed Sample Preparation by Easy Extraction and Digestion-free for Enamel (SPEED-E), building on the Sample Preparation by Easy Extraction and Digestion (SPEED) method and direct stage tip clean-up used in paleoproteomic studies. The SPEED-E protocol is similar in extraction time to acid etching protocols, is overall much shorter than digestion-based protocols, and uses relatively less sample. This new method facilitates a rapid analysis of large sample batches where sample value is high and sample material is very limited. Using SPEED-E, we were able to estimate the sex of 85 of 89 deciduous and permanent teeth from the assemblage of archaeological human skeletal remains from the historic First Baptist Church of Philadelphia, Pennsylvania, USA. The indeterminate teeth had limited or no detected amelogenins because of overprinting larger proteins, likely from sampled dentin.