Extracting proteins from microscopic biominerals: A reproducible method development using oyster larvae

Microscopic biominerals are ubiquitous in the ocean, and several major taxa secrete them during early life stages or as adults. Organisms secrete an extracellular proteome incorporated within the biomineral to guide biomineralization remotely and enhance its material properties. This proteome has attracted the attention of extensive scientific research, but its characterization is challenging due to methodological constraints that limit the overall insight, particularly in small organisms. Therefore, we propose this straightforward and reproducible method development for preparing microscopic biominerals before proteome extraction. The method development can be tailored to other microscopic biominerals, and, importantly, it aims to integrate biomineral cleanliness and integrity without sacrificing proteome completeness. First, we suggest running an in-depth sample exploration to identify key sample characteristics and determine the magnitude of the sodium hypochlorite (NaOCl) treatment. Then, we recommend running a multiple time points experiment for biomineral cleaning treatment with a fixed NaOCl concentration. The time points are evaluated using qualitative (visual assessment) and quantitative methods (biomineral loss, elemental composition, and organic structural components removal). Finally, critical time points are identified for method validation using shotgun proteomics. This approach was tested using Hong Kong oyster larval shells as a model organism. Our study discovered that surprisingly, longer treatments and partial biomineral damage are preferred for Hong Kong oyster larvae and do not lead to protein diversity loss but enrichment. This microscopic biomineral cleaning method development can facilitate harnessing information from increasingly diverse biomineral proteomes.