Quantitative Proteomic Profiling of Pinctada fucata Shell Nacre Defines a Solubility-Based Type Classification of Shell Matrix Proteins

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Shell matrix proteins (SMPs) are key organic components of molluscan biominerals, yet previous nacre proteomics have remained largely qualitative, limiting evaluation of the abundance and fraction association of individual SMPs. Here, we established a quantitative proteomic approach for the nacreous layer of the pearl oyster Pinctada fucata by integrating optimized shell preservation, stepwise fractionation, and data-independent acquisition (DIA) proteomics. SMPs were separated into an ethylenediaminetetraacetic acid (EDTA)-soluble matrix (ESM), an EDTA-insoluble but sodium dodecyl sulfate/dithiothreitol (SDS/DTT)-soluble matrix (SSM), and an SDS/DTT-insoluble matrix (ISM). DIA outperformed data-dependent acquisition in proteome coverage and enabled quantification of 327 SMPs across a broad dynamic range. Fraction-resolved abundance profiling showed that each fraction was characterized by distinct SMP compositions. To summarize these distributions, we introduced a solubility-based type classification that grouped SMPs into four types according to their quantitative partitioning among fractions. Well-known SMPs, including nacrein, Pif 80, and MSI60, were assigned to intuitively consistent types, whereas proteases, protease inhibitors, and tyrosinases also showed biased type distributions. These results support a three-compartment model of the nacreous layer consisting of (i) an insoluble interlamellar membrane core, (ii) a relatively extractable interfacial layer, and (iii) a soluble matrix fraction enriched in proteins potentially involved in ionic regulation and protein maturation. This study provides a quantitative framework for understanding coordinated SMP functions during nacre formation and for comparative analyses of molluscan shell proteomes.

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