In-depth comparative analysis of the chicken eggshell membrane proteome.

The avian eggshell membrane (ESM) is stabilized by extensive cross-linkages, making the identification of its protein constituents technically challenging. Herein, we applied various extraction/solubilization conditions followed by proteomic analysis to characterize the protein constituents of ESM derived from the unfertilized chicken eggs. The egg white and eggshell proteomes (including previous published work) were determined and compared to ESM to identify proteins that are relatively or highly specific to ESM. Merging the results from different extraction/solubilization conditions with various proteomes allowed the identification of 472, 225, and 488 proteins in the ESM, egg white, and eggshell proteomes, respectively. Of these, 163 and 124 proteins were relatively or highly specific to ESM, respectively. GO term analysis of the common proteins and ESM unique proteins generated 8 and 9 significantly enriched functional groups, respectively. Different families of proteins that were identified as ESM-specific included collagens, CREMPs, histones, AvBDs, lysyl oxidase-like 2 (LOXL2), and ovocalyxin-36 (OCX36). These proteins serve as a foundation for the mechanically stable ESM that rests upon the egg white compartment and is a physical barrier against pathogen invasion. Overall, our results highlight the structural nature of the ESM constituents that are relevant to various biomedical applications, such as wound healing. BIOLOGICAL SIGNIFICANCE: The eggshell membranes (ESM) are a highly resilient double-layered fibrous meshwork that is secreted while the forming egg transits a specialized oviduct segment, the white isthmus. The ESM protects against pathogen invasion and provides a platform for nucleation of the calcitic eggshell (ES). ESM is greatly stabilized by the extensive desmosine, isodesmosine and disulfide cross-linkages which make the identification of its protein constituents by standard proteomic approaches technically challenging. Comparative proteomic analyses of ESM, egg white, and ES proteins showed proteins groups that are relatively or highly specific to ESM. These groups of proteins serve as a foundation for the mechanically stable ESM that rests upon the egg white compartment and is a physical barrier against pathogen invasion. These features are essential for eggshell quality and for the prevention of pathogen invasion which reinforce food safety of the table egg.