The protein composition of the insoluble avian eggshell matrix was studied. The determination of these proteins insoluble in water (EDTA-insoluble) was carried out using enzymatic cleavage followed by a high-performance liquid chromatography-mass spectrometry method. The influence of various enzymes on the protein splitting also was studied. The distribution of proteins depends on the type of layer (localization within the eggshell): ovocalyxin-32 was found mainly in the outer layer (the

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... ); ovocleidin-116 and 17 and ovocalyxin-36 were found throughout the whole eggshell, whereas ovalbumin was only found in the inner layer, the mammillary. The pigment (protoporphyrin IX) was mainly found in the cuticle and is incorporated into the protein network. Average values are from three sets. Tryptophan was not tested for, whereas hydroxyproline and hydroxylysine were tested for but not detected. A = cuticle layer, B and C = palisade layers, D = mammillary layer.