Insoluble eggshell matrix proteins – their peptide mapping and partial characterization by capillary electrophoresis and high‐performance liquid chromatography

Abstract

Avian eggshell matrix proteins were studied by two analytical approaches. Peptide mapping was done by trypsin and pepsin followed by collagenase cleavage; analyses were carried out by capillary electrophoresis and reversed-phase high-performance liquid chromatography (HPLC). Comparison of peptide maps obtained by both methods revealed a complex mixture of peptides in the insoluble layers of the eggshell; it was concluded that there are at least three different insoluble protein/peptide layers in the avian eggshell (cuticle, palisade, and mammillary layer). Partial characterization of peptides in each layer was made by HPLC-mass spectrometry analysis. There is an evidence that the eggshell insoluble proteins contain species susceptible to collagenase cleavage, however, the sequences split by this enzyme probably are not those typical for the main triple-helical core of collagenous proteins. It is proposed that the action of collagenase upon eggshell proteins is caused by the side effect of collagenase described previously with synthetic peptides. Some of the proteins present are probably glycosylated. Fatty acid content in the insoluble eggshell layers (after decalcification) was in the range of 2-4% (which reflected both lipid and lipoproteins bound fatty acids). Porphyrin pigments are dominant in the cuticle layer.

Recent developments in capillary electrophoresis and capillary electrochromatography of peptides

“…Both techniques, coupling to a solid support (controlled pore glass), and cross-linking reaction without any solid support, confirmed the advantages of application of immobilized enzymes for peptide mapping: high stability and high activity of the enzyme and noncontamination of protein maps by enzyme fragments due to the suppressed autolysis. CZE and HPLC peptide mapping have been used for characterization of insoluble avian eggshell matrix proteins; better CZE separation and higher number of resolved peaks were obtained in FS capillary dynamically coated with the triblock copolymer Pluronic F127 [77] than in the bare FS capillary [283]. In some advanced systems, CE and CEC peptide mapping is online connected with previous protein digestion by enzymes (trypsin, pepsin) immobilized in microreactors directly coupled to separation capillary [68][69][70].…”

Peptide mapping by capillary electrophoresis with Pluronic F127

“…For example in the previous paper [11] we concluded that in all three layers of the eggshell matrix none of the currently described soluble proteins from the eggshell were present. When we look at the peptide profiles, particularly at the molecular mass of the peptides arising after trypsin cleavage, we found a few peptides the molecular mass of which was identical with peptide fragments of soluble eggshell's ovocleidin 116 [12]; on the other hand some peptides had a similar molecular mass of the peptides/proteins of completely different origin as is, e.g., bovine serum albumin.…”

“…Proteins from avian eggshells have been studied mainly during the last decade, however, these studies were limited to the soluble species only. Recently [11] we made the first attempt to analyze the insoluble protein/peptide-based constituents of the eggshell matrix by capillary electrophoresis and HPLC/MS. It was proven that these matrices are really complex mixtures of proteins/peptides, some of which are glycosylated.…”

Capillary electrophoresis of proteins 2003–2005

“…CZE helped in characterizing insoluble eggshell matrix proteins [270], ribosomoal proteins in Escherichia coli [117], human antithrombin glycoisoforms [271], thaumatin [272], human complement protein C4 [273], intact yeast proteins [162], and identified microbial mixtures based on their marker proteins [274].…”