Proteomic analysis of chicken eggshell cuticle membrane layer

Abstract

The eggshell is a barrier that plays an important role in the defense of the egg against microbial and other infections; it protects the developing bird against unfavorable impacts of the environment and is essential for the reproduction of birds. The avian eggshell is a complex structure that is formed during movement along the oviduct by producing a multilayered mineral-organic composite. The extractable proteins of avian eggshells have been studied extensively and many of them identified, however, the insoluble (non-extractable) proteins have been sparsely studied. We studied the EDTA-insoluble proteinaceous film from the cuticle layer of eggshell. This film consists of three main areas: spots (cca 300 μm diameter), blotches (small spots with diameter only tens of μm), and the surroundings (i.e., the area without spots and blotches) where spots contain a visible accumulation of pigment. These areas were cut out of the membrane by laser microdissection, proteins were cleavaged by trypsin, and the peptides were analyzed by nLC/MS (Q-TOF). This study has identified 29 proteins and a further eight were determined by less specific "cleavage" with semitrypsin. The relative abundances of these proteins were determined using the exponentially modified protein abundance index (emPAI) where the most dominant proteins were eggshell-specific ones, such as ovocleidin-17 and ovocleidin-116. Individual areas of the cuticle membrane differ in their relative proportions of 14 proteins, where significant differences between the three quantification criteria (direct, after normalization to ovocledin-17, or to ovocledin-116) were observed in four proteins.

The calcified eggshell matrix proteome of a songbird, the zebra finch (Taeniopygia guttata)

“…The exact role in mineralization played by the identified proteins is not clear at present and cannot be elucidated by proteomic or transcriptomic studies, although the abundance of particular proteins in different shell compartments [ 34 , 35 ], in uterus fluid [ 39 ] and eggshell matrix [ 40 ] at different stages of mineralization, or the transcriptomic and proteomic exploration of eggshell protein expression differences linked to particular traits, such as eggshell strength [ 40 , 89 , 91 ], may yield initial clues. The same applies to the popular in vitro calcium carbonate precipitation and crystallization assays that have also been applied to isolated eggshell proteins [ 12 , 17 , 27 , 78 ] but seem to be rather tentative and vague and may therefore be complemented by modern electron microscopic techniques, such as atomic force microscopy, to study at higher resolution the assembly of matrix on substrates or mineral nucleation on organic matrices [ 120 ].…”

“…The first studies of this type described the analysis of the proteome and phosphoproteome of the chicken calcified layer acid-soluble matrix [ 30 , 31 ]. This was followed by proteomic analysis of other eggshell compartments such as the acid-insoluble matrix [ 32 , 33 ], the eggshell cuticle [ 34 , 35 ], the soluble fraction of the eggshell membranes in conjunction with the innermost eggshell calcified layer (mammillary cones) [ 36 ], and eggshell membranes alone at different stages of chick embryonal development [ 37 ]. In addition, the proteome of the uterus fluid bathing the egg during shell mineralization was compared to the proteome of the calcified shell [ 38 ] and uterus fluid proteomes at different stages of mineralization were compared to each other [ 39 ].…”

Proteomic analysis of dentin–enamel junction and adjacent protein‐containing enamel matrix layer of healthy human molar teeth

“…The LCM system represents a modified microscopic technique and allows selection of cells within histological sections and their contact‐free laser‐mediated catapult into a test tube. The LCM‐dissected tissues are subjected to protein extraction, reduction, alkylation, and digestion, followed by injection into a nano‐liquid chromatography coupled to tandem mass spectrometry (nLC‐MS/MS) system for chromatographic analysis and protein identification .…”

Protein profiles of hatchery egg shell membrane

“…As expected the HESM contained many proteins such as ovalbumin, ovomucoid, ovocleidin-116, and lysozyme that are inherent to egg proteome [8, 30–32]. Structural proteins such as keratins, collagens, decorin, lumican, and tropomyosin, and cytoskeletal proteins, such as actin, tubulin, vimentin, and their interacting proteins, were present along with several blood (hemoglobin, α-D-globin, annexin, fibronectin) and embryo associated proteins (vitellogenin, zona pellucida sperm-binding proteins, and vitelline membrane outer layer protein).…”