Analytical and Bioanalytical Chemistry

, Volume 406, Issue 29, pp 7633–7640 | Cite as

Proteomic analysis of chicken eggshell cuticle membrane layer

Research Paper

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.

Keywords

Mass spectrometry Bioanalytical methods Cuticle Chicken proteins Insoluble proteins Eggshell 

Supplementary material

216_2014_8213_MOESM1_ESM.pdf (782 kb)
ESM 1(PDF 781 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Institute of Physiology, v.v.iAcademy of Sciences of the Czech RepublicPraha 4Czech Republic

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